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rine Biological

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THE

CHEMICAL ACTIVITIES
OF BACTERIA

ERNEST F. GALE, B.A., Ph.D., Sc.D.

FKI-L,0\V OF ST. JOHN's COLLKGE, CAMBRIDOK

DIRECTOR OF THK MKUICAL RESEARCH COUNCIL UNIT FOB CHEMICAL MICROBIOLOGV

AND READER IN CHE5IICAL MICROBIOLOGY IN THE UNIVERSITY OF CAJIBRIDGE

1951

New York

ACADEMIC PRESS INC.

LONDOX

UNIVERSITY TUTORIAL PRESS LTD.

This bool: is copyrighted. Xo 2^ortion. of it may be
reproduced by any process without written pertiiission.
Enquiries should be addressed to the p^iblishers.

Published 1947

Second Edition 194S

Third Edition 1951

f'HINTED IS (.RKAT HUriATV RY UNIVK.RSITY TUTORIAL PRESS
SKAR CAMRRIDOE

PREFACE TO THE THIRD EDITION

The application of biochemical techniques to the study of
bacteria has thrown considerable light on the mode of existence
of these organisms and, in turn, such studies have assisted the
development of our knowledge of the fundamental biochemistry
of Hving cells. In this book I have attempted to produce an
account of the chemical activities of bacteria which will be
useful to students reading biochemistry, bacteriology, or
chemistry, and which will provide an introduction to more
advanced study. I hope also that the book will prove useful
to research w^orkers who require a concise but not detailed
account of the background to ^^resent research in chemical
microbiology. The treatment is elementary and in no sense
comprehensive, and the bibliography has been limited, for the
inost part, to review articles and textbooks.

A comparatively new subject, such as this is, tends to
develop rapidly and, although experimental facts must remain
true, the accumulation of new facts inevitably alters the
approach towards certain aspects of the subject. For examj)le,
during the last three or four years considerable advances have
been made in our knowledge of synthetic systems and their
control in the bacterial cell by "genes". I have attempted to
incorporate some of these new ideas, and the facts on which
they are based, in this third edition. In the course of making
the changes I have found it desirable to alter the order of
presentation of some of the material and to condense some
sections in order that the book shall remain of a23proximately
the same size.

I wish again to express my indebtedness to the late Dr
Marjory Stephenson, F.R.S., for awakening and encouraging
my interest in bacterial chemistry. I wish also to thank all
those of my colleagues who have helped me to correct and
avoid errors during the preparation of this book.

E. F. G.

Medical Research Council Unit
for Chemical Microbiology,
Biochemistry Laboratory,
Cambridge.
1951

CONTENTS

CHAPTER TAGR

I. Bacteria as Chemical Agents 1

II. The Nature and Identification of Bacteria 10

III. Bacterial Enzymes 24

IV. The Formation of Enzymes in Bacteria ... 58

V. Growth: Synthesis of Bacterial Proto-
plasm ... 82

VI. Bacterial Polysaccharides 119

VII. Provision of Energy: Fermentation ... 122

VIII. Provision of Energy: Oxidation 148

IX. Breakdown of Nitrogenous Material ... 157

X. The Nitrogen Cycle 176

XI. Pathogenicity; Chemotherapy ... ... 186

Index 205

65957

ADDENDUM

While this edition was in the press, Ochoa and co-workers
have published a series of papers [J. biol. Chem., 1950, 187,
849 et seq.) dealing with the fixation of carbon dioxide by
animal and bacterial cells. They have shown that COg will
combine with pyruvic acid in the presence of reduced coenzyme
I (in bacteria) to form malic acid directly, the enzyme con-
cerned being known at present as the "malic enzyme": —

CO2 + CH3 . CO . COOH + CoE . H2

= HOOC.CH2.CHOH.COOH + CoE.

In the schemes put forward on pp. 135 and 137, the first stage
in CO2 fixation is shown as a carboxylation of pyruvic acid by
reversal of oxalacetic decarboxylase. It has not been possible
to demonstrate convincingly that the bacterial oxalacetic
decarboxylase is reversible and the first stage in the fixation
process should be represented as a direct formation of malic
acid by the "mafic enzyme" without the intermediate forma-
tion of oxalacetic acid.

In the "citric acid cycle" outlined in Fig. 12, citric acid is
shown as a side-product not involved in the reactions of the
actual cycle. Evidence has accumulated during the past
year that citric acid itself is involved in the cycle. It is
formed by the condensation of acetyl-phosphate and oxalacetic
acid and then gives rise to m-aconitic acid and the other
substrates shown in the cycle. It is probable, in the light of
the findings concerning the "malic enzyme," that the
oxalacetic acid is formed via malic acid and not directly from
CO2 and pyruvic acid as shown in Fig. 12.

THE CHEMICAL
ACTIVITIES OF BACTERIA

CHAPTER I

BACTERIA AS CHEMICAL AGENTS

The perceptible environment is composed of atoms, some
existing separately, the great majority in constantly changing
molecular combinations. The velocities of reaction between
these molecular combinations vary over a very wide range and
it is probable that only a minute fraction of the chemical
changes occurring in the environment are perceived by man
since the time for which he can make observations is so short.
The chemist makes a study of these changes and endeavours
to speed them up in the laboratory by such tricks as raising
the temperature, adding strong acids or alkalis, introducing
catalysts, etc. But if he studies the world outside his test-
tubes and flasks, he soon becomes aware that biological
material is able to carry out many types of chemical change
with far greater ease and at considerably greater speed than
he is able to achieve in his laboratory. In fact, one of the
properties which differentiate between living and non-living
material is this property of producing rapid and fundamental
change in the chemical environment ("metabolism").
Living material is aggregated in organisms and we divide
organisms for convenience into macro- and micro-organisms.
The term "micro-organism" includes several subclasses such
as the unicellular yeasts, protozoa, fungi, bacteria, etc., and
it is amongst these organisms that we find the widest range
and highest rates of metabolic activities.

As an index of the rate of metabolism we can take
the rate of oxygen consumption which is usually measured
as the c.mm. of oxygen taken up per hour by 1 mg. dry
weight of cell material and is called the Qog of the cells.
If we compare the respiratory activities of various living

CHEM. A.B. 1

BACTERIA AS CHEMICAL AGENTS

cells in this way, we get values for the Q02 of the following

order :

Mammalian liver cells Qog =

2—5

Mammalian kidney cells

4—10

Yeast cells

50—100

Esch. coli ^
Acetobacter^BsLctena,

100—300

ca. 1000

Azotobacter ,

ca. 3000

In this book we shall concern ourselves with bacteria only
as, although it is by no means certain that these are the most
active of the micro-organisms, they have been studied more
intensively up to the present than the other types.

Bacteria have such a wide distribution that there are few
places on or near the surface of the earth, in the waters of
the earth, or in the air near the earth, which are free from them.
They are found in hot mineral springs, in Arctic snows, in
stagnant salt lakes, in oil-saturated soil around oil-wells, in
the acid effluents from gas-works, etc. The only places free
from bacteria are those in which a sterilising influence is at work ;
where heat, sunlight, or caustic chemicals render life impossible,
or in the interior tissues of healthy plants and animals.

Bacterial multiplication takes place under most diverse
conditions and bacterial multiplication involves the formation
of cell-substance or protoplasm which, in turn, involves the
synthesis of all the complicated concomitants of free-living
existence such as proteins, amino-acids, carbohydrates,
lipoids, nucleic acids, growth factors, prosthetic groups of
enzymes, etc. Many of these substances can be synthesised in
the laboratory only with extreme difficulty, if at all. Not only
is there the synthesis of complex molecules to be accomplished
but in many cases these syntheses are further complicated by
considerations of positional isomerism which give rise to the
formation of several substances of the same empirical formula,
only one of which is biologically effective. To take a simple
example, we have the amino-acid R.CHNHg.COOH which,
as it contains an asymmetric carbon atom, exists in two
isomeric forms, one the structural mirror-image of the other.

ISOMERISM OF BIOLOGICAL COMPOUNDS

An examination of the alanine, CHg.CHNHg.COOH, of

bacterial protoplasm will show that it consists almost entirely

of the laevo- form, whereas the alanine synthesised in the

laboratory will consist of equal parts of laevo- and dextro-

forms. If the organism is given synthetic alanine as a source

of nutrient, it will utilise the laevo- form, while leaving the

dextro-alsiiome almost untouched, although it has been shown

recently that very small amounts of D-alanine are taken up

by the growing cells. If we hydrolyse the proteins of bacteria

we find that they are composed almost entirely of the laevo-

forms of the various amino-acids, although in some cases, as

in the antibacterial peptides excreted by certain Bacilli, a

proportion of the constituent amino-acids are found to be of

the dextro- form. Some twenty odd amino-acids have been

isolated and proteins consist of these amino-acids condensed in

various permutations and combinations into peptide chains.

Proteins differ in the order and sequence of the amino-acids in

the chain, but so far the synthesis of any single protein has

eluded the chemist in his laboratory.

Further complications of positional isomerism are met in

structures of the nature of coenzyme I, adenine-nicotinamide-

dinucleotide, which plays a part as a carrier of hydrogen in

cellular respiratory processes. Analysis of hydrolytic and

enzymatic degradation products of this substance leads to a

structural formula:

H
N = C-N

! II

H ^/°\ /^ C = C-C-NH2

C^u n^C OH OH i ii

c— c CH2OP-0-P-OCH2 ,0^ / r.
II II -^c-^ ^/ "

H \^cf H
I I
OH OH

I I

H H

CONH;

but synthesis in vitro is made difficult by the facts that the
whole molecule is essential for biological activity and that
any alteration in the nature or position of the linkages around

BACTERIA AS CHEMICAL AGENTS

the ribose molecules renders the substance inactive. The
power to synthesise proteins, nucleotides, etc, is a property
of many living tissues and in some bacteria we have organisms
which are able to synthesise all these substances from very
simple raw materials and at considerable speed. For example,
the group of bacteria known as the chemosynthetic auto-
trophes synthesise bacterial protoplasm from purely inorganic
sources, utilising COg, NH3, and inorganic salts as the raw
material from which is produced all that chemical complex
forming the multiplying cell.

The synthetic abilities of bacteria form an absorbing
problem for the chemist, although these abilities are not
necessarily exceptional amongst living cells — we do not yet
know sufficient about this aspect of metabolism to say how
exceptional or unexceptional the synthetic powers of bacteria
may be — but when we come to consider the destructive
(catabolic) activities of bacteria we are faced with a bewildering
diversity of chemical potential. It is common experience
that when an organism dies and falls on to the surface
of the earth, it will disappear in the course of time.
Carcases and corpses are buried, dead plants and plant
trimmings are composted, excreta are spread on open fields,
and, in due course, they are altered into some form not
recognisably related to the original. This is mainly due
to the scavenging action of soil micro-organisms which by
their destructive abilities break down the dead material and
convert it into bacterial protoplasm (fungal protoplasm, etc.)
and various soluble products. Think for a moment what this
involves: chemically inert proteins such as keratin; poly-
saccharide complexes such as chitin and cellulose; fats,
hydrocarbons, lipoids, sterols, etc., are broken down into
simpler substances which are assimilated, putrefied, or fer-
mented, with the resultant production of bacterial protoplasm,
salts, ammonia, carbon dioxide, gaseous N2 and H2, etc.
Bacteria which can oxidise sulphur to sulphuric acid exist
in sulphuretted waters, others exist in soil deriving energy
for existence from the oxidation of hydrogen to water, while

RANGE OF CHEMICAL ACTION BY BACTERIA 0

others are found in the soil around oil-wells which oxidise
paraffin hydrocarbons to carbon dioxide and water. It is
probably not unscientific to suggest that somewhere or other
some organism exists which can, under suitable conditions,
oxidise any substance which is theoretically capable of
being oxidised.

Here then we have a small sample of the fascinating field
of chemical activity presented by bacteria. There are many
questions which immediately occur to the chemist. How do
these micro-organisms carry out these reactions which cannot
be achieved in the laboratory ? Is it possible to utilise their
activities to carry out such and such a reaction 1 Can their
metabolic activities be exploited on a commercial scale ?
Why are some bacteria pathogenic to man ? Bacterial meta-
bolism has been studied ever since the initial investigations of
Pasteur, and as new techniques are devised our knowledge is
continually increasing and accumulating, but it is still true
to say that we understand only a very small part of the
activities of bacteria and there is immense scope for research
in this field. In this book an attempt will be made to answer
some of the queries that arise in the mind of the chemist,
and in many cases the answers will be such, that they will
merely indicate our need for further research.

Chemical reactions carried out by living material take place
in simple steps and these steps can often be demonstrated
within the cell either by suitable treatment of the cell, or by
the addition of chemicals which will combine with intermediate
products or with enzymes involved in the formation of these
products, and so break up complete reactions into their
individual steps. The number of basic reactions is few and
include the following:

1 . Reduction : the addition of hydrogen or, alternatively, the
removal of oxygen from the molecule attacked.

2. Oxidation: the removal of hydrogen or, alternatively, the
addition of oxygen.

3. Dehydration : the removal of HgO from the molecule.

BACTERIA AS CHEMICAL AGENTS

4. Hydrolysis: the addition of HgO to the molecule, a step
which is usually followed by a splitting of the molecule at
the link hydrolysed.

5. Deamination: the removal of -NH2 from the molecule.

6. Decarhoxylation : the removal of COg from -COOH.

7. Phosphorylation : the esterification of the molecule with
phosphoric acid — usually accomplished by the transfer of
the phosphate radicle from some substance other than
phosphoric acid itself.

8. Dephosphorylatio7i : the removal by hydrolysis of phos-
phoric acid from phosphorylated compounds.

These eight possibilities may all be utilised in the attack on
a given molecule by different bacteria. It is the fact that
different bacteria can and do utilise different methods of
attack on the same substrate molecule that gives rise to the
varied products of bacterial activity and to the apparently
involved and complicated metabolism of the order as a whole.
The metabolism of the cells of highly organised tissues
living in a constant environment, such as those of the mam-
malian body, seems to be simple compared with that of
bacteria which live in varied environments. The blood of the
rat does not differ greatly from the blood of man and the
metabolism of a rat muscle-cell, rat liver-cell, or rat kidney-
cell does not differ greatly from the metabolism of human
muscle-, liver-, or kidney-cell, or from the metabolism of
similar cells in another rat. But the metabolism of a
cell in a culture of Escherichia coli may differ greatly from
that of a cell of the related Aerohacter aerogenes or even
from that of a cell of another culture of Escherichia coli
grown under different conditions. The metabolism of the
bacterial cell is dependent not only on the intrinsic or
potential composition of the organism but also on the
environmental conditions holding during its division from the
mother-cell. To take an example, consider the molecule
of pyruvic acid, CH3.CO.COOH. The muscle-cells of

DIFFERENT METHODS OF ATTACK

man, rat, frog, etc., will reduce this to give lactic acid,
CHg . CHOH . COOH. A culture of Aerobacter aerogenes grown
at pH 8 will attack it by hydrolysis to give acetic and
formic acids:

CHg . CO . COOH -f H2O = CH3 . COOH + H . COOH.

The same organism grown in the same medium but adjusted
to ^H 6 will utilise a third method of attack by decarboxyla-
tion :

2CH3.CO.COOH= CH3.CO.CHOH.CH3 + 2CO2,

while another organism, Propionihacterium, will reduce
pyruvic acid to propionic acid CH3. CHg. COOH. The fact
that a given culture of a bacterium will attack a certain
substance in a certain way means nothing more than that a
culture of that identical organism grown and tested under
identical conditions will attack that substance in that way;
vary the growth conditions, the experimental test conditions,
the strain, species, genus, or family of organism and we cannot
say, without further experiment, anything about the reaction
that will occur. At first sight it would seem as though we
have here a biological problem which is uncontrollable from a
chemical point of view by reason of its possible variations.
But the situation is not as hopeless as it may first seem;
the nature of the attack on a given substance by a bacterium
depends upon

1. the bacterium,

2. the conditions under which it grows,

3. the conditions under which it is tested.

By taking typical organisms and studying their chemical
reactions under various growth and experimental conditions,
we have already acquired a considerable amount of knowledge
concerning the factors governing the variations under (2) and
(3). Once we have covered the ground with one organism,
we can repeat with others closely and distantly related, and,
fortunately, we often find that there are certain fundamental
principles underlying the variation of activity with environment.

8 BACTERIA AS CHEMICAL AGENTS

It will be seen as we go along that it is often possible to
predict how a given organism may react to a given chemical
environment or how to arrange the chemical environment in
such a way that bacteria might be expected to carry out a
desired chemical task — although only experiment will tell
whether they will actually do so.

Before embarking on this problem we must first have some
understanding of the reasons why bacteria attack their
environment at all. When an organism is inoculated into a
suitably nutrient medium, it begins to grow, synthesising
new bacterial protoplasm with consequent increase in size
until eventually division takes place with the formation of
two cells from one. The rate at which subsequent divisions
occur depends to a large extent upon the nature of the medium
but an organism such as Escherichia coli living in a rich
medium such as a tryptic digest of casein can divide once
every ten or fifteen minutes. At this rate one organism can
give rise to over one million organisms in five hours. Con-
sequently one organism can synthesise over one million times
its own weight of bacterial protoplasm in five hours. This
high rate of synthesis must take place at the expense of the
environment which has to supply all the raw materials
including major requirements of carbon and nitrogen; minor
requirements of phosphorus, sulphur, and iron, and traces of
many other elements. Since these elements may be present
in the medium in a form not primarily utilisable by the
organism, it must attack the complex substances present in
the medium so as to render the raw materials available in an
assimilable and utiHsable form. Secondly, the synthesis of
this chemical complex of the bacterial cell involves the
expenditure of energy and this the organism obtains by the
degradation of energy-rich substances in the environment.
Thirdly, if the physico-chemical properties of the environment
vary to any significant extent during the synthesis, then the
organism reacts by speeding up those reactions tending to
stabilise the internal environment. For example, the decom-
position of carbohydrate for energy purposes usually results in

REASON FOR CHEMICAL BREAKDOWN OF ENVIRONMENT 9

the formation of acid end-products and a consequent fall in
the pH of the medium, which may be of such dimensions as
to steriUse the activities of the growing organism ; under such
conditions some organisms are capable of catalysing neutralisa-
tion reactions which either have alkaline end-products or
result in the alteration of acid products to neutral ones, so
that some degree of stabilisation of the internal environment
is accomplished. The organisms therefore attack their
environment to obtain material for growth, energy for
synthesis, and stabilisation against unfavourable conditions.

CHAPTER II

THE NATURE AND IDENTIFICATION OF BACTERIA

Bacteria

Bacteria are simple unicellular organisms which, multiply,
often very rapidly, by binary fission. The majority possess
no chlorophyll, though bacterial chlorophyll does occur in
the photosynthetic organisms (see p. 86). No nucleus is
visible in the bacterial cell if this is examined by the usual
methods, although the application of paiticular staining
techniques will reveal the presence of what are usually called
" nuclear structures " in the cytoplasm.

Size

Large bacteria such as Clostridium welchii may have a
length as great as S-bfi (1/x = 0-001 mm.) and, at the other
end of the scale, we have various Micrococci with a diameter of
300-500 m/x (Im/x = 0-001 /z). The larger organisms thus
approximate in length to half the diameter of tJie red blood-
corpuscle or of the yeast-cell. At the other end of the scale
it is difficult to draw a line between the smaller bacteria and
the larger viruses, especially if we take into account the
Rickettsia which, in size and properties, fall between the true
bacteria, which can exist outside the cells of a host, and the
viruses which can multiply only within the cells of a host.
The frontispiece shows the gradation in size from CI. welchii,
streptococci, rickettsia, and viruses large and small, to protein
molecules. Most bacteria can be cultivated in laboratory
media of varying complexity, but in general it is true to say
that the smaller the organism, the poorer its synthetic powers
and, consequently, the more parasitic it becomes, until in the
ultimate stages shown by the viruses a sufficiently nutrient
medium is supplied only in the interior of the living cells of
a host. A particle having the size of the Foot-and-Mouth-
disease virus is sufficiently large to accommodate about 50-100
protein molecules only.

PROPERTIES USED FOR IDENTIFICATION 11

IDENTIFICATION
General

An organism is identified by a consideration of many
properties including its shape, staining reactions, biochemical
reactions, pathogenicity, etc. Many of the properties of an
organism tend to change with conditions of cultivation, such
as the nature of the growth medium, the age of the culture,
the temperature, the degree of aerobiosis, etc., and conse-
quently classification must be based as far as possible upon
stable properties tested under standard conditions. There
is no point in this book in attempting a detailed account of
the theory or practice of systematic classification, but the
non-bacteriologist requires some guidance concerning the
identification of particular organisms, so the following
represents a brief account of the properties which are
investigated for purposes of classification.

Morphology

Some bacteria are spherical, some rod-shaped, comma-
shaped, or twisted like a spiral, and all varieties of intermediate
shapes occur. The shape, where it is constant, is easily
observed through the microscope and formed the basis of
many of the earlier systems of classification. Thus spherical
organisms were called " Cocci," rod-shaped organisms
*' Bacilli," and spiral-shaped " Spirilla." This simple
morphological grouping has nowadays been complicated by
subdivision of the groups on a basis of other characteristics.

Spore formation

Some bacteria possess the capacity to produce spores which
are a resting or non-vegetative form considerably more
resistant to heat, desiccation, or unfavourable chemical
environments than the vegetative forms. It was thought at one
time that these organisms form spores when their environ-
ment becomes unsuitable for continued vegetative existence
but this is not necessarily the case, as it is known that some

12 THE NATURE AND IDENTIFICATION OF BACTERIA

organisms will form spores only if their environment is
nutritionally rich, and spore formation then appears to be
part of the normal life-cycle of the organism. Whatever
may be the cause of their formation, spores constitute a form
in which the organism can survive for long periods under
adverse conditions. When the environment again becomes
suitable for vegetative existence, the spores germinate to
form normal cells capable of multiplication as usual.

Staining reactions

A useful test that can be applied to bacteria as an aid in
diagnosis is their reaction to the staining technique invented
by Christian Gram. The dried organisms (in the form of a
smear on a microscope slide) are stained with a dye of the
pararosaniline series (see p. 203) such as crystal violet and
then treated with iodine solution. The preparation is then
washed with alcohol until no more violet dye washes off the
slide and finally counterstained with a dye of contrasting
colour — usually a red dye such as carbolfuchsin. Under
this treatment some organisms retain the violet dye and are
said to be " Gram-positive," whilst the violet dye is washed
out of others by the alcohol, these are stained red by the
counterstain and are said to be " Gram-negative." It has
been shown recently that the staining complex in Gram-positive
organisms is a nucleoprotein which can be extracted from the
cells which then stain Gram-negative. For some reason
not yet understood, those organisms which are Gram-positive
differ in general — there are individual exceptions — from the
Gram-negative organisms in being more exacting nutritionally
(see Chap. V), having more restricted chemical activities,
and in being more sensitive to the action of chemotherapeutic
agents such as penicillin, the sulphonamides, the triphenyl-
methane dyes, and the acridine dyes.

Cultural characteristics

The nature and composition of the medium in which the
organism will or will not grow may aid its identification (see

PROPERTIES USED FOR IDENTIFICATION 13

Chap. V). Wlien a satisfactory growtli medium lias been
obtained, further assistance in identification can be obtained
from the investigation of colony form. The medium is mixed
with agar-agar, sterilised, and poured while hot into Petri
dishes ; on cooling, the medium solidifies as a sheet of nutrient
jelly. The organisms, in high dilution, are streaked on to the
surface of the solid medium, the dish covered, and then
incubated. Each organism on the medium proceeds to
multiply and to form a small pile or " colony " which, after
24-4:8 hours, is visible to the naked eye as a tiny stud or con-
vexity on the surface of the medium. Each single colony may
represent a pure culture in that it has arisen from a single
organism and, if the cells are far enough apart at inoculation,
then discrete and distinct colonies will appear on the plate.
Colonies of different organisms have different appearances:
Esch. coli gives smooth, round, translucent colonies; Strepto-
coccus faecalis on media containing glucose forms small, round,
white colonies; Staphylococcus aureus forms round, raised
colonies which turn golden-yellow or orange after 48 hours
incubation; Serratia marcesce^is gives blood-red colonies;
while Proteus vulgaris, which is highly motile, forms big, flat,
spreading colonies that look like mountain ranges on a contour
map.

Oxygen requirement

Bacteria fall into four main groups according to the oxygen
tension they can tolerate for growth:

1. Strict aerobes: organisms which can multiply only in
the presence of oxygen.

2. Facultative anaerobes : organisms which can live equally
well in the presence or complete absence of oxygen.

3. Microaerophilic organisms: organisms which can live in
the absence of oxygen or in the presence of very low
oxygen tensions, high tensions being inhibitory.

4. Strict anaerobes: organisms which can multiply only in
the complete absence of oxygen.

14 THE NATURE AND IDENTIFICATION OF BACTERIA

The nature of the metabolism of an organism is closely con-
nected with the aerobic or anaerobic nature of its growth
conditions.

Biochemical characteristics

This book is mainly concerned with the variety of chemical
changes that bacteria can produce in their environment.
Where these changes can be detected easily they can often
be used to separate individuals which appear to be alike in
morphological and other characteristics. This will be dealt
with in greater detail below.

Serological characteristics

When a foreign body, particularly if it is of protein nature,
is introduced into the blood-stream of an animal it may there
act as an antigen and stimulate the animal to produce
antibody. The serum of the animal will then contain the
antibody which will react specifically with the antigen.
Bacterial cells are antigenic and if we inject bacterial cells
into an animal, the serum of that animal will eventually
contain antibody which will react with the cells in vitro so as
to produce a visible result such as agglutination. Since the
antibody formed in response to the injection of an antigen
is specific for that antigen, the reaction can be used as a
delicate test for that antigen. The surface of the bacterial
cell may contain several different antigens and the composition
of the surface varies from one organism to another. Conse-
quently the serum prepared as a result of the injection of an
organism A will react with cells of A itself or of organisms
possessing the same antigen in their surface. By preparing
the antibody to A we can therefore determine what other
cells belong to the same antigenic group or, alternatively,
we can divide a collection of organisms into groups according
to their antigenic reactions. For example, the species
Streptococcus haemolyticus has been divided by Lancefield
into a number of groups, known as the Lancefield Groups
A, B, C, D, etc., by serological methods. By a modification

SYSTEMATIC CLASSIFICATION 15

of the method it has been possible to divide the groups still
further into serological types so that, for example, Lancefield
Group A streptococci have been divided into some thirty- two
serological types.

Animal inoculation

The medical bacteriologist dealing with pathogenic
organisms has a further possibility of characterising an
organism by the lesions it produces after inoculation into a
suitable animal host. Thus the tubercle organism can be
identified by the lesions it produces after injection into a
guinea-pig.

CLASSIFICATION

By combinations of the tests outHned above it is possible to
separate organisms into groups and sub-groups. There will
always be individuals which will not fit cleanly into any set
grouping, but the majority can be assigned to various pigeon-
holes in a systematic classification. In some cases the out-
standing characteristics will be morphological, cultural, or
pathogenic, while in others differentiation will be based upon
finer investigation of a multiplicity of biochemical reactions.
Many systems of classification have been used in. the past and
there is, unfortunately, no definite agreement upon any one
system at the present time. The nomenclature used in this
book is that adopted by the Society of American Bacteriologists
and detailed in Bergey's Manual of Determinative Bacteriology.
In Table I the names of the main Families, Tribes, and Genera
used in this book are outlined, but for details concerning the
rationale of the classification and for the differentiation of the
groups, reference must be made to the Manual and other
standard textbooks of bacteriology. -

ORDERS AND FAMILIES

The whole group of micro-organisms which come within our

description of "bacteria" is strictly termed Schizomycetes

^(fission-fungi) and is divided into several orders. Most

THE NATURE AND IDENTIFICATION OF BACTERIA

TABLE I

Outline of Systematic Classification of the Order
Etjbacteriales

Family

Tribe

Genus

Nitrobacteriaceae

Nitrobacterieae
Thiobacilleae

Nitrosomonas

Nitrobacter

Thiobacillus

II.

Pseudomonadaceae

Pseudomonodeae
Spirilleae

Pseudomonas

Acetobacter

Vibrio

Desulphovibrio

Spirillum

III.

Azotobacteriaceae

Azotobacter

IV.

Rhizobiaceae

Rhizobium

Micrococcaceae

Micrococcus

(Staphylococcus)

Sarcina

Neisseriaceae

Neisseria
Veillonella

VII.

Lactobacteriaceae

Streptococceae
Lactobacilleae

Diplococcus

Streptococcus

Leuconostoc

Lactobacillus

Propionibacterium

VIII.

Corynebacteriaceae

Cory nebacteriu m

Enterobacteriaceae

Eschericheae

Serrateae

Proteae

Salmonelleae

Escherichia

Aerobacter

Serratia

Proteus

Salmonella

Shigella

Eberthella

XII.

Bacteriaceae

Bacterium
Methanobacterium

XIII.

Bacillaceae

Bacillus
1 Clostridium

SYSTEMATIC CLASSIFICATION 17

biochemical studies have been carried out with members of
the order Euhacteriales in which the organisms exist as
separate individuals and do not show any form of mycelium
or filaments. The order is subdivided into families, partly
on a morphological basis and partly on a chemical basis.
Eleven of the families differentiated by Bergey are included in
Table I. The Nitrobacteriaceae comprise organisms which can
carry out an oxidation of inorganic material as source of energy.
The Azotohacteriaceae and the Rhizobiaceae are both capable
of utilising atmospheric nitrogen as nitrogen source, the latter
carrying out the fixation process only when living in symbiosis
with a host-plant (see Chap. X). The Micrococcaceae are
usually Gram-positive spherical organisms, while the Gram-
negative coccal organisms are placed in the Neisseriaceae ; the
spherical organisms which divide to form chains, the Strepto-
cocci, are not included in these groups but are classed in the
Lactobacteriaceae since they carry out a simple lactic acid
fermentation of glucose, and generally resemble the Lacto-
bacilli in their nutritional and chemical characteristics. The
rod-shaped organisms are divided into several families: the
Bacillaceae being those organisms which can form spores under
suitable conditions, the non-sporing rod-shaped organisms are
subdivided and most of the organisms of this group with which
we shall be dealing in this book are classed in the Entero-
bacteriaceae which comprises many of those bacteria normally
found in the intestinal flora.

TRIBES AND GENERA

In many cases families include organisms which fall clearly
into sub-groups. Thus the Lactobacteriaceae are subdivided
into spherical organisms, Streptococdeae, and rod-shaped
organisms, Lactobacilleae. Again, within the family Nitro-
bacteriaceae, we have organisms using inorganic nitrogenous
substances as oxidation substrate, the Nitrobacterieae, and
other organisms, obtaining energy from the oxidation of
sulphur compounds, which are consequently placed in a

cHEM. A. B. 2

18 THE NATURE AND IDENTIFICATION OP BACTERIA

separate " tribe " called the Thiohacilleae. The Entero-
bacteriaceae are divided into a number of tribes, some of which
are shown in Table I; the divisions in this case are based on
less well-defined characteristics and are still the subject of
debate amongst taxonomists.

The next subdivision of the tribe is into genera. Again the
basis of subdivision may be morphological as in the separation
of Streptococci which are spherical organisms dividing about
one axis to form long chains, from Diplococci in which the
organisms occur in pairs rather than in chains. The separation
of genera within the Micrococcaceae is similar: when division
takes place evenly about three axes to give cubical packets the
organisms are called Sarcina ; when division occurs about two
axes to give plates the organisms are called Micrococci ; a third
genus. Staphylococcus, used to comprise organisms dividing
unevenly to give " bunches of grapes," but the latest edition of
Bergey includes the Staphylococci within the Micrococci as
" variants." In the Bacillaceae two genera are differentiated
by oxygen tolerance, thus the Bacilli are strict aerobes and the
Clostridia, strict anaerobes. In many cases the differentiation of
genera within a tribe is based upon biochemical characteristics;
thus the Lactobacilli ferment glucose to produce lactic acid
only, while the Propionibacteria have a more varied array
of fermentation products including propionic acid. The
differentiation between Escherichia and Aerobacter also rests
largely on differences in fermentation (see Chap. VII).

SPECIES
An organism can be allocated to a family and genus along
the lines already indicated, but the differentiation of species
within many genera is mainly a matter of biochemistry, that
is, the species are separated by their reactions in a number of
simple biochemical tests. If we examine the fermentation of
a number of sugars such as sucrose, glucose, maltose, fructose,
etc., by different organisms, we shall find, without going into
any detailed examination of the products, that some organisms
can ferment some sugars but not others. Further, if we add

SYSTEMATIC CLASSIFICATION 19

indicator to our sugar media and immerse a small inverted
tube full of media in the culture-tube, then we shall find
that the fermentation of a given sugar by one organism gives
rise to acid as shown by a change in the colour of the indicator,
while fermentation of the same sugar by another organism
gives rise to acid and also gas as shown by the accumulation
of bubbles inside the inverted tube. If we extend our range
of fermentation substrates to include such as xylose, mannitol,
dulcitol, glycerol, salicin, etc., we shall find that we have
already a method for the separation of certain individuals
from others by the range and nature of their fermentations.
With these tests we can combine others which involve simple
manipulation and observation, such as those for the formation
of indole from protein or amino-acid media (see p. 172) or
for the formation of acetylmethylcarbinol during the fermen-
tation of glucose (see p. 136). By using a series of such tests
we find that it is possible to differentiate many common
organisms, as can be seen from the selection of organisms and
their reactions in Table II (see p. 22).

The following are some of the common species mentioned
in this book:

Family I

NiTROSOMONAS species all utilise ammonia as source of nitrogen and

energy. These organisms are very difficult to isolate in pure

culture and no methods of separating species have yet been

described.
NiTROBACTER specics utilise nitrite as nitrogen source and cannot

utilise ammonia.
Thiobacillus thio-oxidaxs is found in sulphur- containing soils and

waters, and obtains energy by the oxidation of sulphur to sulphuric

acid, to which it is very resistant.

Family II

PsEUDOMONAS AERUGINOSA (pyocyanea) is a common soil organism
which produces a distinctive blue-green pigment, pyocyanine,
which is excreted into the medium. It is non- pathogenic but
proves to be extremely diflScult to eradicate from wounds.

AcETOBACTER spccies are found in the vinegar industry and as con-
taminants in brewing vats. Acetobacter xylinum produces a form of
capside made of cellulose.

20 THE NATURE AND IDENTIFICATION OF BACTERIA

Destjlphovibrio desulphuricans can be isolated from mud and
sulphur-containing waters. It is a comma-shaped organism which,
in some media, grows to give spiral-shaped organisms of con-
siderable length and marked motility. It reduces sulphate to
hydrogen sulphide.

Family V

Micrococcus pyogenes var. aureus (Staphylococcus aureus) is
the common organism producing pus in Wounds, boils, etc. It is a
spherical organism producing orange colonies on solid media. It is
one of the organisms which cause septicaemia and ostiomyelitis.

Family VI

Neisseria intracelluxaris (Meningococcus) is the causal organism

of meningitis.
Neisseria gonorrhoeae (Gonococcus) is the causal agent in

gonorrhoea.

Family VII

Streptococcus haemolyticus, the causal organism of scarlet fever,

streptococcal septicaemia, puerperal fever, and streptococcal throat.

The organism causes lysis of red blood-cells by secretion of a

haemolysin and the species has been divided into serological groups

and types; the human pathogens belong mainly to group A.
Streptococcus eaecalis, one of the common intestinal inhabitants.

It is non-pathogenic although some variants are haemolytic and

belong to the haemolytic group D.
Streptococcus lactis, the common non-pathogenic streptococcus

of milk. This organism can be clearly differentiated from S.

faecalis by biochemical and serological tests. Both S. faecalis and

S. lactis are used for nutritional assay procedures (see p. 110).
Lactobacillus caset, one of several species used for nutritional

studies and normally found in milk.

Family VIII

CoRYNEBACTERiUM DiPHTHERiAE, the causal Organism of diphtheria.

Family X

Escherichia coli, numerically the most common intestinal bacterium.
This organism is easily grown in large quantities, is non-pathogenic,
has very wide chemical activities, and has consequently been
subjected to more intense biochemical investigation than any
other bacterium.

Aerobacter aerogenes, an organism' whose chemical activities are
similar to those of EscJi. coli but which is more commonly found in
association with soil and plant materials than in intestinal contents.

Proteus vulgaris, a highly motile soil organism usually found in
association with putrefying material. It often proves a nuisance
when it becomes established in wounds since, although it is non-
pathogenic, it is insensitive to almost all the present antibacterial
agents used in chemotherapy.

SEPARATION OF STRAINS 21

Salmonella species are food-poisoning organisms which produce
toxins when growing in protein- containing media such as meat
products, egg powders, etc.

Shigella species cause dysentery if they become established in the
intestinal flora.

Eberthella typhosa is the causal organism of typhoid,

Fanuly XIII

Bacillus subtilis is a common air and soil inhabitant. Organisms
of this and related species are now proving fruitful sources of new
antibiotics.

Bacillus anthbacis is the causal organism of anthrax.

Clostridium tetani is the causal organism of tetanus.

Clostridium Botulinum is the food-poisoning organism causing
botulism. Its exotoxin is the most toxic substance known.

Clostridium welchii is an intestinal organism which, if it becomes
established in a wound, produces a number of toxins and gives rise
to the condition known as gas gangrene.

Clostridium acetobutylicum is non-pathogenic and is used com-
mercially for the production of acetone and butyl alcohol.

STRAINS

Using the chemical, morphological, and cultural tests
described above it is possible to divide organisms into genera
and species, but we cannot identify any particular strain of
organism with certainty. For example, any organism having
the characteristics in Table II (plus such others as are com-
monly used for finer differentiation) of, say, Esch. coli will be
called ''Esch. coli'' But organisms conforming to the Esch.
coli test form the bulk of the flora of faecal matter and
we can isolate millions of ''Esch. coli " from a particle of
faeces. Although these organisms all give the characteristic
tests it does not follow that they are all identical or have
sprung from one common stock. When we come to examine
enzyme systems other than those involved in the systematic
tests, or if we examine the rates at which the various sugars,
etc., are attacked, we shall find wide differences between the
various organisms that have been isolated and called "Esch.
coli " and we say that these different organisms are different
" strains " or " variants " of Esch. coli. Strains are often

<11

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<I1

<ii

<I3

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2.2

wth
anaer.

<s>

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2 2

P. 3

o
P5

-d

OQ w

C/JIJ

••

•

_«0

•

•«»

s
S

:§

-o

ft.

Ijq

a o .s

a a ^
I I I

p • s

I I I I

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<1 ^

^ 73

i^73 ^

-^ i s ^

o £ S bO
^ Ti ,^

p^^ 2 c

I I I I

Ph><iJO
> <5

ABBREVIATIONS 23

identified by the name of the person who first isolated them,
thus: CI. septicum Pasteur. Whenever we isolate a new
Esch. coll we must assume that it is a new strain different
from any other, as two cultures cannot be said to be of the
same strain unless they have identical chemical properties,
qualitatively and quantitatively, under all possible conditions
of growth. In practice we never assume that two organisms
are of the same strain unless we know that they have both
been cultivated from the same mother-culture (or ideally
from the same mother-cell), and even then it is not uncommon
for an organism to give rise to two or more strains by mutation
in the course of serial subcultivation. The chemist working
with bacteria must be careful to specify not only the species
but also the strain of any organism used for a given purpose,
and it does not follow that a published experiment can be
repeated unless the identical strain used in the original work
is used for the repetition. The relation between serological
types and biochemical strains has not yet been sufficiently
clarified for any general statement to be made ; it is probable
that a group of organisms belonging to one serological type
would contain strains separable on biochemical grounds.

Abbreviations

The standard abbreviations for generic names have been
adopted in this book: Bad. for Bacterium, B. for Bacillus,
CI. for Clostridium, Esch. for Escherichia, Pr. for Proteus,
Ps. for Pseudomonas, Staph, for Staphylococcus, S. for
Streptococcus. Where genera are mentioned or species not
included in Table I, these have been given their full titles.

FOR FURTHER READING

Manual of Determinative Bacteriology, Bergey (Bailliere,
Tindall and Co.).

Handbook of Practical Bacteriology, Mackie, T. J., and
McCartney, J. E. (Livingstone).

The Bacterial Cell, Dubos, R. (Harvard University Press).

Fundamentals of Bacteriology, Frobisher, M. (W. B. Saunders).

CHAPTER III

BACTERIAL ENZYMES

Enzyme action

The chemical activities of bacteria and other living tissues
are due to the catalytic action of enzymes. Enzymes are
organic substances which are produced by living cells and
which act as catalysts of specific reactions. They have
properties similar to those of catalysts used in chemical
processes in that they speed up the velocity of a reaction
without altering the nature or proportions of the products
and without adding any energy to the reacting system.
Other things being equal, the velocity of the reaction is
proportional to the concentration of the catalyst or enzyme.
Enzymatic activity is dependent upon certain physical con-
ditions being suitable; thus an enzyme is active over a small
range of pH only, the value at which it displays maximal
activity being known as the " optimal joH." For the
majority of enzymes this optimal ^H value lies between
pH 5 and 8, but in individual cases it may be within wider
limits of 2-10. A catalyst can speed up a reaction which is
already proceeding slowly, but it cannot by itself initiate a
reaction, and it is as yet uncertain whether this is or is not the
case with enzymes. Many reactions which take place in the
presence of living tissues will not noticeably take place in their
absence, but, as has already been pointed out, many chemical
reactions occur at too slow a rate to be perceived by man
through his senses. It is not illogical therefore to regard
enzymes as catalysing reactions which are normally occurring
at an insensible rate, but many authorities are of the opinion
that enzymes, by straining the structure of the molecule upon
which they act and so rendering it more susceptible to change,
can initiate new reactions. Enzymes also differ from most

SUBSTRATE SPECIFICITY 25

chemical catalysts in that they are thermolabile or destroyed
by heat. A number of enzymes have now been isolated in a
pure and even crystalline state and are found to be proteins
the structure of which is altered (or denatured) by heat.

The substance whose chemical change is catalysed by an
enzyme is said to be the '' substrate " of that enzyme and
the majority of enzymes display a strict specificity towards
their substrate. This specificity may be such that the
enzyme can catalyse the alteration of one substance only;
thus succinic dehydrogenase is an enzyme which will catalyse
the removal of hydrogen from succinic acid to form fumaric
acid:

CH2.COOH Succinic CH.COOH

I - 2H > II

CHo . COOH dehydrog. CH . COOH

but the enzyme is specific towards succinic acid and will
dehydrogenate no other substance. Where the substrate
exists in two or more isomeric forms the enzyme is usually
specific for One isomer only; thus L-lysine decarboxylase will
decarboxylate L-lysine to cadaverine but is unable to attack
D-lysine or any other amino-acid:

H2N.CH2.(CH2)3.CHNH2.COOH - CO2 >

L-lysine

H2N . CH2 . (CH2)3 . CH2 . NH2

Cadaverine

Such optical specificity has been used in the past for the
resolution of racemic mixtures since the enzyme will attack
one isomer and leave the other intact. The specificity may,
on the other hand, be less restricted so that an enzyme may
catalyse the alteration of any substance containing a certain
chemical grouping; thus some proteases may hydrolyse any
substance having the linkage — CO — NH — in its structure
and will hydrolyse proteins and peptides down to amino-acids.
Other proteases, however, will attack the peptide link only
if the residues R and S on either side of the link R — CO —
NH — S have specific structures.

26 BACTEEIAL ENZYMES

This specificity of enzyme action is important in that it
means that an organism such as a bacterial cell must possess
enzymes for each process it carries out at a rate greater than
that which will occur in the absence of living cells. Consequently
an organism which carries out numerous different chemical
reactions must contain numerous enzymes in its constitution.
If an organism possesses succinic dehydrogenase it will
dehydrogenate succinic acid to fumaric acid (or, alternatively,
since the reaction is a reversible one, will hydrogenate fumaric
to succinic acid in the presence of a hydrogen-donator). If it
possesses an enzyme called " fumarase " it will hydrate
fumaric acid to L-malic acid; if it possesses L-malic dehydro-
genase, malic acid will be dehydrogenated to oxalacetic acid;
if it possesses transaminase then oxalacetic acid can be con-
verted to aspartic acid, etc.

CH2.COOH Succinic CH.COOH Fumarase CH2.COOH

CH2 . COOH dehydrog. CH . COOH CHOH . COOH

Succinic acid Fumaric acid Malic acid

Malic CHg.COOH Transaminase CH2.COOH
> I > I

dehydrog. CO. COOH CHNHg.COOH

Oxalacetic acid Aspartic acid

So we get a chain of reactions in which each step is catalysed
by a specific enzyme. If any one of these enzymes is missing
then the chain stops at the substance forming the substrate of
the missing enzyme. The distribution of enzymes among
bacteria is very varied and, taking this chain of reactions as
example, bacteria exist in which four, three, two, one, or none
of these enzymes are present and consequently the end product
of succinic acid metabolism in various organisms may be
succinic acid, fumaric acid, L-malic acid, oxalacetic acid, or
L-aspartic acid. Here then lies the key to the variety of
chemical reaction displayed by bacteria ; every substance that
is metabolised passes through a chain of small alterations,
each one involving a simple chemical change and each catalysed

REACTION ■ CHAINS 27

by a specific enzyme the distribution of which is by no means
universal.

The nature of enzyme action means that if a reaction is a
reversible one, then the enzyme-catalysed reaction is also
reversible and the back reaction will be catalysed to the same
extent as the forward reaction so that the final equilibrium
mixture is the same whether the whole reaction is catalysed
or not. This means that the degree to which a given meta-
bolite in a chain of reactions will accumulate will depend upon
the velocity of the forward and backward reactions of the
various steps in the chain, and all the substances in one chain
may be side-tracked into another chain by an alteration in
the conditions governing the various equilibria. Where a
given molecule may be dehydrogenated in organism A, it
may be hydrolysed in organism B with the production of a
different chain of reactions. Taking the above series of
reactions as example again, Esch. coli possesses an enzyme
" aspartase " which brings about a reaction between fumaric
acid and ammonia to produce aspartic acid without the other
intermediate steps:

CH.COOH Aspartase CH^.COOH

II +NH3 ^=^ I

CH.COOH CHNH2.COOH

Other organisms possess an oxalacetic decarboxylase which
decarboxylates oxalacetic acid to pyruvic acid and that opens
up an enormous number of possible reaction chains (see
Chap. VII).

CHg . COOH Oxalacetic CH3

I - CO2 > I

CO. COOH decarboxylase CO. COOH

The final product of succinic acid metabolism in an organism
will depend therefore upon, first, the enzymes present in the
organism and, second, upon the various side reactions occurring
at the particular moment studied. The chemical variety of
bacterial action is therefore based upon the permutations and

28 BACTERIAL ENZYMES

combinations of the enzymes of the organism and their inter-
play with the external environment.

THE STUDY OF ENZYMES IN BACTERIA

When organisms are growing in a medium, their chemical
activities involve the building of cellular material and the
breakdown of substances in the medium ; thus we may have
one sort of protein being hydrolysed outside the growing cell
and another sort being synthesised within the cell. The
number of enzymes concerned may be very great and their
integrated activities too complex to disentangle. The first
step in the simplification of the system is to eliminate synthetic
reactions by preventing growth ; this is performed by removing
the cells from the growth medium, washing them free from
traces of medium, and then suspending the washed cells in
distilled water or a suitable salt solution. Investigations of
the activity of bacterial enzymes are usually carried out in the
first place with such " washed suspensions." Bacteria are
seldom susceptible to osmotic rupture when suspended in
water, and washing the cells in water often has no deleterious
effect upon their chemical activities, but if the properties of
the cell-wall are involved in these activities, then it is preferable
to wash the cells in a salt solution of composition similar to
that of the medium in which they were grown. Since many
organisms retain their enzymic activities unimpaired in such
" washed suspensions " they can be used for the investigation
of metabolic changes, and the system further simplified by
incubating the suspension with a single substrate in the
presence of a known buffer solution. It does not follow that
washed suspensions can be used to study all the enzyme
systems of an organism, as it is sometimes found that some
activities " decay " rapidly after, or during, the preparation of
the suspension. It is seldom that suspensions can be kept in
an active state for more than twenty-four hours, although some
enzymes, e.g. formic dehydrogenase, will remain active for
weeks even in autolysing suspensions.

EXPERIMENTAL STUDY OF BACTERIAL ENZYMES 29

The enzymatic activities studied in intact cell suspensions
may be complicated by such factors as the rate of passage of
the substrate through the cell-wall, the removal of reaction
products by other enzyme systems, and differences between
the physico-chemical conditions holding inside and outside the
cell. Also with complex reactions which may involve many
small changes, each catalysed by a specific enzyme, it is
difficult to determine whether a given change is the result of
the action of one or more enzymes. This can only be decided
by preparing the enzymes in a cell-free state, studying their
action m vitro, and separating them by methods of protein
separation and purification. Where bacteria produce extra-
cellular enzymes, these can be easily prepared in the cell-free
state by filtering the cells from the medium and then removing
the enzyme from the filtered medium by precipitation or by
adsorption on to a suitable adsorbant such as alumina or
calcium phosphate gel. The study of intracellular enzymes
involves rupture of the cell-wall prior to purification pro-
cedures. The cell-walls of bacteria are very resistant and
special methods have been devised to rupture them in such a
way as to liberate the enzymes in an active state. The methods
that can be used depend to a certain extent upon the relative
resistance of the enzyme concerned and of the cell- wall to the
treatment. The following are some of the more common
methods used:

1. Thick washed suspensions of cells are treated with
denaturing agents such as toluene, acetone, acetone-ether
mixtures, Or by simple drying. If the enzymes survive such
treatment they can often be extracted from the denatured
cell debris by incubation in buffer solutions.

2. The cell suspensions are incubated with proteolytic
enzymes such as pepsin, trypsin, or papain and the debris
extracted with buffer solutions.

3. Mechanical disintegration in some form of ball-mill.
The simple ball-mill is usually ineffective but an effective
crushing-mill consisting of steel cylinders rotating in a race

30 BACTERIAL ENZYMES

under pressure has been devised by Booth and Green.^ A
simpler mill has recently been produced in which a steel ball
runs in a closely fitting channel in a steel bowl, the whole
being immersed in solid COg ; the combined effect of pressure
and freezing accomplishing disintegration of the cells.

4. Disintegration by friction between fine hard particles.
Werkman and his colleagues^ first showed that thick pastes
of bacterial cells can be disintegrated by grinding with finely
powdered glass in a mortar. They later evolved a mechanical
mortar to deal with large quantities of organism. Other
workers have found that powders of carborundum or alumina
are as effective as glass.

5. Disintegration by vibration. Exposure of cell suspen-
sions to supersonic vibration of a certain range of frequency
results in very effective breakdown of the cell structure, in
fact, care has to be taken to prevent breakdown of the enzyme
structures themselves. Vibration of lower frequency is often
effective {i.e. sonic vibration of 50-60 cycles/sec.) but the
efficiency is usually increased by addition of small glass beads
or carborundum particles to the cell material.^

6. Specific treatment can be applied to certain organisms.
Thus the enzymes of Micrococcus lysodeikticus and some strains
of Staphylococcus can be liberated after disintegration of the
cell-wall with preparations of lysozyme.

THE NATURE OF ENZYMES

Such studies often lead to knowledge concerning the nature
of the enzymes concerned and it has been found that although
all enzymes have the properties of proteins, many of them
consist of two parts, one protein in nature and the other,
called the prosthetic group, of a simpler non-protein nature.
Prosthetic groups can often be detached from the protein
moiety — in which case the enzymatic activity ceases — and
their structure determined. The link between the, prosthetic

1 Booth and Green, Biochem, J., 1938, 32, 855.

2 Werkman et al, J. BacL, 1945, 49, 595.

3 Curran and Evans, J. Bad., 1942, 43, 125.

PROSTHETIC GROUPS 31

group and the protein varies in strength so that some prosthetic
groups are firmly fixed to the protein whilst others are in such
a loose combination that they may wander from one protein
molecule to another. In this second case the non-protein
moiety is called a " coenzyme." There is still some doubt
whether there is any difference between prosthetic groups and
coenzymes other than in the strength of the link with the
protein, or whether there is a fundamental difference in that
the prosthetic group is an integral part of the enzyme
structure while the coenzyme acts as a separate carrier of
hydrogen ions, etc., from one enzyme to another. This
controversial point is outside the scope of the present dis-
cussion so we shall discuss all prosthetic groups and
coenzymes under one heading.

PROSTHETIC GROUPS
The prosthetic groups (and coenzymes) which have been
identified up to the present are:'
Ademne-nicotinamide-dinucleotide (Coenzyme I)

N= C— NHp

I I

HC C— N^ O O CONH2

II II >H II II .^ ' ^
N — C — N^ RIBOSE— P-o-P-0— RIBOSE— N,

OH O"

This acts as the coenzyme for certain dehydrogenases and
these enzymes display a specificity towards coenzyme I as
the hydrogen acceptor in the same way as they display
specificity towards their substrate as hydrogen donator.
The molecule appears to act as a carrier of hydrogen by
alternate reduction and oxidation of the nicotinamide group.
By accepting hydrogen from one dehydrogenase system and
transferring it to another dehydrogenase system working
in reverse, the coenzyme acts as a hydrogen carrier between
what are called " coenzyme-linked-dehydrogenase systems "
(see Chap. VII).

32 BACTERIAL ENZYMES

Coenzyme II

Coenzyme II has a structure similar to that of coenzyme I,
with an additional phosphate group in the molecule; the
position of the third phosphate in the molecule is not yet
certain. The coenzyme acts as hydrogen acceptor towards
certain dehydrogenase systems in a manner analogous to
that of coenzyme I.

Adenylic acid, Adenosine-diphosphate, Adenosine-tri-phosphate

K. r MW i OH OH OH

N=C-NH2 I I I I

II Q l_p_0-p-o-P-OH

HC C-N<^ I ]J I I

" '^^ -n. CH,0-P-OH I O O O

N- C-N

H H

OH OH

I I
-P-O-P-OH

II II

Adenosine-tri-phosphate (ATP) acts as a donator of phosphate
in phosphorylation reactions such as those that occur in
fermentation cycles (see Chap. VII). The phosphate is linked
in ATP by an energy-rich bond so that its rupture gives rise
to the liberation of energy. Adenylic acid and adenosine-di-
phosphate can act as phosphate acceptors, being synthesised
to ATP. Since the glycolysis cycle depends upon phosphoryla-
tion and dephosphorylation (see Chap. VII) ATP and adenylic
acid act as coenzymes in the cycle.

Adenine-riboflavin-dinucleotide , Flavine-adenine-dinucleotide

O H

II 1

N = C-NHo

I 1

HC C-Nx

II II ^CH

N-C-N^

-RIBOSE-

-0-P-O-
OH

-P-O-

1
OH

-RIBITYL N N

CH3CH3

PROSTHETIC GROUPS 33

This forms the prosthetic group of enzymes known as
flavoproteins and again acts as hydrogen carrier by
alternate reduction and oxidation of the double bond in
the isoalloxazine ring.

Riboflavin-phosphate

Consists of riboflavin with a single phosphate radicle;
occurs as the prosthetic group of a flavoprotein enzyme known
as cytochrome reductase (see below).

Thiamindiphosphate, Aneurindiphosphate, or Cocarboxylase

N==C — NHi CH3 0 0

I I CI c==c-CH2-CH20— P-O-P-OH

H3C— C C CH2 N^ I OH OH

II II ^c-1

N CH H

Thiamindiphosphate was first identified as the prosthetic
group of yeast carboxylase, the enzyme which decarboxylates
pyruvic acid to acetaldehyde. It is found in many tissues
and bacteria which do not possess carboxylase and plays a
part in many of the reactions involving pyruvic acid (see
Chap. VII), such as oxidative decarboxylation. It also acts
in some as yet undefined way in the oxidation of certain
fatty acids.

Pyridoxal phosphate, Pyridoxamine phosphate

CHO 9 ^^2^^2 ?

^ I

\.J OH

tCH^O-P-OH H0/\CH^0-P-0H

3 N

(a) Pyridoxal phosphate. (6) Pyridoxamine phosphate.

Pyridoxal phosphate has the probable structure shown above,
although the exact position of the phosphate group is not yet
certain. It acts as the prosthetic group of the amino-acid
decarboxylases (see Chap. IX) and of bacterial transaminase.

CHRM. A. B.

BACTERIAL ENZYMES

In the latter case it probably reacts with the — NHg group
of an amino-acid and is converted to pyridoxamine phosphate,
leaving the keto-acid corresponding to the amino-acid.
Pyridoxamine phosphate can then react with a suitable
keto-acid converting it to the corresponding amino-acid
while being itself restored to the pyridoxal form.

Haematin

HOOC.CH2.CH2

CH=CH2

CH=CH.

This forms the prosthetic group of haemoglobin and of enzymes
such as catalase, peroxidase, and cytochrome oxidase. Side
chains may differ in different enzymes, etc.

Metals

Some enzymes contain a metal in their structure, while
others are activated by the presence of metal coenzymes.
Thus phosphatase is activated by magnesium ions, while in
other cases metals such as copper (polyphenol oxidase) or
zinc (carbonic anhydrase) appear to be an essential part of
the enzyme structure.

PROSTHETIC GROUPS OF INCOMPLETELY
DETERMINED STRUCTURE

Coenzyme A

Lipmann, in the course of studies on the acetylation of
sulphanilamide by pigeon liver, discovered that the acetylase
involved possessed a prosthetic group which could not be

PROSTHETIC GROUPS 35

replaced by any known coenzyme. Preparations of the new
coenzyme A, as it was called, were found to contain panto-
thenic acid and their activity could be correlated with their
pantothenate content. The complete structure of coenzyme A
has not yet been determined and the complete molecule is
more complex than pantothenic acid alone. Coenzyme A is
now known to act in many reactions involving acetic acid, or
" acetyl phosphate," and is required by bacteria for the
acetylation of choline. Recently it has been shown that a
cell-free extract of Esch. coli will catalyse the condensation of
acetyl phosphate and oxalacetic acid to give citric acid
(see p. 153) and that the extract is activated by coenzyme A.

CH3 H

I I

HOCH2— C C— CO— NH— CH2— CH2— COOH

I I

CH3 OH

Pantothenic acid

Biocytin

The aspartic deaminase (see p. 162) of Esch. coli has been
shown to require a coenzyme which contains biotin and is
probably a compound of biotin and adenylic acid. The
complete structure is not yet known. A crystalline prepara-
tion of biocytin, which is a compound of biotin present in
yeast extracts, has now been made but it is not yet certain
whether this is identical with the aspartic co-deaminase or not.
0

HN NH

I I

HC CH

I I

HgC CH— (CH2)4— COOH

Biotin

36 BACTERIAL ENZYMES

NATURE OF ENZYME CATALYSIS

Knowledge of the nature and function of the prosthetic
groups becomes of importance in understanding the nutrition
of exacting bacteria (see Chap. V). Several enzymes may
have the same prosthetic group but, nevertheless, have
different substrate specificities. It is probable that the
prosthetic group plays an active part in the decomposition
of the substrate, but that the protein moiety of the enzyme
is responsible for the specificity towards the substrate. It is
thought that a type of loose combination takes place between
the substrate and the enzyme protein before catalysis occurs.
For instance we often find that the enzyme action is dependent
upon the presence, not only of the chemical group whose
alteration is catalysed, but also of other groups in the substrate
molecule. Lysine decarboxylase cannot catalyse the decar-
boxylation of lysine unless both a and e — NHg groups of
the lysine molecule H2N.CH2.CH2.CH2.CH0.OHNH2.COOH
are intact and unsubstituted. Further, the enzyme cannot
attack the dextro-is,om.ei of lysine nor can it attack L-ornithine,
which differs from L-lysine in having one less C atom in
the carbon chain. Before L-lysine decarboxylase can attack
its substrate, this must possess :

1. an unsubstituted — COOH group,

2. an intact alpha — NHg group in the laevo- position,

3. an intact — NHg group in the terminal position,

4. the distance corresponding to — CHg . CHg . CH2 . CHg . CH—
between the two amino-groups.

The — COOH and — NHg groups are chemically reactive
or " polar " groups, and it is thought that a combination
between these groups and corresponding groups on the
surface of the enzyme protein must take place before the
decarboxylation is catalysed. L-Lysine decarboxylase can
thus be thought of as combining with the two amino-groups
of lysine as a preliminary to the removal of the — COOH
group. Substitution of — CH3, etc., in either — NHg would

ENZYME-SUBSTKATE COMBINATION 37

prevent the enzyme-substrate attachment, as would alteration
of the carbon chain length between them as this would place
the two — NHg groups at the wrong distance apart to orientate
and attach to the combining groups of the enzyme surface.
Such a substrate-enzyme relation would also explain optical
specificity as it is obvious that the laevo- and dextro- forms
of the substrate would not " fit " on to the same combining
group structure. This hypothesis that the substrate and
enzyme-surface have to fit or interlock in an exact position
has given rise in the past to the analogy of a lock and key,
in that the one must fit the other before any further action
can occur.

If the combining groups on the surface of the enzyme are
almost, but not quite, in the right position to " fit " the polar
groups of the substrate, then the resulting enzyme-substrate
combination will introduce a strain into the structure of the
substrate and so render it more unstable. Haldane has
suggested that the properties of enzyme action can be explained
by such enzyme-substrate combinations which result in the
production of a strain in the substrate molecule and facilitate
its chemical alteration.

COMPETITIVE INHIBITION
i The substrate of an enzyme therefore is any substance
which can combine reversibly with the right groups on the
enzyme surface. The reversible nature of the combination
is important, for if we can find a substance whose structure
is such that it can combine with the combining-groups on
the enzyme surface but which is not strained, altered, and
released as is the true substrate, then this substance will
remain on the surface of the enzyme, block the essential links,
and so prevent the true substrate froln combining. The net
result of this is that the breakdown of the substrate is inhibited
and such a substance is called a " competitive inhibitor."
An example of a competitive inhibitor is malonic acid,
HOOC.CHg.COOH, which combines with succinic dehydro-
genase and inhibits the dehydrogenation of the true substrate,

38 BACTERIAL ENZYMES

succinic acid, presumably by reason of the similarity of the
structures — especially the spatial relations of the two polar
groups in the — COOH groups — of substrate and inhibitor.
It is characteristic of this type of inhibition that the degree
of inhibition depends upon the relative proportions of sub-
strate and inhibitor present. It will be seen later that the
bacteriostatic action of the sulphonamide drugs has been
explained in terms of their competitive inhibition of the
essential metabolism of structurally similar ^^-amino-benzoic
acid.

CH2.COOH COOH

I CH,(

CH2.COOH ^COOH

Succinic acid Malonic acid

H2N /~\ COOH H2N ^"^SOoNHg

^-Amino-benzoic acid Sulphanilamide

TYPES OF ENZYMES IN BACTERIA
Enzymes are classified according to the reactions which
they catalyse. It will have been obvious from the above that
an enzyme catalysing a dehydrogenation is called a
" dehydrogenase," one catalysing a decarboxylation a
" decarboxylase," etc., the name of the substrate usually
being specified as well. Bacteria possess a great variety of
enzymes and these will be discussed briefly under the general
headings set out in Chap. I as indicating the basic reactions
carried out by bacteria (pp. 5-6).

1, 2. Reduction and Oxidation

Most biological oxidations are of the nature AHg -f B
= A + BH2, where the substance AHg is oxidised to A and
the substance B reduced to BHg. The oxidation of AHg is
catalysed by a dehydrogenase specific for that substrate.
Many dehydrogenases have been obtained in a cell-free state
from yeasts and animal tissues, but until recently the problem

OXIDATIVE MECHANISMS 39

of rupturing the bacterial cell-wall has niade the study of
cell-free bacterial enzymes a matter of considerable difficulty.
However, using the recently invented methods of breaking
bacterial cells by grinding with glass particles, shaking with
minute glass beads, exposure to supersonic vibration, etc.
(see p. 30), it has been possible to obtain a number of
dehydrogenases in a cell-free state and they do not appear to
differ significantly from their counterparts in other cells.
Amongst the enzymes isolated from Esch. coli we have those
which will specifically dehydrogenate formic acid to COg,
L-malic acid to oxalacetic acid, ethyl alcohol to acetaldehyde,
triosephosphate to phosphoglyceric acid, succinic acid to
fumaric acid, etc. The action of the dehydrogenase can be
written

AHg = A H- 2H,

and the dehydrogenation cannot take place until a hydrogen
acceptor B is available:

AHg + B = A + BH2.

The dehydrogenases are specific towards the hydrogen acceptor
as well as towards the substrate. In some cases the hydrogen
acceptor is oxygen, in which case the reaction is either

AH2 + O2 = A + H2O2 or AHg + 0 = A + HgO,

but we find in practice that only relatively few dehydrogenases
can utiHse oxygen as hydrogen acceptor. An example of
such an enzyme is the D-amino-acid oxidase of animal tissues
which reacts

K.CHNH2.COOH + 0 > R.C:NH.COOH -f HgO,

followed by spontaneous hydrolysis of the imino-acid to the
corresponding keto-acid with liberation of ammonia.

The majority of the dehydrogenases effect a transfer of
hydrogen from the substrate to an intermediate carrier
represented by B in the above equation. The investigation
of such oxidation-reduction reactions has been carried out

40 BACTERIAL ENZYMES

mostly with animal tissues or yeast-cells, so it will be best to
summarise the findings for enzymes from these cells and then
outline their application to bacteria. The general findings
are summarised in Table III.

In animal tissues and yeasts the transfer of hydrogen from
substrate to oxygen passes through several intermediate
oxido-reduction reactions with oxygen as the final hydrogen
acceptor in the chain.

AHj + B

BH,+ C

^enzyme 3
CH2+ D — - — ^C + DH2

enzyme 4

DH^+E >- D + EH2

The ultimate carrier (D) is a haematin-protein called cyto-
chrome which is capable of alternate oxidation and reduction.
Eeduced cytochrome becomes oxidised again by the action
of the enzyme cytochrome oxidase. Oxidised cytochrome can
be reduced directly by the action of certain dehydrogenases
transferring hydrogen from their substrates to cytochrome.
The chain in this case (substrate type 83, Table III) can be
represented :

/' AH2 '

A.H2 + Cytochrome > A + Reduced cytochrome

dehydrogenase

A Cytochrome ^^^^"^

Reduced cytochrome + 0 > Cytochrome + H^O

Oxidase

The cytochrome is thus alternatively reduced by the action of
the dehydrogenase and oxidised by the action of cytochrome

OXIDATIVE MECHANISMS

u.'u|0

< UJ ^

> 2 O
_lOZ

< UJ
CO •-

^1-

S 8
I o

<>>

u "

Z UJ

OH
X ^

fcl

■t

<•■)

iLl

LfiL

i-i

(z;

f-H

WP^

BACTERIAL ENZYMES

oxidase, the net result being the oxidation of the dehydro-
genase substrate.

Enzymes which transfer hydrogen direct from their sub-
strate to oxygen are called "oxidases"; the next simplest
system is the direct cytochrome system described above.
The majority of dehydrogenases are unable to transfer
hydrogen from their substrate to cytochrome without the
intermediation of a further carrier in the form of a coenzyme.
Dehydrogenases of this third type can be looked upon as
enzymes with prosthetic groups so loosely attached that the
latter can become detached from the protein and act as
hydrogen carriers between one enzyme system and the next.
In this case the action of the dehydrogenase is to transfer
hydrogen from the substrate to the coenzyme. The formulae
of coenzymes I and II have been given on p. 31. These
substances act as hydrogen acceptors by reduction of one
of the double bonds in the pyridine ring of nicotinamide.
The reduced coenzyme is not autoxidisable but acts as the
specific substrate for a coenzyme dehydrogenase (" diaphorase "
for coenzyme I, " cytochrome reductase " for coenzyme II)
which transfers the hydrogen to cytochrome. In this case
the reaction chain is (substrate types S3 and S4, Table III) :

AH2 + Coenzyme

C0E.H2+ Cytochrome

Cytochrome

Reduced Cyt. + 0 — >■ Cytochrome+H^G

oxidase

and the hydrogen passes through two intermediate carriers

before linking with oxygen.

OXIDATIVE MECHANISMS 43

In this manner oxidations involving large changes of energy
are split up into a number of steps, each involving smaller changes
of energy, and each catalysed by a specific enzyme. There are
yet more complex respiratory systems involving further carriers
in the chain between initial substrate and oxygen, but we need
not discuss these here as our knowledge of bacterial oxidation
has not yet progressed beyond the stages so far outlined.

In order of increasing complexity then we have:

(a) Oxidase systems: substrate type S■^_, Table III.

In animal tissues we have oxidases attacking D-amino-acids,
amines, uric acid, etc., but few oxidases have so far been
identified in bacteria. Some organisms oxidise amines to the
corresponding aldehydes, but no analysis of the enzyme
systems involved has yet been made. Aerobic organisms
and those possessing cytochrome (Table IV) possess cytochrome
oxidase. An L-amino-acid oxidase has been found in Proteus
vulgaris and obtained in a cell-free condition by supersonic
disintegration of the cells : it carries out the reaction

R . CHNH2 . COOH + 0 > R . CO . COOH + NH3

in which, presumably, the first step is a dehydrogenation to
the unstable imino-acid

R . CHNH2 . COOH - 2H > R . C :NH . COOH.

The oxidation of their substrates by certain mammalian
oxidases gives rise to the production of HgOg, but this L-amino-
acid oxidase of Pr. vulgaris is. said not to produce peroxide
and no evidence has been presented concerning the nature of
the protein, whether it has a flavine prosthetic group or not.
Hydrogen peroxide is highly toxic to living cells and many
organisms possess an enzyme, catalase^ which destroys peroxide
by breaking it down to water with the liberation of oxygen

Catalase
2H2O2 ^2H20 + 02.

Catalase is a haematin-enzyme and recently it has been shown

44 BACTERIAL ENZYMES

that catalase can also catalyse the oxidation of certain sub-
strates utilising HgOg as hydrogen acceptor,

Catalase
AH2 + H2O2 > A + 2H2O.

Such a reaction is said to be a " coupled oxidation " and the
result is again the removal of hydrogen peroxide.

(6) Cytochrome systems: substrate type Sg, Table III.

The formic dehydrogenase of Esch. coli belongs to this class.

H.COOH + Cytochrome »► CO2 + Reduced Cyt.

dehydrog- ^^^

enase ^ -^

^ ^ Cytochrome

Reduced Cyt.+ 0 -; >- Cytochrome + H,0

' oxidase ' ^

The cytochrome system of animal- and yeast-cells consists
of at least three components which are distinguished by the
position of their absorption bands in the visual spectrum;
these components are known as cytochromes a, b, and c.
Cytochrome b is slowly autoxidisable but cytochromes a and
c cannot react with oxygen in the absence of cytochrome
oxidase. The cytochrome systems of bacteria differ from
those of animal- and yeast-cells in that bacteria may have
several or none of the components. Esch. coli has one com-
ponent only and this has absorption bands corresponding to
those of cytochrome b, but differs in that it is not autoxidisable ;
it is usually referred to as cytochrome b^. The distribution
of the cytochrome components as identified by their absorption
bands in various bacteria is given in Table IV: the letters
a, b, and c are given as for animal tissues, but it is by no means
certain that these bacterial cytochromes are identical with
those in other tissues.

It can be seen from the table that certain species possess
no cytochrome components, and consequently cannot carry

CYTOCHROME SYSTEMS

out oxidation mechanisms of the cytochrome type. It is
possible that some other carrier might take the place of
cytochrome but the only one of these organisms which is
known to produce a pigment which can be reversibly oxidised
and reduced, other than cytochrome, is Ps. jpyocyanea
{Ps. aeruginosa). This elaborates a blue pigment, pyocyanine,
which is capable of acting as a hydrogen- carrier with certain
dehydrogenase systems when tested in vitro. This organism
has, however, a full complement of cytochrome components.

TABLE IV
Distribution of CYTOcimoME Components in Bacteria

Strict aerobes :

Mycobacterium tuberculosis

A zotobacter chroococcum

—

Facultative anaerobes :

Ps. pyocyanea

Staph, aureus

—

Esch. coli

—

Pneumococcus

S.faecaUs

—

Lactobacillus acidophilus

—

Strict anaerobes :

CI. tetanum

—

' —

CI. welchii

—

The organisms devoid of cytochrome are either strict
anaerobes such as the Clostridia, or microaerophilic such as the
Streptococci or Lactobacilli; this suggests that the absence
of cytochrome components leads to the inability of these cells
to utiHse oxygen. Isolated dehydrogenase systems can be
made to react in vitro by replacing,^ cytochrome with certain
"redox" indicators such as methylene blue or cresyl blue;
the dehydrogenase catalyses the transference of hydrogen
from substrate to the dye, and the reduced dye is autoxidisable.
When cytochrome is replaced in this way the final product
of the oxidation is HgOg and not HgO, so that the complete

46 BACTERIAL ENZYMES

reaction is AHg + Og = A + HgOg, resembling some of the
oxidase mechanisms discussed above. The distribution of
catalase is not universal amongst bacterial species, and those
organisms which are devoid of cytochrome are often devoid
also of catalase. It has been suggested that organisms such
as the Clostridia, the Streptococci, etc., owe their sensitivity
to the presence of oxygen to the fact that, being devoid of
catalase, they become poisoned by the formation of H2O2
under aerobic conditions. The amounts of HgOg which would
be formed and which might be toxic are so small as to be
beyond present methods of detection, and this point has yet
to be satisfactorily investigated. It is suggestive, however,
that Pneumococci can be protected in the presence of air by
pyruvic acid, and it is known that pyruvic acid and H2O2
react together chemically in such a way as to destroy the
H2O2.

(c) Coenzyme systems: substrate types S3 and S4, Table
III. In Esch. coll we have coenzyme systems which are
apparently identical with those in animal- and yeast-cells;
thus L-malic acid dehydrogenase:

CH2.COOH Malic CH2.COOH

I + Coenzyme I ^^ |

CHOH . COOH dehydrogenase qq cqOH

L-Malic acid Oxalacetic acid

+ Keduced coenzyme I

and alcohol dehydrogenase:

Alcohol
CH3.CH2OH -f Coenzyme I ^ -^ CH3.CHO +

dehydrogenase

Eeduced coenzyme 1.

The coenzyme I of Esch. coli has never been isolated in
sufficient quantity and purity for its chemical structure to
be determined, but we know that (1) the alcohol and L-malic
dehydrogenases of Esch. coli will not reduce cytochrome or
methylene blue in the absence of a coenzyme which can be

COENZYME SYSTEMS 47

replaced in vitro by coenzyme I isolated from yeast, and (2) the
malic dehydrogenase of animal tissues is likewise inactive
in the absence of a coenzyme which can be supplied by the
autogenous coenzyme I or by partially purified extracts of
Esch. coli. Quantitative studies of these relationships leave
no doubt but that the coenzyme I of yeast and animal tissues
and the L-malic codehydrogenase of Esch. coli are identical.

Coenzyme II systems also exist in bacteria; for example,
the L glutamic acid dehydrogenase of Esch. coli :

CH2 . CH2 . COOH CH2 . CH2 . COOH

I + Coenzyme II ^ -^ |

CHNHo.COOH . C : NH.COOH,

+ Keduced coenzyme II

and in this case the dehydrogenase cannot be activated by
coenzyme I, although the corresponding L-glutamic acid
dehydrogenase of animal tissues is specific for coenzyme I.
These coenzyme specificities are worked out with isolated
enzymes in vitro, and it is probable that the intact bacterial
cell can interconvert coenzymes I and II. The reduced
coenzymes cannot react with cytochrome without the inter-
vention of the coenzyme dehydrogenases ; little work has been
done on the coenzyme dehydrogenases of bacteria, and there
is no evidence that these are any different from the similar
enzymes of other cells.

Linked oxidation-reduction reactions: So far in this
section we have discussed the oxidation of various substrates.
Many of the dehydrogenases are reversible and can carry out
the general reaction

A + 2H > AH2

in the presence of a suitable hydrogen donator. Keduced
coenzyme can act as H-donator in this way and so can act
as H-carrier between one dehydrogenase and another.

AH2 -f Co ^ A + C0H2 C0H2 + B > BH2 + Co.

In this case AH2 has been oxidised anaerobically by the

48 BACTERIAL ENZYMES

reduction of B. Several such " coenzyme-linked " oxido-
reductions have been demonstrated in fermentation reactions
(Chap. VII). In the Clostridia we find an oxido-reduction
occurring between two amino-acids:

E . CHNH2 . COOH K . CO . COOH

+ H2O > + 2NH3

X . CHNH2 . COOH X . CH2 . COOH.

The enzymes in this reaction have not as yet been investigated
in detail, although it is possible to demonstrate the presence
of the specific dehydrogenases for K.CHNH2.COOH and
X.CHNH2.COOH (Chap. IX). The Methanobacteria carry
out an interesting oxido-reduction reaction, in which alcohols
are oxidised anaerobically with CO2 acting as the H-acceptor,
and being reduced to methane (p. 154).

2CH3 . CH2OH + CO2 > 2CH3 . COOH + CH4.

Hydrogenase: Many bacteria are able to activate
molecular hydrogen as H-donator by the possession of a potent
enzyme, hydrogenase, which catalyses the reaction:

Hydrogenase
H2 --- --^ 2H.

The presence of this enzyme can be demonstrated in Esch. coli
by shaking a suspension of cells with methylene blue in the
presence of gaseous hydrogen when the dye becomes reduced
and decolourised. Boiling of the organisms or replacing the
hydrogen by any other gas stops the reduction. In this
reaction methylene blue acts as H-acceptor for hydrogen
made available by hydrogenase and, similarly, other suitable
H-acceptors can be reduced in the presence of hydrogen.
Esch. coli possesses an enzyme, nitratase, which activates
nitrate as H-acceptor, so in the presence of the cells and
hydrogen, nitrate is reduced to nitrite:

HNO3 + 2H > HNO2 -f H2O.

The reduction does not stop at this point but continues all

dehydration; hydrolysis 49

the way to ammonia with the probable intermediate formation
of hydroxylamine,

HNO2 + 4H > NH2OH + H2O

NH2OH + 2H > NH3 + H2O,

the sum total of the reduction being represented,
HNO3 + 4H2 > NH3 + 3H2O.

3. Dehydration

The removal of HgO from a substrate molecule is com-
paratively rare within our knowledge. In those cases where
such a reaction is the result of the action of a single enzyme,
that enzyme is called a dehydrase. Such an enzyme has been
postulated to explain the breakdown of L-serine to pyruvic
acid by Esch. coli :

CH2OH CH2 ^^3 CJHg

I Serine ll | |

CHNH2-H2O > C . NH2 ^^ C :NH ^-^ CO + NH3

I dehydrase | | H2O |

COOH COOH COOH COOH

The first step in the postulated breakdown being a dehydration
of serine to the corresponding imino-acid which hydrolyses
spontaneously to pyruvic acid. A very similar reaction takes
place with cysteine, in which the first step is the removal of
H2S (instead of HgO) from the molecule, after which the course
of the breakdown is the same (Chap. IX).

4. Hydrolysis

Hydrolytic enzymes are responsible for the processes known
as digestion, whereby proteins are broken down to amino-
acids, fats to fatty acids and glycerol, aomplex polysaccharides
to simpler polysaccharides and monosaccharides, etc.

Proteolytic enzymes of many types are known, and have in
common the power to hydrolyse the linkage — CO — NH — ,
splitting the peptide containing that linkage into two sub-
stances, one with a free — COOH and the other mth a free

CHEM. A. B. 4

50 BACTERIAL ENZYMES

— NH2 group arising from the broken peptide link. Pro-
teolytic enzymes differ in specificity towards tlie chemical
groups on either side of the — CO — NH — link, towards the
length of the peptide chain they can attack, and towards the
nature of the terminal groups of that chain. Some proteases
are able to attack large protein molecules in a native state,
others can attack only after the protein has been denatured,
others can attack relatively short polypeptide chains, and others
are specific for peptides of two, three, or four amino-acid
residues of definite structure. Some peptidases display speci-
ficity towards the nature of the particular amino-acids on
either side of the peptide link to be hydrolysed, and much
of our knowledge concerning stereo-specificity and the " lock
and key " nature of enzyme action has been deduced from
studies of particular peptidases and the structure of the
peptides they can attack. The proteases and peptidases of
animal tissues have been studied in considerable detail, but
our knowledge of the proteolytic enzymes of bacteria is so
far meagre.

In order to digest large protein molecules which cannot pass
through the cell-wall, bacteria excrete extracellular proteases
into the surrounding medium, and the power to do this
seems to be restricted to comparatively few species. The
majority of bacteria can hydrolyse the simpler molecules of
peptone and polypeptides, but with a few notable exceptions
no detailed studies or separation of the enzymes involved has
been undertaken. Some organisms excrete an enzyme which
specifically hydrolyses gelatine and consequently these
organisms bring about a liquefaction of gelatine media on
which they are grown — this property is used as a diagnostic
test in systematic bacteriology — but the power to form
gelatinase is not necessarily accompanied by the ability to
excrete proteases.

The ability to hydrolyse polysaccharides again involves the
excretion of extracellular enzymes, and the ability to do this is
subject to the same degree of species variation as any of the
fermentation reactions used for characterisation tests in

HYDROLYTIC ENZYMES

!>5

o
o

SIl

(tidase

-CH-
1

-o —

-a

I i

:2 ^

03 O

>; z

^ o

-2

J^ *->

O 1

U —
1

fvJ

■<:

3 6

ce
O

Pepsin
CO-NH

^8
1

t« c«

'c3

•1

r-4

+=> o

(4-(

-*^

- !

C3 o

O 14-1

CL— > 1

2? O

-1:^ .2

i r

-o —

u —

-o

O
1

.^ 1

Ch ^

-tJ

lypept
-NH

1 1

Aminopo!

M— CH — CO
1

-^\ X

It
111

1 .

cs .a

P <1 o

52 BACTERIAL ENZYMES

systematic bacteriology (Chap. II). The specificity of thfe
polysaccharide-splitting enzymes depends upon the nature of
the linkages between the sugar units of the polysaccharide
chain. CI. welchii, for example, cannot hydrolyse starch
unless it is first grown in the presence of starch which then
evokes the production of the enzyme. The organism grown
in the presence of starch can also hydrolyse maltose and
glycogen. Likewise, if the organism is grown in the presence
of maltose, then it gains the power to hydrolyse starch and
glycogen. Consequently the enzyme or enzymes necessary
for the hydrolysis of starch, glycogen, or maltose cannot be
produced unless growth takes place in the presence of any one
of these carbohydrates. Growth in glucose does not produce
the enzyme. Full investigation has not been made, but the
assumption is that we are dealing with the production of an
adaptive enzyme (Chap. IV), specific for the hydrolysis of the
maltose linkage. The breakdown of starch by CI. aceto-
hutylicum is accomplished by the production of two enzymes ;
first, an amylase which hydrolyses the starch to maltose, and,
secondly, a maltase which completes the hydrolysis of maltose
to glucose. Cellulose is attacked by a variety of organisms
normally found in the rumen and on plant tissues. Cellulo-
bacillus myxogenes produces two enzymes responsible for the
hydrolysis of cellulose, the first, cellulase, hydrolyses cellulose
(see p. 120) to cellobiose, and the second, cellobiase, completes
the hydrolysis of cellobiose to glucose.

5. Deamination

The removal of — NH2 from the molecule of an amino-acid,
amine, etc., is seldom achieved in a single step. We have
already had two examples of deamination occurring in two
steps: the amino-acid oxidase of Pr. vulgaris, and the
L-glutamic acid deaminase of Esch. coU, where the first step
is a dehydrogenation of the amino-acid to the corresponding
imino-acid which then undergoes spontaneous hydrolysis to
the corresponding keto-acid liberating ammonia:

DECAEBOXYLASES 53

R . CHNH2 . COOH ±=:^ R . C : NH . COOH + 2H (-> coenzyme)

JH,0
E.CO.COOH + NH3.

Deamination of amino-acids can also take place by reduction,
desaturation, or hydrolysis (Chap. IX), but in the majority
of these cases the intermediate steps, if any, are not known.

6. Decarboxylation

The removal of CO2 from the molecule has been observed
with two types of compound, keto-acids and amino-acids.
In yeasts a-keto-acids are decarboxylated by the enzyme
carboxylase which has been isolated in a cell-free condition
and studied in a highly purified state. The enzyme consists
of a protein and a loosely attached coenzyme or prosthetic
group identified as thiamindiphosphate (see p. 33). Carbo-
xylase attacks a-keto-acids, decarboxylating them to the
corresponding aldehydes,

Carboxylase

R. CO. COOH >R.CHO-f CO2.

Pyruvic acid, CH3.CO.COOH, is attacked more rapidly than
other acids of this group and, in general, the longer the carbon
chain of the R group, the slower the rate of attack by the
enzyme. Although thiamindiphosphate would seem to bear the
same relation to carboxylase that coenzyme I does to L-malic
dehydrogenase, we have as yet no definite knowledge of the way
in which it functions in the decarboxylation of keto-acids. Carbo-
xylase itself has not been found in the enzyme constitution of
organisms such as Esch. coli, and it is possible that it does not
enter into bacterial metabolism. The breakdown of pyruvic
acid by bacteria is not by simple decarboxylation to acetalde-
hyde, but involves other mechanisms which also, however,
require the presence of thiamindiphosphate (see Chap. VII).

Yeast carboxylase is specific for the decarboxylation of
a-keto-acids, but in some bacteria we find enzymes which

54 BACTERIAL ENZYMES

remove COg from other keto-acids. Azotobacter and Micro-
coccus lysodeikticus contain an enzyme which decarboxylates
oxalacetic acid to pyruvic acid:

Oxalacetic

HOOC . CHg . CO . COOH > CH3 . CO . COOH + COg.

decarboxylase

CI. acetohutylicum possesses an enzyme which decarboxylates
acetoacetic acid to acetone:

Acetoacetic

CH3 . CO . CH2 . COOH > CH3 . CO . CH3 + CO2.

decarboxylase

These enzymes have all been studied in a cell-free state and
do not appear to involve thiamindiphosphate.

Some bacteria also carry out a decarboxylation of certain
amino-acids to the corresponding amines.

R . CHNH2 . COOH > R . CH2 . NH2 + CO2.

The amino-acid decarboxylases are specific for a single amino-
acid and, so far, enzymes have been isolated in a cell-free
state which are specific for the natural isomers of lysine,
arginine, histidine, ornithine, tyrosine, and glutamic acid. All
these amino-acids have a polar group other than the a-NH2
and the 1-COOH groups, and it has been found that
substitution of the third polar group, i.e. the second — NHg
group in lysine, ornithine, or arginine, the — OH in tyrosine,
or the second — COOH group in glutamic acid, results in
complete inhibition of the decarboxylation. This suggests
that the enzyme and substrate must combine through at
least two polar groups — other than the — COOH attacked —
before decarboxylation can occur. The product of the
decarboxylation is the corresponding amine or, in the case
of glutamic acid, y-amino-butyric acid. The decarboxylases
of lysine, arginine, ornithine, tyrosine, and glutamic acid
consist of a protein portion and. a prosthetic group which can
be replaced in vitro by pyridoxal phosphate (see p. 33).

PHOSPHORYLATION 55

7. Phosphorylation and dephosphorylation

Tiie anaerobic breakdown of carbohydrate in yeast and in
muscle involves the initial phosphorylation of the carbo-
hydrate by a series of reactions involving adenosine-tri-
phosphate and inorganic phosphate. The phosphorylated
compounds then undergo a series of changes resulting in the
formation of phosphopyruvic acid which is then dephos-
phorylated before the final stages of fermentation take place.
The fermentation of glucose by Esch. coli and related organisms
has now been investigated in considerable detail (Chap. VII) ,
and appears to involve the same basic cycle of reactions as
those occurring in yeast fermentation, so that phosphorylation
of glucose to hexosediphosphate precedes breakdown to
simpler molecules. The intermediate stages of the phosphory-
lation and the enzymes involved have not yet been worked
out with bacteria, but it is highly probable that the first step
is a transfer of phosphate from adenosine- tri -phosphate to the
6-position of glucose by the enzyme Hexokinase:

-\ Adenosine-di-phosphate

Many other bacterial fermentations will occur only in the
presence of phosphate and are accompanied by an uptake of
phosphate from the medium by the fermenting cells. The
changes have seldom been investigated in detail and it is not
possible to say whether phosphorylation is an invariable step
in anaerobic carbohydrate breakdown. Fermentation of
carbohydrate represents one of the main sources of energy for
anaerobic existence. In those cases which have been worked
out in detail, it appears that the incorporation of phosphate at
low energy levels into organic compounds, followed by its
removal at a later stage of the fermentation process from
compounds in which the phosphate bond has become

56 BACTERIAL ENZYMES

'' energy-ricli," is the source of this utiUsable energy (see
Chap. VII). Considerable evidence is now accumulating that
the energy obtained from oxidation processes also arises from
the formation and breaking of energy-rich phosphate bonds;
thus the oxidation of pyruvic acid by pyruvic oxidase of
Lactobacillus delbreuckii is accomplished only in the presence of
phosphate and involves the formation of acetyl phosphate as
the first stage in the reaction :

CH3 . CO . COOH + H3PO4 + 0 =

CH3.COOPO3H2 + CO2 + H2O.

Phosphorylation may occur in many reactions other than
those involved in fermentation and oxidation. Examples of
phosphorylated intermediates are still being discovered in
many metabolic changes as the biochemistry of living tissues
is further probed.

General

The metabolism of a cell consists of many chemical changes
catalysed by various enzyme systems. The biochemist has
concerned himself with the isolation of these enzyme systems
in an endeavour to identify the steps by which the changes
occur and to interpret the mechanism of these changes in
terms of the chemistry of the enzyme molecules. The
systems outlined in this chapter summarise the types of
enzymes that have been discovered in the course of these
studies, but it must be realised that the metabolism of the
intact cell is far more complicated than any of the separate
reactions which can be studied in vitro, as we have not only
the interplay of the various enzymes on each other and on
the reactions catalysed by each other, but also the effect of
environmental conditions on the formation of the enzymes
themselves. We shall proceed to the discussion of this aspect
of bacterial metabolism in the next chapter.

FOR FURTHER READING 57

FOR FURTHER READING

Dynamic Aspects of Biochemistry, Baldwin, E. (Cambridge

Univ. Press).

"A Classification of Proteolytic Enzymes," Bergmann, M.,
Advances in Enzymology, 1942, 2, 49.

" Biological Oxidations and Reductions," Dixon, M.,
Ann. Rev. Biochemistry, 1939, 8, 1.

Multi-Enzyme Systems, Dixon, M. (Cambridge Univ. Press).

Mechanisms of Biological Oxidations, Green, D. E. (Cam-
bridge Univ. Press).

Enzymes, Haldane, J. B. S. (Longmans, Green and Co.).

" The Enzymatic Properties of Peptidases," Johnson, M. J.,
and Berger, J., Advances in Enzymology, 1942, 2, 69.

" Metabolic Generation and Utilisation of Phosphate Bond
Energy," Lipmann, F., Advances in Enzymology, 1941, 1, 99.

" Acetyl Phosphate," Lipmann, F., Advances in Enzymology,
1946, 6, 231.

CHAPTER IV

THE FORMATION OF ENZYMES IN BACTERIA

The manifold chemical activities of bacteria are catalysed
by enzymes formed within the bacterial cell. Some bacterial
species can exist under widely different chemical and physical
environments, and require different types of enzymes in order
to deal with the differing external conditions. Esch. coli
utilises different enzymes for anaerobic existence from those
utilised for aerobic existence, and needs different neutralisation
mechanisms when growth takes place in an alkaline medium
from those required when growth takes place in an acid
medium. We find that an organism does not possess all the
enzymes necessary for dealing with all possible environments
at any one time, but that the actual enzymic constitution,
as opposed to the potential enzymic constitution, is determined
to a large extent by the external conditions holding during
the formation of the individual cell. Consequently the cell
grown aerobically is equipped with the mechanisms for
oxidative metabolism, while the cell grown anaerobically is
deficient in those mechanisms which can be utilised only
under aerobic conditions but possesses highly developed
anaerobic mechanisms. The actual enzymic constitution of
a cell of a given species may thus vary widely. The identifica-
tion of bacterial genera and species is based upon certain
biochemical tests, but these are always carried out under
standardised growth conditions and represent cross-sections
of the potential enzymic constitution of the organism con-
cerned. If an organism can ferment sucrose, then the fermen-
tation will occur under the test conditions of growth in a
fully nutrient medium containing sucrose, but it does not
follow that the same organism can ferment sucrose if it is
first grown in a medium free from sucrose or, say, nicotinamide.

CONTROL OF POTENTIAL ENZYMIC CONSTITUTION 59

THE POTENTIAL ENZYMIC CONSTITUTION
It is obvious that all organisms cannot form all enzymes.
Otherwise any attempts at classification would fail. Although
the enzymic constitution of an organism can and does undergo
vast changes with alterations in the growth environment, theie
is still a limit to the changes that can occur and the enzymes
that can be produced by any one organism. In higher
organisms the enzymic constitution is controlled by the genetic
composition of the cell. Genes are hypothetical units which
determine the carry-over of characteristics from mother- to
daughter- cell. In nucleate cells it is possible to observe
changes in the form of the nucleus during division of the cell ;
the nucleus forms a skein instead of a solid body, the skein
breaks up into short rod-like structures known as chromosomes
and, in normal division, each chromosome divides into two
before division, one of each pair passing into each daughter-
cell; after the daughter cell has spUt off, the chromosomes join
up again into a skein which then collapses to form the new
nucleus. The genes occupy definite positions on the chromo-
somes and damage to a chromosome in a certain place will be
accompanied by loss of the property associated with possession
of the gene lying at that place. Chromosomes have not yet
been clearly demonstrated in 'bacteria, although structures
allied to chromosomes have been described in some of the
filamentous organisms. However, the general behaviour of
bacteria, the inheritance of enzymic properties from one cell to
another, and the occurrence of "mutants" suggest that some
form of genetic control of enzyme constitution occurs in these
organisms.

Neurospora crassa, a mould which commonly occurs on
bread, has certain characteristics concerning the arrangement
of its spores which make it easy to study from a genetical point
of view. It has been found that it is possible to alter the
genetic constitution of the mould by irradiation with X-rays.
When this is done an occasional " hit " is made by the radiation
on the chromosome and the absorption of a quantum of energy

60 THE FORMATION OF ENZYMES IN BACTERIA

results in an alteration of the gene at tlie site of the " hit."
It has been found experimentally that whenever a gene is so
altered, there is a loss of one enzyme from the enzymic con-
stitution. From this work, and from other investigations
carried out with yeast cells, it can be concluded that the
formation of an enzyme by a cell is controlled in the first
place by the presence of the correct gene and that one gene
controls one enzyme. When a gene is altered and the enzymic
constitution of the resulting cell changes, the new cell is said
to be a " mutant." Mutants arise spontaneously, and, as a
result of many studies which have been carried out on micro-
organisms, it appears that any given gene may alter and give
rise to a mutant about once in every 10^ to 10^ generations.
Consequently a spontaneous mutant is a very rare thing but
when we are dealing with large populations, and in bacterial
cultures we normally deal with populations of 10^-10® cells/ml.,
it is probable that mutants will be present and any change in
the environmental conditions which favour the growth of the
mutant rather than that of the unaltered mother culture,
will give rise to a progressive selective growth of mutant
cells. Likewise if the environment is not suitable for growth
of the mutant, then it will not multiply and will not exert
any significant effect upon the properties of the culture as
a whole.

Biologists working with moulds and higher organisms are
accustomed to thinking in terms of genes and mutations, but
it is only during recent years that the application of these
terms to bacteriology has been investigated in detail. The
presence of a heritable factor involved in enzyme control has
been shown by some masterly studies by Avery and his
colleagues on the conditions governing the formation of the
polysaccharide capsule of the Pneumococcus (see jDhap. VI).
Pneumococci are divided by serological methods into a number
of types, and type specificity is conferred by the chemical
structure of the polysaccharide capsule of the organism. Thus
the capsule of a Type III Pneumococcus is composed of a

TRANSFORMING PRINCIPLE 61

polysaccliaride of different chemical structure from the poly-
saccharide of Type II Pneumococcus. Capsulated pneumo-
cocci will give rise under certain conditions to the growth of
non-capsulated or " Eough " strains. Avery and his colleagues
have shown that a rough non-capsulated Type II Pneumococcus
will grow as a capsulated Type III Pneumococcus [i.e. acquire
the enzyme necessary for the synthesis of the Type III
polysaccharide) if an extract of Type III organisms is added
to the medium. Careful investigation of the nature of the
" transforming principle " in the extract shows that it is a
desoxyribonucleic acid and it is active in a dilution of 1 part
in 6 X 10^ parts of medium. Further, once the Type II
organism has been transformed into a Type III organism,
it then continues to grow as a Type III organism, even when
grown in the absence of the desoxyribonucleic acid. In this
case the potential enzymic constitution of the organism has
been altered by the addition to the organism at a certain
stage of a minute amount of the nucleic acid, and it is tempting
to think that this is equivalent to adding a gene to the genetic
make-up of the organism.

The studies, mentioned above, with Neurospora have shown
that alteration of a gene will result in the loss of an enzyme —
and, presumably, reconstitution of the gene will result in the
reappearance of the enzyme. The enzyme may thus be lost,
or gained, by spontaneous mutation or the process may be
artificially accelerated by irradiation or by treatment with
"mutagenic" substances such as mustard gas. This sort of
phenomena is well known in bacterial chemistry. Perhaps
the earliest case to be studied was that of Escherichia coli
mutabile: this is a variant of Esch. coli which will not ferment
lactose and when grown on lactose plates containing indicator,
produces white colonies, indicating no acid formation from
lactose. If, however, the incubation is continued, small red
papillae appear on the white colonies, indicating that new
cells are growing which have the ability to ferment the sugar.
If the non-fermenting culture is serially subcultivated several
times in lactose-containing medium, then the power to ferment

62 THE FORMATION OF ENZYMES IN BACTERIA

the sugar is slowly acquired in the course of subcultivation, and
after several such passages, the culture behaves as though it
were a normal lactose-fermenting Esch. coli. Detailed
investigation of the individual cells in the cultures (by plating
out a high dilution on lactose- and -indicator-plates) shows that
all cultures contain a number of fermenting cells and a number
of non-fermenting cells. The initial non-fermenting culture is
found to contain, on the average, one fermenting " mutant "
for every 10^ non-fermenting cells. In the course of cultivation
in the presence of lactose, the proportion of fermenting cells
increases, since the medium will obviously favour the growth
of these mutants but the final fermenting culture will still
contain a small proportion of non-fermenting cells.

It is not certain what is the difference between the fermenting
and non-fermenting cells. It is probable that the fermenting
cells possess the enzyme lactase, whereas the non-fermenting
cells are mutants which have lost this enzyme. However,
one investigator has claimed that both types of cell possess
lactase but the non-fermenting one has a cell-wall impermeable
to the disaccharide. Whatever may be the true difference, it
is clear that growth in a lactose medium results in selective
growth of the mutant able to utilise the sugar.

In the next chapter we shall be considering organisms which
have lost the ability to synthesise certain amino-acids. A
simple example is that of Eberthella typhosa which, when
freshly isolated, is unable to synthesise tryptophan. The
primitive type is able to synthesise the amino-acid; in the
course of multiplication, mutants arise and, in approximately
10^ generations, a cell arises which has lost one of the enzymes
involved in tryptophan synthesis ; if the organism is growing
in the tissues of a host it will find tryptophan supplied in the
medium and the synthetic disability will therefore not impose
any restriction on growth. The synthesis of a substance
such as tryptophan involves the expenditure of energy ; con-
sequently if the organism can grow by the assimilation of
preformed tryptophan its growth process will be energetically
more efi&cient and, in the course of many generations, the

MUTATION 63

slightly more efficient growth of the non-synthesising mutant
will have the result that it will outgrow the synthesising cells. In
the course of evolution the primitive cell, capable of tryptophan
synthesis, has been lost and now only arises as a " back
mutant " from the tryptophan-requiring organism. Fildes
has shown that such back mutants can be demonstrated in
cultures of Eberthella typhosa, and growth in a tryptophan-free
medium will selectively grow the mutants and so give rise to
cultures of the organism which are capable of synthesising their
own tryptophan.

The spontaneous production of mutants can become highly
important in considerations of drug resistance. A chemo-
therapeutic drug such as sulphanilamide acts by inhibiting an
essential enzyme process (see Chap. V). If a mutational
alteration occurs which renders that particular enzymic
process non-essential, then the resulting organism is no longer
sensitive to the drug. One method of selecting such insensitive
mutants is to culture the organism in the presence of an amount
of drug which limits the growth of all the sensitive cells. Such
a procedure may take place accidentally during the clinical
treatment of a patient infected with the organism and the
appearance of sulphonamide-resistant strains of organisms
such as Staph, aureus is well known to medical scientists.
Selection of the mutants may arise accidentally, as described,
or as a result of deliberate cultivation in the laboratory.
Eesistance may arise in small steps or cells may rapidly become
completely resistant to a drug. One of the main drawbacks
to the clinical use of streptomycin is that many organisms
acquire a complete resistance to it within a very short period
of cultivation in its presence.

The actual enzymic constitution of a cell is that portion of
its potential enzymic constitution that is selected by the
conditions under which it has been grown. Amongst the
factors controlHng this selection we may list the following:
(a) the chemical constitution of the medium, (6) the physico-
chemical conditions holding during growth, and (c) the "age"
of the culture.

THE FORMATION OF ENZYMES IN BACTERIA

CHEMICAL CONSTITUTION OF THE MEDIUM .
Presence of substrate

This subject has been studied in considerable detail by
Karstrom, who found that the ability to ferment certain
sugars is often acquired only if growth takes place in the
presence of those sugars. Table V shows the variation of
the fermentation abilities of Betacoccus arabinosaceus with the
nature of the sugar present during growth. From the table
we can see that this organism ferments glucose and sucrose
whether these sugars are present in the growth medium or
not, but the fermentation of galactose, maltose, lactose, and
arabinose will take place only if growth has occurred in the
presence of galactose, maltose, lactose, or arabinose respectively.
Galactose can be fermented if growth has taken place in the
presence of lactose, since growth in lactose results in the
liberation of galactose from the lactose molecule.

TABLE V

Relation of Febmentative Properties to Nature of the Sugar
Present during Growth (Betacoccus arabinosaceus)

Sugar in
Growth
Medium

Fermentation Occurs with

Glucose

Galact-
ose

Sucrose

Maltose

Lactose

Arabin-
ose

No sugar ...

Glucose

Galactose . . .

Sucrose

Maltose

Lactose

Arabinose ...

Tr„ „i. ^•

xl,„ „i?„

„ J* ,;j

„j i,„„i.

Karstrom therefore divided bacterial enzymes into two
classes :

ADAPTATION AND SELECTION 65

1. Adaptive enzymes, which are formed only when growth
takes place in the presence of the specific substrate, i.e. are
formed only when required.

2. Constitutive enzymes, which are formed whether
growth occurs in the presence or absence of the substrate.

The application of quantitative studies to enzymes has now
shown that adaptive enzymes are usually formed to a small
degree even when growth occurs in the absence of the sub-
strate, and that the presence of the substrate during growth
results in a marked stimulation of the enzyme formation.
One explanation that has been put forward of this difference
between the enzymes is that the adaptive enzymes are unstable
in the absence of their substrate and consequently lose their
activity if growth takes place in the absence of the substrate.
It is important to realise that this distinction between the
two classes of enzymes is an experimental one, as in normal
existence an organism will but rarely meet, after growth has
ceased, with substances with which it has not been in contact
during growth. The adaptive nature of an enzyme can only
be shown by taking an organism after growth has taken place
in the absence of the substrate, placing it in contact with the
substrate, and comparing its activity with that of an organism
grown in the presence of the substrate.

Adaptation of this nature takes place rapidly, for if an
organism can ferment galactose adaptively, or, in other words,
if it has an adaptive galactozymase, then a single cultivation in
the presence of galactose will be sufficient to evoke the enzyme
to its full extent. A single subsequent cultivation in the
absence of galactose will result in the loss (or marked decrease
in the activity) of the enzyme. The difference from an experi-
mental point of view between adaptation and selection of
mutants is illustrated in Fig. 1 .

We can summarise the position regarding the formation of
an enzyme in the growing cell as follows : the capacity to form
the enzyme depends upon the presence of the corresponding
gene ; if the gene is present, then the actual formation of the

CHEM. A. B. 5

THE POEMATION OF ENZYMES IN BACTERIA

enzyme may further depend upon the presence of the substrate.
The mechanism of adaptation may be that the gene controls
the formation of an inactive enzyme-precursor which is only
activated by the presence of the substrate; alternatively, it
may be that the enzyme is formed in the presence of the gene
but is itself unstable in the absence of its substrate.

SELECTION

+ = present

- = absent

during

growth

ADAPTATION

Fig. 1. Diagram to illustrate the difference between adaptation and
selection processes in enzyme formation.

The presence of substances other than the substrate

Adaptation involves a relation between the organism, the
enzyme, and the substrate, but sometimes substances other
than the specific substrate may play a part in the formation
or activity of an enzyme. The most marked example of this
is found when fermentable carbohydrate is added to the
medium. This problem has been studied chiefly with respect
to the effect of the presence of glucose during growth on the
production of enzymes concerned in the breakdown of proteins

EFFECT OF CARBOHYDRATE 67

and amino -acids, but the effect is by no means restricted to
these enzymes alone. If we study the formation of a deaminase
such as alanine deaminase, which carries out the reaction

Alanine

CH3 . CHNH2 . COOH + 0 > CH3 . CO . COOH + NH3,

deaminase
in Esch. coli which has been grown in an amino-acid mixture
with and without glucose, we find that the amount of deaminase
produced in the absence of glucose is some twenty times
greater than that produced in its presence. No really satis-
factory explanation of this effect has yet been put forward.
At one time it was suggested that the inhibitory action of the
presence of glucose on the formation of some enzymes could
be attributed to the production of fermentation acids, but in
many cases this has now been disproved. A further suggestion
that has been put forward is that the presence of glucose
during growth has a " protein sparing " action similar to that
postulated in mammalian nutrition. The addition of glucose to
washed suspensions of Esch. coli has no effect on the deaminase
activities of the cells, so that glucose has no effect once the
enzymes have been formed in the cell and the inhibitory
action must affect enzyme formation during growth.

An alteration of the _pH of the medium during growth does
have a marked effect on enzyme production, but the suppres-
sion of deaminase formation, for instance, by the presence of
glucose is greater than can be explained by the fall in ^H
due to acid formed by its fermentation. Table VI shows
the action of the presence of glucose during growth on the
formation of various enzymes of Esch. coli, and it can be seen
that in some cases the effect can be satisfactorily explained
by fermentation acidity, while in others the effect is greater
than, or sometimes even opposed to, that produced by an
equivalent acidity during growth. Only in the case of
glucozymase (the enzyme system responsible for the first
stage of glucose fermentation) does the presence of glucose
during growth result in an enhanced activity over and above
that due to acidity.

THE FORMATION OF ENZYMES IN BACTERIA

TABLE VI

Comparison of the Activities of Esch. coli when Grown in Casein
Digest: (1) adjusted to pH 7, (2) adjusted to pH 5, and (3) containing
2 per cent glucose and attaining a final pK = 5-2.
Activities are expressed as Q units = fil. Og or CO2 or NH3 or
methylene blue (MB), etc., formed or reduced/hr./mgrm. dry weight
of organism.

Activity of Organism Grown

Enzyme

Unit

(1)
SitpB. 7

(2)
atj9H5

(3)
in Glucose

Glucose
Effect

Hydrogenase

Catalase

Arginine

decarboxylase
Lysine

decarboxylase
Alanine

deaminase
Glutamate

deaminase
Aspartase
Serine

dehydrase
Tryptophanase
Alcohol

dehydrogenase
Succinic

dehydrogenase
Eormic

dehydrogenase
Glucozymase

MB
O2

CO2

NH3

NH3
NH3

NH3

Indole

MB
MB

Glucose

240
4200

12
127

855
5-4

110
38-5

126
6360

338

194

3
247

656
1-6

179

138
31

146
6310

272

198

1
15

167
0-2

None
None

None

Inhibition

Inhibition
Inhibition

Inhibition

Inhibition
Stimulation

The presence of fermentable carbohydrate during growth
has four known effects: (1) the production of acid and con-
sequent alteration of the medium ^H; (2) the production of
gas with consequent anaerobiosis ; (3) a considerably increased
crop of organisms; and (4) the transient formation of poly-
saccharide within the growing and fermenting cell. Despite
various attempts, no one has yet succeeded in linking any of
these effects with the inhibition of formation of certain
enzymes. Monod has investigated the effect of growing the

ENZYME SUPPRESSION 69

organism in a mixture of sugars. For example, if Esch. coli
is grown in a mixture of glucose and galactose, lie finds that
the organism utilises all the glucose before it begins to attack
the galactose. Galactose is attacked by means of an adaptive
enzyme, galactozymase, which catalyses the phosphorylation
to galactose-1-phosphate, and galactozymase is not formed by
the organism until all the glucose is removed from the medium.
If the organism is first grown in galactose so that it contains
galactozymase, and then inoculated into a mixture of glucose
and galactose, the galactozymase activity disappears until all
the glucose is again used up. In other words, the formation
of the constitutive glucozymase suppresses the formation of
the adaptive galactozymase. Monod suggests that the effect
is due to a definite " enzyme suppression." There is still
no clear explanation of the mechanism of this suppression,
although it can be postulated that both enzymes arise from a
Hmited supply of a common protein precursor : the formation
of the constitutive glucozymase thus uses up the available
precursor so that the adaptive galactozymase cannot be
produced. This hypothesis involves, in turn, a further
supposition that the active enzyme is produced by some
reaction between substrate and precursor, and that a substrate
such as glucose has a higher affinity for the precursor than a
substrate such as galactose. In this connection, Spiegelman
and his co-workers have shown, in yeast, that the formation of
galacozymase in washed cells is accompanied by a fall in the
glucozymase activity, but that if the cells are provided with
available nitrogen so that they can synthesise proteins without
drawing on their internal reserves, then the formation of the
new enzyme can occur without reduction in other activities.
All these findings emphasise that the living cell is a very
dynamic system with its enzymes continually undergoing
breakdown and resynthesis.

Gram-positive bacteria differ from Gram-negative organisms
in that they are able to assimilate certain amino-acids and
concentrate them in the free state in the internal environment.
The assimilation of certain amino-acids, such as glutamic acid

70 THE FORMATION OF ENZYMES IN BACTERIA

and histidine, can only take place if energy is provided by
some metabolic activity such as glucose fermentation. Some
Gram-positive organisms also differ from many of the Gram-
negative species in that they are unable to synthesise glutamic
acid, etc., from ammonia, whereas many Gram-negative
species can synthesise all their amino-acid requirements from
ammonia. Yeasts again synthesise their amino-acids from
ammonia, but cannot assimilate ammonia unless fermentation
is occurring simultaneously. Further research is necessary
to clarify the relation of these various findings, but there is a
suggestion that the presence of glucose during growth may
alter the assimilatory processes of the cells, and this, in turn,
may be reflected in an alteration of the enzyme constitution,
especially with regard to those enzymes concerned with the
breakdown of amino-acids to ammonia.

Further examples of substances, other than the specific
substrate, having an effect on enzyme formation, are found in
the case of certain growth factors (see Chap. V) which act
as coenzymes. For instance, Haemophilus parainjluenzae is
unable to synthesise coenzyme I, and is unable to grow in
its absence. If, however, sub-optimal amounts of coenzyme
are provided in the growth medium, then we find that the
organisms grow, but are unable to oxidise at a normal rate
those substances forming the substrates of coenzyme I
dehydrogenase systems. The deficient organisms can oxidise
L-malic acid slowly, but if coenzyme is added to a suspension
of these organisms they can then oxidise malic acid at the
normal rate. This means that the organism has synthesised
its normal complement of enzyme-protein, but has been unable
to saturate it with coenzyme due to its inability to synthesise
this substance.

PHYSICO-CHEMICAL CONDITIONS OF GROWTH
Aerobiosis or anaerobiosis

Facultative anaerobes can grow under strictly aerobic or
strictly anaerobic conditions and develop a different enzyme
constitution in each case. When we examine the activities

EFFECT OF pB. DURING GROWTH 71

of individual enzymes we find that those that can function
under aerobic conditions only, are produced only when growth
is aerobic, being suppressed when growth is anaerobic, and
vice versa. Taking the deaminases of Esch. coli as examples,
the formation of the oxidative L-alanine deaminase is 5-6
times greater under aerobic conditions than under anaerobic,
while the formation of the anaerobic serine dehydrase is
2-3 times greater when growth is anaerobic than when aerobic.

pB. of the growth medium

Esch. coli can grow in a casein-digest medium adjusted to
any pH between the approximate limits 4-2 and 9-5. The
formation of an enzyme within the bacterial cell is dependent
to a large extent upon the pH of the medium at the time of
formation of the cell. The effect of the ^H varies with the
type and function of the enzyme concerned, and we can
distinguish three types up to the present:

{a) Neutralisation mechanisms: bacteria are able to
grow in media covering a wide range of pH by the production
of mechanisms whose action is to neutralise the external
acidity or alkalinity and so tend to stabilise the internal
environment. Thus growth in an acid medium promotes
the formation of enzymes catalysing reactions with alkaline
end-products, and inhibits the formation of enzymes having
acid-forming actions. When Esch. coli grows in an acid
medium containing amino-acids, it attacks certain of the
amino-acids by decarboxylation liberating COg with the
formation of alkaline amines; when it grows in an alkaline
medium the amino-acid decarboxylases are no longer formed,
but, instead, enzymes attacking amino-acids by deamination
are formed and these liberate NHg with the formation of
acid products. Other organisms react to acid growth conditions
by the formation of enzymes catalysing the formation of
neutral substances from acids — as, for example, the reduction
of butyric acid to butyl alcohol by CI. acetohutylicum, and the
formation of acetylmethylcarbinol from pyruvic acid by
Aerobacter aerogenes (see Chap. VII). In all these cases

THE FORMATION OF ENZYMES IN BACTERIA

investigation of the amount of enzyme formed in the cell at
various growth ^H values shows a direct relation between
the formation of the neutralising mechanism, suppression of
the non-neutralising mechanisms, and the ;pH of the environ-
ment (see Fig. 2).

(6) Protective mechanisms: the function of some
enzymes such as catalase is to destroy metabolites which,
if allowed to accumulate, would prove toxic to the cell. All
enzymes are optimally active at a definite ^H and, conse-
quently, as the environment pH diverges from this pH of

< 60-

•^ 40-

-100^

80°
<

pH OF MEDIUM DURING GROWTH
Fig. 2. Variation of formation of glutamic acid decarboxylase
and deaminase of Esch. coli with the pB. of the medium
during growth.

optimal activity, the effectiveness of each enzyme unit
decreases. This means that in the case of catalase, which
has optimal activity at ^H 6-5, the enzyme unit is considerably
less effective during growth occurring at pH 9 than at pH 6-5.
In such cases we sometimes find that the organism compensates
for this loss of efficiency per enzyme unit by the production of
more enzyme so that the effective activity (= No. of enzyme
units X activity of each unit at the environmental pH) is
roughly constant whatever the pB. in the medium. Enzymes
whose formation is affected by pH in this way are urease,
catalase, formic, and alcohol dehydrogenases — enzymes whose

EFFECT OF ^H DURING GROWTH

substrates are toxic to the organism. Fig. 3 shows the
variation of the formation of alcohol dehydrogenase of Esch.
coli with growth jpH. The potential activity is that activity
estimated at the optimal activity pH (8-0) of the enzyme
and represents the total formation of enzyme within the cell ,
the effective activity is the activity estimated at the _pH of the
environment in which the cell was grown.

90H

Qm8

60 H

GROWTH
;jH=9

GROWTH

4-5 5 6 1

pW OF MEDIUM DURING GROWTH
(a)

45 5 6 7

REACTION joH
(b)

Fig. 3. (a) Variation of potential activity (•— •) and effective
activity (x - - - x) of alcohol dehydrogenase of Esch. coli with
_pH of medium during growth.
(6) Variation with reaction ^H of activity of alcohol dehydrogenase
of Esch. coli grown at various pH values — showing that jjH of
optimum activity does not vary with growth 7>H. Qmb = M^-
methylene blue reduced/hr./mg. dry weight of organism.
(After Gale and Epps, Biochem. J., 1942, 36, p. 609.)

value of their ^^H of optimum activity. It is remarkable
that, as far as we know, there is no enzyme whose formation
is not affected in some way or other by the environmental ^H
during growth. Enzymes of this third group, having functions
neither neutralising nor protective, are formed to a significant
extent over a hmited range of growth pH values centred about
the value of the optimal activity pH. The growth pH value
giving maximal formation is not necessarily the same as the
optimum activity ^H, as can be seen in Fig. 4 for the case of

THE FORMATION OF ENZYMES IN BACTERIA

hydrogenase. In this example the optimum activity plcL is
6-0 (Fig. 46), but maximal formation of the enzyme occurs
at ^H 8-0 (Fig. 4a) and investigation of the effective activity
between growth pH values of 6 and 8 shows that the greater
formation of the enzyme between these values compensates
for the loss of activity per enzyme unit over this range.
As a result the effective activity is approximately constant
over the middle of the growth range but falls off rapidly

GROWTH
pH-d

120-

100-
80-

■A

Qm8

X
\

60-
40-

\ I

20-

t
X

5 S 6

7 8 9

Qme

20-

45 5 6 7 8 9

pH OF MEDIUM DURING GROWTH REACTION^H

(O) (b)

Fig. 4. (a) Variation of potential activity (•-•) and effective
activity (x---x) of hydrogenase of Esch. coli with ^H of
medium during growth.
(6) Variation of reaction pH of hydrogenase activity of Esch. coli
grown at various pB. values. (After Gale and Epps, Biochem.
J., 1942, 36, p. 612.)

outside these limits. In these cases, then, we get a restricted
amount of compensatory formation over the neutral part of
the growth range but no compensation towards the ends of
that range. Enzymes in this group include hydrogenase,
succinic dehydrogenase, glucozymase, and tryptophanase.

Growth temperature

It has become customary to study many organisms after
growth at 37° C, presumably since this is the temperature of

GROWTH PHASES 75

parasitic existence in man. It does not follow that this is
the optimum temperature for bacterial metabolism. In fact,
many soil organisms cannot grow successfully at temperatures
as high as 37° C. A few studies have been made of the effect
of growth temperatures on enzyme constitution, and it has
been shown, for example, that the amino-acid decarboxylases
of some strains of Escli. coli are formed to a greater extent
when growth occurs at 20° C. than when at 37° C. Several
workers have shown that the efficiency of protein synthesis
increases as the temperature falls.

THE AGE OF THE CULTURE

When an organism is inoculated into a suitably nutrient
medium, it begins to increase in size until, in due course, the
enlarged organism divides into two daughter- cells apparently
similar to the mother-cell. This process will go on until
some nutrient in the medium is exhausted. The growth
process can be followed experimentally in two main ways:
by counting the number of cells per ml. of medium, or by
determining the mass of cell-material (measured as cell-
nitrogen, cell-carbon, dry weight of cell-material, etc.). If we
record the amount of growth against time, we find that we
get curves of different shape if we measure growth by cell-
numbers or by cell-mass, as shown in Fig. 5.

If we follow the increase in cell-numbers with time we find
that the curve can be divided into a number of phases.
Starting from the time of inoculation, we get

1. an initial stationary phase during which no increase in
cell-numbers takes place;

2. a lag phase during which the rate of multiplication
increases with time;

3. a phase of logarithmic growth when the rate of multiplica-
tion is constant;

4. a phase of negative growth acceleration during which
the rate of growth decreases with time;

5. a maximum stationary phase.

THE FORMATION OF ENZYMES IN BACTERIA

If we count the number of viable cells {i.e. the cells capable
of further division) rather than the total number of cells
present, we find a similar curve (Fig. 5), though the number
of viable cells is always less than the total number of cells
present. Following the maximum stationary phase, during
which the number of new cells is balanced by the number of
dying cells, we get a falling off in numbers as the cells die
at an increasing rate.

If, however, we estimate growth by cell-mass rather than
by cell-numbers, we get a different curve, as shown in Fig. 5,

PHASE I PHASE 2 PHASE 3 PHASE 4 PHASES
AGE OF CULTURE »-

Fig. 5.

which shows no initial stationary or lag phases but a steady
increase in mass until growth ceases. The difference between
the two curves lies in the fact that the size of the cells is not
the same throughout the growth period. When the inoculum-
cells enter the new medium they begin to grow in size, i.e. in
cell-mass, but do not divide and so give rise to the stationary
phase of cell-numbers. The cells do eventually divide, but
whereas they may divide at a limiting size x in the logarithmic
phase of growth, they will grow to a size considerably larger
than X before division occurs in the lag phase of growth.
Consequently we have a steady increase in cell-mass, but

"age of culture" effects 77

the cells themselves are, ou the average, larger in phases
1 and 2 than they are in phases 3 and 4, and it is this difference
in cell-size during various growth phases that gives rise to
the different shapes of the growth curves. If the inoculum-
cells have to undergo any form of adaptation before growth
can take place in the new medium, then stationary and lag
phases may be shown by the cell-mass curves as well as by
the cell-number curves.

If we wish to investigate the development of an enzyme
system with the growth of a culture, the results we shall obtain
if we correlate enzyme activity with cell-mass will obviously
differ from those we shall obtain if we correlate activity with
cell-numbers. Many of the early investigations of this problem
were calculated on a basis of enzyme units per cell, and curves
were obtained which showed very high enzymatic activities of
cells during the early phases of growth. Since the cells are
larger during these phases than in the later stages of growth,
it follows that they will contain more protoplasm than older
cells and may well therefore contain more enzyme. We can
only follow the development of the enzyme if we relate it to
the amount of cell-substance present without reference to
cell-numbers. Of more recent years the estimation of cell-
mass has become a relatively simple matter owing to the
development of photo-electric and turbidimetric methods,
and nowadays the enzymatic activity of bacteria is usually
expressed as enzyme units per mgrm. dry weight of organism
or per mgrm. nitrogen content. Whichever form of expression
is used we find marked variations of enzyme content with the
age of the culture. Since the culture is formed by continued
binary fission of cells, each division apparently similar to the
last, it is not immediately obvious why the enzyme content
of the cells should vary with the time elapsing since inoculation,
but we must remember that the physico-chemical nature of
the environment is changing throughout the growth period as a
result of the metabolic activities of the growing cells, and it has
already been shown that the enzyme content of a cell is largely
dependent upon the environment at the time of its formation.

THE FORMATION OF ENZYMES IN BACTERIA

When the enzyme content of the culture is expressed on a
basis of enzyme units per mgrm, dry weight of organism, we find
two main types of variation with the age of the culture (Fig. 6).

In the case of enzymes giving the Type I variation (Fig. 6),
cultures taken as early as possible in the growth period have
high activities, and these activities decrease as the culture
grows, usually falling off rapidly after cell division has ceased.
In the Type II variation, cells taken early in the growth period
have little or no activity and the enzyme is formed during
growth, reaching a maximum at about the time of cessation

AGE OF CULTURE

Fig. 6.

of cell division. After the end of the growth the activity may
fall off, due to death of the cells, oxidation or digestion of the
enzyme protein, etc. It is probable that we have not yet
studied all the types of enzymes present in bacteria and the
majority of those so far reported in the literature give a Type II
variation with age of culture. These enzymes — deaminases,
decarboxylases, dehydrogenases, etc. — are concerned with the
breakdown of substrates with the liberation of carbon, nitrogen,
energy, etc., and it does not follow that these enzymes have
any direct connection with the synthetic processes of growth.
Consequently it is possible that this Type II form of variation

''age of culture effects 79

with age is characteristic of enzymes concerned mainly with
catabolic or protective mechanisms. There is also the
possibility that some, at any rate, of these variations are
artefacts produced by the permeability of the cell-wall to
substrates varying with age of culture. That this is not always
the case, however, has been demonstrated with extracellular
enzymes such as proteases, whose formation in the external
environment follows just such a curve as that shown for
Type II enzymes in Fig. 6. In the case of certain amino-acid
decarboxylases, showing a Type II variation, it has been
possible to estimate the amount of enzyme formed in the cells
by breaking these down with acetone and ether, when the
amount of enzyme formed within the cell is found to vary with
the age of the culture in the same way as the Type II variation
found with the intact cells. If the Type II variation is
characteristic of catabolic systems, it may be that Type I
variation is characteristic of anabolic systems which the cell
must possess for growth to take place. Our knowledge is
not yet sufficiently extensive for any such generalisations to
be made. In Streptococci we find enzymes showing both
types of variation : tyrosine decarboxylase showing Type II ;
arginine dihydrolase (see p. 171) and the enzymes involved
in glucose fermentation showing Type I variation.

Where the formation of enzymes within the cell is also
conditioned by ^H, the Type II variation may be modified if
growth occurs in the presence of fermentable carbohydrate.
Thus an enzyme whose formation is optimal only when the
growth pH approximates to the optimum activity ^H {e.g.
hydrogenase) may give a Type II variation with age, but
the activity may decrease again before the end of growth
owing to the pH of the medium becoming considerably acid.
These variations are very important in the experimental
study of bacterial metabolism. For example, CI. aceto-
hutylicum possesses hydrogenase and acetoacetic acid
decarboxylase (acetoacetic acid — ^acetone), both of which show
a Type II variation with age of culture. If we studv the
formation of these enzymes in suspensions of organisms

THE FORMATION OF ENZYMES IN Bx\CTERIA

harvested at different ages of culture we find that {a) cells
harvested very early in the growth period have neither
enzyme ; (6) cells harvested during the phase of linear growth
possess a very active hydrogenase but no acetoacetic
decarboxylase ; and (c) cells harvested at the time of cessation
of growth have no hydrogenase activity but a very active
acetoacetic decarboxylase. These differences in time of
formation can probably be correlated with the fact that the
^H of optimum activity of hydrogenase is 8-0, while that of
acetoacetic acid decarboxylase is approximately 5, and the

ct>>-

NUMBERS

TIME OF GROWTH &»

Fig. 7.

joH of the medium falls from 7 to ca. 4-5 during the growth
period.

It might be thought that some changes in the chemical
composition of the cell during its growth should be demon-
strable in view of the marked alterations in enzymic constitu-
tion. Changes in enzymic constitution involve changes in
protein constitution, but these may relate to the organisation
of the amino-acids within the molecules, rather than to gross
changes in their proportions. However, in recent years,
workers in Sweden have applied the technique of ultra-violet
spectrophotography to bacteria and have found very significant
changes in the composition of the cell during growth. Sub-
stances containing purines'and pyrimidines, such as nucleotides

"age of culture" effects 81

and nucleic acids, have a very marked absorption in the ultra-
violet at 265 m/x, and changes in the amount of such substances
in the cells can be shown by photographing the cells in light
of that wave-length. If this is done, it is found that cells from
old cultures have very little " nucleic " material but that this
increases markedly during the growth phases corresponding to
the late lag and early logarithmic periods; the concentration
then decreases steadily throughout the phases of negative
growth acceleration and stationary growth. The changes in the
concentration of nucleic material {i.e. measured by its U-V
absorption at 265 m/x) are related to the growth of the organism
as shown in Fig. 7. It can be seen that there may well be
correlation between these alterations in composition and those
variations in enzymic activity described above.

FOR FURTHER READING

Genetics of Micro-organisms, Catcheside, D. G. (Pitmans).

" Enzymatische Adaptation bei Mikroorganismen,"
Karstrom, H., Ergebnisse de Enzymforschung, 1938, 7, 350.

" Factors Influencing the Enzymic Activities of Bacteria,"
Gale, E. F., Bacteriological Reviews, 1943, 7, 139.

La Croissance des Cultures Bacteriennes, Monod, J., 1943
(Hermann et Cie, Paris).

Original paper: " Studies on the Chemical Nature of the
Substances Inducing Transformation of Pneumococcal Types,"
Avery, 0. T., MacLeod, C. M., and McCarty, M., J. exp. Med.,
1944, 79, 137.

CHEM. A. H.

CHAPTER V

GROWTH: SYNTHESIS OF BACTERIAL PROTOPLASM

When a bacterium is inoculated into a nutrient medium
it first begins to increase in size, and this increase in cell-
material is eventually followed by binary fission, the two
daughter-cells proceeding to increase in size until they divide,
and so on. The speed at which this process takes place
depends upon the particular organism concerned and on the
physico-chemical constitution of the environment. Esch. coli
will divide once every twenty minutes when inoculated into a
nutrient broth at ^H 7 and 37° C. This means that each cell
synthesises its own weight of protoplasm including proteins,
enzymes, prosthetic groups, essential metabolites, etc., in
twenty minutes. In this chapter we intend to survey the
synthetic abihties of various organisms.

For synthesis to occur an organism requires (1) inorganic
salts, (2) a source of carbon, (3) a source of nitrogen, and (4) a
source of energy. Table VII summarises sources of carbon,
nitrogen, and energy which are either commonly available in
nutrient media or which are known to be essential for the
growth of particular organisms. Some organisms can
synthesise all their protoplasm from simple sources such as
carbon dioxide and ammonia, plus a source of energy, and
must therefore be equipped with all the enzymes necessary
for the formation of the essentials of their existence from
these simple sources. This is not the case with all bacteria,
as many organisms are lacking in enzymes necessary for
certain synthetic processes. When this occurs the organism
in question is unable to synthesise some essential constituent
and is consequently unable to grow unless and until that parti-
cular constituent is supplied ready made in the environment.
When an organism has such a synthetic disability it is said
to be nutritionally "exacting" towards the substance which
it is unable to synthesise. It is by a study of the synthetic
disabilities of the more exacting organisms that we gain our

NUTRIENT FUNCTION OF INORGANIC SALTS 83

knowledge of the synthetic abilities of the nutritionally non-
exacting.

NUTRITIONAL REQUIREMENTS
Inorganic salts

All bacteria require the presence of certain inorganic ions
for growth. Salts are required for the regulation of osmotic
pressures, for the maintenance of membrane equilibria, and
for the action of enzymes. Enzymes such as catalase, cyto-
chrome oxidase, and polyphenol oxidase, possess a metal as
part of their structure, phosphatases require the presence of
magnesium as coenzyme, while pyrophosphatase is optimally
active only in the presence of a definite ratio of magnesium
and calcium ions. Traces of metals such as zinc, cobalt,
molybdenum, vanadium, etc., are found to be essential for
certain activities in some organisms. The presence of phos-
phate plays an essential role in energy transfer and many
fermentation reactions. A nutrient salt mixture which will
support growth must contain Na+, K+, Fe++, Mg++, Cu++,
NH4+, Cr, SO/', PO4'", CaCOg, and traces of other metals
which are usually present in sufficient quantities as impurities
in the commercial salt preparations. Recent investigations on
the properties of Esch. coli and Aerohact. indologenes grown in
iron -deficient media have showm that the organisms grown
under such conditions are deficient in certain enzymes such
as formic dehydrogenase, hydrogenase, and formic hydro-
genlyase (see p. 132), with the result that they carry out an
acid fermentation of glucose without the usual formation of
gas (see Chap. VII). If the iron deficiency is severe, the
organisms may show a decreased content of catalase and
cytochrome.

Autotrophic bacteria

The least exacting group of organisms is the autotrophic
group, members of which are able to multiply in a purely
inorganic environment, synthesising their carbon substances
from CO2 or HCO3', their nitrogenous material from ammonia
or nitrate, and obtaining their energy in one of two ways.

84 growth: synthesis of bacterial protoplasm

Chemosynthetio autotrophes: organisms of this group
synthesise all their protoplasmic constituents from CO2 or
HCO3' and NH3 or NO3', and obtain the energy for the synthesis
by oxidation of an inorganic substrate which is specific for the
particular organism, and by means of which the various
organisms can be identified. For example, there are two
chemosynthetio autotrophes present in soil which carry out
the nitrification of ammonia. The first step in the oxidation
is carried out by Nitrosomonas, which obtains its energy from
the oxidation of ammonia to nitrite :

2NH3 + 3O2 = 2HNO2 + 2H2O + 79 Cals.

Nitrite is then oxidised to nitrate by Nitrobacter utilising
this reaction as a source of energy for its synthetic processes:

HNO2 + 0 = HNO3 + 21-6 Cals.

The oxidation substrate is specific ; Nitrobacter cannot oxidise
or grow on ammonia as source of energy, while Nitrosomonas
cannot oxidise nitrite; neither organism can oxidise sulphite
or carbon compounds. Some of the chemosynthetio organisms
are inhibited by the presence of organic matter; thus the
nitrifiers are inhibited by the presence of meat broth but
the inhibitory substances can be removed by extraction of the
broth with ether or ethanol. The growth of Nitrobacter is
accelerated by the addition of 0-1 per cent peptone to the
medium but is inhibited by the presence of asparagine,
gelatine, or urea. Consequently the nitrifying organisms
cannot be isolated by plating out on solid media containing
gelatine or agar-agar, but are usually isolated by growth on
inorganic media solidified in silicic acid gel.

A further example of a chemosynthetio autotrophe is the
iron bacterium, Leptothrix ochracea, which lives in iron-con-
taining streams and obtains its energy by the oxidation of
ferrous carbonate to ferric hydroxide. The complete
reaction is

4FeC03 -f O2 + 6H2O = 4Fe (0H)3 + ^CO^ + 81 Cals.

PHOTOSYNTHETIC BACTERIA 85

Iron bacteria can play an important part in the corrosion of
iron pipes, etc.

An organism of particular interest is Thiohacillus thio-
oxidans, which utilises as energy source the oxidation of
elementary sulphur to sulphuric acid:

2S + 3O2 + 2H2O = 2H2SO4 + 141-8 Cals.
This organism has an exceptionally high tolerance of acid,
and is unaffected by a pH value as low as 0-6, while it grows
most rapidly at a ^H between 3 and 4. In this case the
oxidation of sulphur provides the energy for the assimilation
of CO2 and its reduction to cell-carbon. The processes can
be separated ; the organism will oxidise a certain amount of S
in the absence of COg and then, if exposed to CO2 later, will
take up and reduce an amount of COg corresponding to the
initial oxidation. The oxidation of S is accompanied by an
uptake of inorganic phosphate from the medium and this
phosphate is again liberated during CO2 reduction; this
suggests that the energy obtained from the oxidation process
is stored as a form of " energy-rich " phosphate until it is
utilised in the reduction process. Umbreit has isolated
adenosine-tri-phosphate from Thiohacillus thio-oxidmis, which
suggests that this is the organic phosphate in which the energy
is stored, thus Unking the energy-systems of the autotrophic
bacteria with those demonstrated in heterotrophic organisms
(see Chap. VII).

PHOTOSYNTHETIC AUTOTROPHES: It is obvious from the
nature of their metaboHsm that the chemosynthetic auto-
trophes must be strictly aerobic in their habitat. The photo-
synthetic autotrophes, on the other hand, are strict anaerobes
and obtain the energy for their synthetic activities by photo-
chemical utiHsation of hght energy. They obtain their
nitrogen from ammonia or nitrate, their carbon from
bicarbonate, and reduce the bicarbonate to organic carbon by
a linked oxidation of an inorganic substrate. The organic
carbon so produced may be of carbohydrate nature in the
first instance and can be conveniently represented by

growth: synthesis of bacterial protoplasm

(HCOH). Important members of this group are the sulphur
bacteria Thiorhodaceae which are abundant in soil, mud, and
sulphuretted waters. Some varieties of the group are purple
and some green, the colour in each case being due to a mixture
of pigments including a magnesium-porphyrin pigment with
the structure of chlorophyll, differing from that of plants
only in the nature of certain of the side-chains.

H H

COCH

CH, COOCH,

I '

COOC^H,^ BACTERIAL CHLOROPHYLL
20 39

These organisms reduce CO2 to organic carbon by a linked
oxidation of hydrogen sulphide:

CO2 + 2H2S = (HCOH) + H2O + 2S.

In the presence of large amounts of hydrogen sulphide, the
elementary sulphur is deposited inside the cell in the case of
the purple sulphur bacteria, or outside the cell in the case of
the green sulphur bacteria. If the supply of hydrogen sulphide
is limited, a further oxidation-reduction takes place, the
elementary sulphur being oxidised to sulphuric acid :

2CG2 + H2S + 2H2O = 2 (HCOH) + H2SO4.

Again the autotrophic organisms are characterised by oxida-
tion of an inorganic substrate. In the photosynthetic group
the oxidation occurs anaerobically as a means of reducing

COMMON SOURCES OF CARBON AND NITROGEN 87

available COg, but in the chemosyntlietic group the oxidation
occurs aerobically as a source of energy.

The strict autotrophes can multiply only in the presence
of inorganic matter, and may be inhibited by the presence of
organic matter. There are, however, some organisms which
lead an autotrophic-like existence, in that they utilise COg
or bicarbonate as carbon source and ammonia or nitrate as
nitrogen source, but are able to obtain energy from the break-
down of certain organic substances. These organisms thus
form a bridge between the true autotrophes and the hetero-
trophes, and are consequently called " autotrophic hetero-
phants "; they do not represent any large proportion of the
organisms in common experience.

An example of an organism whose metabolism is inter-
mediate between that of the autotrophes and that of the
heterotrophes is B. methanicus which obtains its energy (and
possibly its carbon) by the oxidation of methane.
CH4 -f- 2O2 = CO2 + 2H2O + 195 Cals.

We also find organisms whose metabolism is intermediate
between that of the photosyrithetic autotrophes and the
heterotrophes. Thus the Athiorhodaceae are photosynthetic
organisms closely related to the Thiorhodaceae but use organic
acids as hydrogen-donators whereby to reduce the CO2. They
can be distinguished from the heterotrophes in that growth
will only occur anaerobically and in the light. COg is essential
to the growth and the organism appears to obtain most of its
carbon material from the assimilation of COg.

TABLE VII
Common Sources of Carbon and Nitrogen

Sources of Carbon

Sources of Nitrogen

CO2
HCO

NH,
NH2OH
Carbohydrates : ! NOg'

Glucose ' NO,'

growth: synthesis of bacterial protoplasm

Sources of Carbon

Sources of Nitrogen

Fructose
Lactose
Sucrose
Maltose
Starch
Glycogen
etc.

Fatty acids :

Amino-acids (natural isomers):
Glycine, CH2NH2.COOH
Alanine, CH3.CHNH2.COOH
Serine, CH2OH.CHNH2.COOH
Cysteine, CH2SH.CHNH2.COOH
Cystine, CH2.CHNH2.COOH

S
1

Acetic acid
Propionic acid
Butyric acid

Keto-acids:
Pyruvic acid
Acetoacetic acid
Oxalacetic acid
Ketoglutaric acid

Hydroxy-acids :
Lactic acid
Malic acid

Alcohols
Glycerol
Hydrocarbons
Amino-acids

CH2

Threonine, CH3.CHOH.CHNH2.COOH
Methionine, CH3 . SCHg . CH2 . CHNHg . COOH
Valine, (CH3)2.CH.CHNHo.COOH
Leucine, (CH3)2 . CH . CHg . CHNHg . COOH
Norleucine, CH3 . CH2 . CH2 . CH2 . CHNHg . COOH
Isoleucine, CH3.CH2.CH(CH3) .CHNH2.COOH

Phenylalanine,
Tyrosine, HO

Tryptophan,
Histidine,

CHo.CHNH,.COOH

CH^.CHNH^.COOH

Lysine,
Arginine,

HN,

HC==C— CH2 . CHNH2 . COOH

I I

Nv /NH

H
H2NCH, . (CH2)3 . CHNH2 . COOH

^C— NH.CH2.(CH2)2.CHNH2.COOH

Glutamic acid, HOOC . CHg . CH2 . CHNH2 . COOH
Aspartic acid, HOOC . CHg . CHNH2 . COOH
H2C CH2

I I

Proline,

H,C

CH.COOH

This table is by no means comprehensive and includes only those
substances mentioned in this book.

TABLE VIII

TABLE VIII
Growth Factors
COOH

Nicotinic acid, N

CONH,

Nicotinic amide, N )>

Coenzyme I (see p. 31)
Riboflavin (see p. 33)

Haematin (see p. 34)
Thiamin (see p. 33)

j8- Alanine,

H2N.CH2.CH2.COOH

Pantothenic acid,
CH3 H

I I

HOCH2— C C— CO— NH— CH2-

I I CH2— COOH

CH, OH

Pimelic acid,

HOOC.(CH2)5.COOH Biotin, 0

Uracil, HN— CO

I I •
OC CH

I I
HN— CH

Pyridoxin, CHgOH

Ho/^.CHaOH

H,C

'N'

p- Amino-benzoic acid,

HoN/~~^COOH

Folic acid [casei factor),

COOH O

I II

HOOC-CHj-CHj-CH • NH • C

HN^ \NH

I I

HC CH

H,C. /CH-

-(CH2)4.COOH

Hypoxanthine, N=C — OH

HC C— NH.

II 11 >CH

N— C N^

Pyridoxal, CHO

HgC^

NH-CH

90 growth: synthesis of bacterial protoplasm

Heterotrophic bacteria

The other major group of bacteria is the heterotrophic
group; these organisms obtain their carbon mainly from
organic sources, their nitrogen from either or both inorganic
and organic sources, and the energy for their synthetic processes
by the degradation (oxidation, fermentation, etc.) of energy-
rich organic material. Heterotrophic bacteria are thus
related in their general metabolism to animals, while the
autotrophic bacteria are related to plants. It is possible that
the two higher forms of existence may have evolved from the
related groups of micro-organisms.

Heterotrophic bacteria can be subdivided on nutritional
grounds as follows (see Table IX) :

(a) Organisms able to utilise atmospheric nitrogen:
The ability to trap atmospheric nitrogen and transform it into
inorganic or organic nitrogenous compounds within the cell
is called "nitrogen fixation," and is a property possessed'
by comparatively few species. Such organisms are of great
importance in agriculture, since the natural fertilisation of
the soil is a result of their activities. The most important
member of this group is Azotobacter, a strict aerobe found
free-living in the soil. This organism can grow in the complete
absence of " fixed-nitrogen " as long as it is provided with
atmospheric nitrogen and a source of carbon in the form of
fermentable carbohydrate. When growth occurs under such
conditions there is a quantitative relation between the amount
of carbohydrate fermented and the amount of nitrogen fixed.
Despite much research, we still have little or no definite
knowledge of the chemistry of the fixation process, and the
primary product of fixation has not yet been identified. Claims
have been made that atmospheric Ng is first reduced by the
organism to either NH3 or NHgOH but these claims have yet
to be satisfactorily substantiated. Studies in which Azoto-
bacter has been exposed to gaseous nitrogen, enriched with
isotopic-Ng, have shown that the isotope appears in the amino-
acids of the organism within a few minutes of exposure. The

2 (i

r s a

c3 o

1 + 4-

Ji ©

Ofl

W)J

<4H '

1 1 1

-►3

+ 1 I

V hJ

-H 1 1

s.^

I 1 1

(-(

-t^

'S ■

+ + ±

l^*

1 1 +

' .^

§,o

1 1 +

«H-1 j

O '

^_^

,_^

+ + +

■ +

: : :

to
1

o ; :

.si
ti

i -2

-£3 ^

« «

o o

■+J ^

0 --r-

,^ j_j qj ^

^< ws

"S-

c<i CO

•S

<m' CO

O V.

•<s> CO

rH CO

92 growth: synthesis of bacterial protoplasm

various amino-acids have been isolated and their content of
the isotopic-N determined; those containing the highest
amount of isotope must be those which are formed first, as a
result of the fixation process. Glutamic acid is found to
contain the highest content of isotopic-N and so, presumably,
is the first amino-acid formed after fixation and must act as
precursor of the other amino-acids. The organisms will grow
on ammonia as N-source but if such fixed-nitrogen is provided
in the medium, then fixation immediately ceases. If the
organism is supplied with ammonia enriched with isotopic-N,
it can utilise this immediately and the isotope is found in the
cell-protein within three minutes of contact. The distribution
of the isotope in the amino-acids of the cell is the same as that
obtained when the cell is fixing isotopic-Ngi this result
suggests that ammonia is a primary product of the fixation
reaction. When growth of Azotobacter is occurring by fixation
of atmospheric nitrogen then the presence of traces of iron,
calcium, and molybdenum are essential. The greater require-
ment of molybdenum for growth on gaseous nitrogen than for
growth on ammonia-nitrogen suggests that this metal has a
function in the fixation process.

Nitrogen fixation is not confined to Azotobacter, but is also
a property of some photo synthetic bacteria and certain
Clostridia such as CI. pastorianmn, which was the first nitrogen-
fixing organism to be isolated from soil. In addition, the
Rhizobaceae, the root-nodule bacteria, can perform nitrogen-
fixation, but only when living in symbiosis with the host-plant
(see Chap. X).

(6) Non-exacting organisms : This is probably the largest
of the sub-groups and consists of those organisms which can
synthesise their nitrogen requirements from ammonia or
nitrate, their carbon from a simple organic source such as
glucose or lactate, and obtain their energy from the degradation
of organic matter. Esch. coli is a typical example, in that it
can grow luxuriantly in a medium consisting of nutrient salts,
including ammonium ions, and either glucose or lactate as

SYNTHESIS OF AMINO-ACIDS

carbon and energy source. The organism can be maintained
indefinitely by serial subculture in such a medium.

The bacteria belonging to the sub-groups so far discussed
are able to synthesise all their amino-acids from a source of
inorganic nitrogen and a suitable source of carbon. Analysis
of the proteins of bacteria shows that they resemble all other
proteins in being composed of some twenty-odd amino-acids,
all of which can be synthesised by these organisms from
ammonia and a source of carbon such as glucose.

Aspartic acid can be synthesised from fumaric acid and
ammonia by the enzyme aspartase (see p. 162), while glutamic
acid can be synthesised from a-ketoglutaric acid and ammonia
by reversal of the glutamic dehydrogenase system (p. 47). In
mammalian tissues glutamic acid, and to a smaller extent
aspartic acid, act as the starting point for the formation of
other amino-acids by a process of transamination whereby
the amino-group of the dicarboxylic amino-acid is transferred
to the a-position of an a-keto-acid:

COOH

CH,

CHo

I
CHNR

R
I

I
COOH

Transaminase

CH2 R

I I

CH2 -f CHNH2

I I
C=0 COOH

COOH

and the a-ketoglutaric acid so formed is then resynthesised
to glutamic acid through the reversal of the glutamic dehydro-
genase system. This reaction was originally discovered by
Braunstein and Kritzmann, and it has been suggested that any
a-keto-acid can enter into the reaction, so that transamination
opens up a way for the general synthesis of other amino-acids
from glutamic acid and the corresponding keto-acids. Since

growth: synthesis op bacterial protoplasm

the original work, other investigators have obtained cell-free
preparations of transaminating enzymes, and these appear to
catalyse, m vitro, transamination between glutamic acid and
oxalacetic acid or between glutamic acid and pyruvic acid, but
not to carry out a general transamination as first suggested.

COOH

CH2 COOH

I I

CHg + CHg

I I

CHNHa C=0

I I

COOH COOH

Glutamic Oxalacetic

COOH

CH2 COOH

I I

CH2 + CH2

I I

C=0 CHNH2

I I

COOH COOH

a-ketoglutaric Aspartic

COOH

CH,

CHo

CH2

I
CHNH2

I
COOH

Glutamic

+ c=o

COOH

Pyruvic

CH2 + CHNH2

I I

C=0 COOH

I
COOH

a-ketoglutaric Alanine

The only " new " amino-acid formed in this way is alanine
from pyruvic acid. The prosthetic group of transaminase is
pyridoxal phosphate and if an organism has lost the ability to
synthesise pyridoxin (see p. 33) then it cannot produce
active transaminase. Recent work with Lactobacilli, which
have lost the abihty to synthesise pyridoxin, has shown that the
organisms can grow in a medium which contains D-alanine
even in the absence of pyridoxin. Organisms grown in this
medium are devoid of pyridoxin and its derivatives, whereas
organisms grown in media containing pyridoxin but no

TRANSAMINATION 95

D-alanine are found to syntliesise D-alanine. D-alanine cannot
be the precursor of pyridoxin but it is probable that pyridoxin
mediates the synthesis of the unnatural isomer of alanine.
This finding has led to the discovery of a new enzyme called
"racemase" which produces DL-alanine from L-alanine.
D-alanine is essential for growth and is synthesised by trans-
aminase followed by racemase, both enzymes having pyridoxal
phosphate as prosthetic group. It appears improbable that
transaminase is concerned with the general synthesis of
amino-acids, as first suggested, but it is always possible that
the cell-free preparations and the studies carried out with them
represent only a part of a more complex system within the
living cell.
The transamination reactions:

Glutamic acid + oxalacetic acid -> a-ketoglutaric acid +

aspartic acid,

Glutamic acid + pyruvic acid -^ a-ketoglutaric acid -|- alanine,

have now been demonstrated for many bacteria including
species of Escherichia, Shigella, Eherthella, Proteus, Pseudo-
monas, Azotohacter, Staphylococcus, Streptococcus, and Pneu-
mococcus. A cell-free transaminase has been prepared from
S. faecalis and resolved into specific protein and a prosthetic
group replaceable by pyridoxal phosphate.

The biosynthesis of other amino-acids has been elucidated
by the application of a new technique which is peculiar to
microbiology. It has been mentioned above (see p. 60)
that the formation of enzymes in the cell is determined by the
presence of the controUing gene and that alteration of that
gene results in the loss of that enzyme. The rate of alteration
or mutation of genes can be accelerated by irradiation with
X-rays and this method has been used by Tatum, Bonner,
Beadle, and their co-workers to produce a very large number of
artificial mutants of the bread mould Neurospora crassa. The
same method was later applied to other moulds, such as species
of Penicillium and Aspergillus, and to bacteria such as

96 growth: synthesis of bacterial protoplasm

Escherichia coli, but tlie early work which involved genetical
analysis of the mutants produced was carried out with
Neurospora. The synthesis of cellular material occurs as the
end-result of a chain of reactions, each catalysed by a specific
enzyme. If an essential cell constituent D is synthesised from
the raw food-stuff A by a series of steps A^B-^C-^D, then
the cell will be able to grow if it is supplied with any of the sub-
stances A, B, C, or D. If, however, the enzyme catalysing the
formation of C from B is inactivated, then the organism will be
able to grow provided it is supplied with either D or C but not
if supplied with A or B. If we can find three mutants of
the organism, each of which has lost one of the enzymes
involved in the synthesis of D from A, then, by studying the
nature of substances necessary for growth of the mutants,
it should be possible to reconstruct the chain A->B->C->D.
This is the principle of the method of " biochemical
mutants."

Irradiation of Neurospora results in the production of
mutants; the absorption of one quantum of radiant energy
causes an alteration of one gene which causes the loss, in turn,
of one enzyme. Some of the mutants produced will have lost
an enzyme involved in the synthesis of an amino-acid X.
All the " wild type" organisms can grow on a medium which
contains ammonium ions as N-source but the mutants will
only grow if the amino-acid X is added to the medium.
Consequently the organisms obtained after irradiation are
examined for their ability to grow (1) on a medium containing
ammonia as sole N-source, and (2) on a medium containing
ammonia -|- X as N-source. All those organisms which grow
on (1) can be discarded, while those which grow only on (2)
are further investigated. In the first place the number of
genetic types present is determined ; this gives an idea of the
number of genes, and consequently enzymes, involved in the
synthesis of X. Next the possible precursors of X are tested
as nutrients in place of X.

For example: seven different mutants of Neurospora were
isolated by Srb and Horowitz and were found to require

BIOCHEMICAL MUTANTS 97

arginine for growth ; of the seven, one would grow on addition
of nothing but arginine, two would grow on citrulline or
arginine, and four would grow on ornithine, citrulline, or
arginine. This indicates that the biosynthesis of arginine from
some precursor N must take place according to the sequence :

N -^ (four separate steps) -> Ornithine -> ? ->

Citrulline -> Arginine.

The same synthetic series was investigated by Bonner, using
mutants of Penicillium, and he was able to elucidate some of
the earlier stages of the sequence and to show that glutamic
acid is a precursor of arginine thus :

O, ,NH^ HN NH,

\/ ^ /

C C

I I

COOH CH^NH^ CH^NH CH^NH

CHz >► ? >- CHj ^ ? ^ CH2 ^ CHj

CHNH2 Ih CHNH^ CHNHj CHNH;

I J I ' I I

COOH ^1 COOH COOH COOH

HX — CHp ^ ^ A

Glutamic | | Ornithine Citrulline Arginine

acid H,C CH-COOH

'\ /

N
H

Proline

Thousands of such biochemical mutants of various micro-
organisms have now been isolated and the nutritional require-
ments of a small fraction of them discovered. Many of these
show a disability in the synthesis of an amino-acid and their
detailed investigation is yielding much information on the
biosynthetic precursors of substances such as valine, isoleucine,
methionine, tryptophan, lysine, etc. ^ It is not known yet
whether results obtained with one organism also apply to
another, although it is fairly certain that the biosynthesis of
tryptophan is very similar in Neurospora and Escherichia coli.
The synthesis of methionine from sulphate has been worked

98 growth: synthesis of bacterial protoplasm

out witli Esch. coli and tlie last four steps confirmed in
Neurospora:

SO4

t ?"=

" ?^ Pyruvic acid

SO3 ! '

I COOH

->► SH CH2OH

I I

CHj CHj L,

I -L I Momosenne

Cysteine chnh, chnh,
II

COOH COOH

CH,-S CH, CHjSH CHj-S-CHj

II I I

Cu Ki i_i CH7 CH? CH?

HNH2 I ^ j,^ I ^ 3^ I

I CHNH^ CHNH2 CHNH2

COOH I I I

COOH COOH COOH

Cystathionine Honnocysteine Methionine

Biocliemical mutant studies with Neurospora have shown
that the biosynthetic precursors of tryptophan are anthranilic
acid and indole, and that the last step involves the condensation
of indole with serine. This condensation has been further
proved by the preparation of a cell-free enzyme which accom-
plished the synthesis in the presence of pyridoxal phosphate as
prosthetic group :

COOH >^ CH,OH

I ^^" r n iCH^-CHNHj-COOH

CHNH, ^ ' " '

COOH

KAJ

Anthranilic Indole Serine. Tryptophan.

acid.

AMINO-ACID SYNTHESIS 99

These syntlieses have been deduced from studies with
artificially-induced biochemical mutants but mutation also
occurs spontaneously and, consequently, mutants of bacteria,
etc., will arise which have lost the ability to synthesise amino-
acids. This is the case with freshly isolated strains of Eberthella
typhosa which are unable to synthesise tryptophan and con-
sequently cannot grow in a tryptophan-free medium. If a
trace of tryptophan is added to the basal medium of salts,
ammonia and glucose, then normal growth and subculture is
possible. The organisms are able to grow if provided with
indole so the enzyme which has been lost catalyses a step in
the biosynthesis of indole rather than of tryptophan itself.
Fildes has shown that it is possible to select tryptophan-
synthesising mutants from the non-synthesising culture and
so, presumably, obtain the primitive synthetically competent
strain (see p. 63). Eberthella typhosa is exacting, if at all,
to tryptophan alone, but other species, especially Gram-
positive cocci, are unable to synthesise other amino-acids and
consequently will grow only in a medium rich in preformed
amino-acids.

If a cell has lost the ability to synthesise its amino-acids, it
necessarily becomes dependent upon the supply of these
substances in the medium. The concentration and relative
proportions of the various amino-acids in the medium will
rarely be those optimal for protein sjmthesis by the organism,
and it has been shown recently that certain Gram-positive
bacteria, such as the Streptococci and Staphylococci, have
acquired a concentration mechanism which compensates for
this loss of synthetic ability. These organisms possess a cell-
wall or membrane which enables them to take up amino-acids
from the external environment and to concentrate them inside
the cell prior to metabolism or condensation into protein.
Basic amino-acids such as lysine are able to diffuse through
this cell-wall, but acidic amino-acids such as glutamic acid
cannot penetrate the wall unless energy is supplied to the cell
through a metabolic process such as fermentation. If the cell
ferments glucose, then glutamic acid passes rapidly through the

100 growth: synthesis of bacterial protoplasm

cell-wall and becomes concentrated within the cell to an extent
such that the internal concentration may be over a hundred
times that in the medium. Gram-negative organisms do not
possess this capacity to concentrate amino-acids in the free
state inside the cell.

(d) Organisms exacting towards growth factors:
Some species of bacteria are able to grow in complex media
such as blood-serum, yeast extract, etc., but are unable to do
so in a salt-ammonium-glucose medium, even if a mixture of
pure amino-acids is added. In such cases, fractionation of
the blood or yeast medium leads to the isolation of a substance
or substances whose presence in minute quantities is essential
to growth, and which is known as a " bacterial vitamin "
or " growth factor." Table VIII gives a list of some of the
bacterial growth factors that have been identified. They
have no common chemical nature, A simple example is
given by the organism Pr. vulgaris, which is unable to grow
in a salt-ammonia-glucose medium unless nicotinic acid or
nicotinic amide is added. The presence of 1 X 10~^ grm.
nicotinic acid per ml. medium is sufiicient to support full
growth. From Table VIII it can be seen that several of the
growth factors are either prosthetic groups or parts of pros-
thetic groups of proteins and enzymes, and it would seem that
some bacteria find difiiculty in synthesising these chemically
complex active groups of enzyme systems. Pr. vulgaris is
unable to synthesise nicotinic acid and consequently cannot
manufacture coenzymes I and II, essential for the action of
certain oxidation mechanisms. In the case of Haemophilus
farainfluenzae, the synthetic disability extends to the nico-
tinamide-nucleoside part of the coenzyme molecule, and growth
cannot take place in the presence of nicotinic acid or amide,
but only if the nucleoside or the complete coenzyme molecule
is added to the medium. A sub-maximal growth of this
organism can occur in the presence of sub-optimal amounts
of coenzyme in the medium, and such " deficient " organisms
have an impaired oxidation mechanism, in that the rate of
oxidation of certain substrates is considerably less than normal.

DEFICIENT CULTURES 101

If washed suspensions of these deficient organisms are prepared
and their rate of oxidation of malic acid studied (malic acid
dehydrogenase requires coenzyme I), it is found that the
addition of coenzyme I to the suspension during the test
results in a greatly increased rate of oxidation. This demon-
strates that the organism has synthesised the enzyme malic
dehydrogenase, but the activity of the enzyme is not fully
effective as the coenzyme part of the system is deficient, and
the addition of coenzyme repairs the deficiency.

In this last example it was possible to grow an exacting
organism in the presence of sub-optimal quantities of a growth
factor whose function could be guessed with reasonable
certainty, and then demonstrate a metaboHc impairment in
that function. A similar technique can be used to determine
the metabolic function of other growth factors; to do this a
culture is grown in the presence of sub-optimal quantities
of growth factor and a control culture in the presence of
excess growth factor. A survey is then made of the metabolic
activities of the two cultures in an attempt to discover an
activity affected by the deficiency of growth factor in the
deficient culture. If such an impaired activity is found,
the effect on the activity of adding growth factor to the washed
suspension of the deficient organism is studied. Staph,
aureus, for example, is exacting towards thiamin. Thiamin-
deficient organisms metabolise pyruvic acid at a rate signifi-
cantly less than that of normal organisms, and the addition
of thiamindiphosphate makes good the deficiency. It follows
that thiamin plays some part in the metabolism of pyruvic
acid by these organisms.

Studies on the impairment of metabolism in growth-factor-
deficient cultures have assisted in the elucidation of the
function of pyridoxal which is the biologically active form
of pyridoxin (see Table VIII). If we study the growth of
streptococci in media containing increasing amounts of
pyridoxin, but otherwise fully nutrient, we get a curve relating
the amount of growth to the pyridoxin content of the medium
as shown in Fig. 8. We can distinguish three types of culture :

102 growth: synthesis of bacterial protoplasm

cells grown in medium A, rich in pyridoxin; cells grown in
medium B, containing just sufficient pyridoxin to allow full
growth; and cells grown in medium C, which is deficient in
pyridoxin to such an extent that the growth is seriously
restricted by the pyridoxin deficiency. If we investigate the
activity of the two enzymes, tyrosine decarboxylase (p. 168)
and transaminase (pp. 93-4), in the three cultures, we find that
both enzymes are fully developed in culture A ; in culture B
the transaminase system is fully developed, but the

PYRIDOXIN CONTENT OF MEDIUM

Fig. 8. Relation of growth of streptococci to
pyridoxin content of growth medium.

decarboxylase has about 5 per cent of the activity of culture A ;
in culture C the transaminase activity is considerably less
than that of cultures A and B, and no decarboxylase activity
is demonstrable. If, however, we take the cells from cultures
B and C and re-estimate their activities in the presence of
added pyridoxal, we find that both the tyrosine decarboxylase
and transaminase activities are restored to normal. This
suggests that the organism has synthesised the protein portions
of the enzymes in all three cultures, but that the enzymes are
inactive in the absence of their prosthetic groups or coenzyme

FUNCTIONS OF PYRIDOXAL 103

moieties which are related to pyridoxin in structure. Detailed
studies of the cell-free enzymes have now proved that both
enzymes have the same prosthetic group, and this can be
substituted in vitro by pyridoxal phosphate. The actual
identity of the natural prosthetic group with pyridoxal
phosphate has yet to be established, although there is little
doubt but that they are the same.

Some at least of the growth factors thus seem to function
as prosthetic groups of essential enzyme systems. The
synthetic disability may refer to the whole prosthetic group,
as in the case of riboflavin for S. haemolyticus, or to -a part of
the essential molecule as in the cases of nicotinic acid for Pr.
vulgaris and of thiamin for Staph, aureus. It is highly probable
that other growth factors are also needed by the organism as
parts of essential prosthetic groups. The nature of the
essential metabolism involved can be shown by the deficient
culture technique, and then, later, studies of the cell-free
enzymes can establish the complete structure of the prosthetic
group containing the growth factor. This type of investigation
is now being used to elucidate the structure of the active forms
of the growth factors pantothenic acid and biotin. Panto-
thenic acid-deficient cultures of organisms, which require this
substance as growth factor, are found to have impaired meta-
boHsm of acetic acid and impaired ability to carry out acetyla-
tion reactions. Thus deficient cultures of L. plantarum have
a greatly impaired ability to acetylate choline and this ability
can be restored by the addition of pantothenic acid to the
washed cells. Lipmann (see p. 34) has isolated a sulpha-
nilamide acetylase from animal tissues and shown that it
possesses a new prosthetic group, which he calls " coenzyme
A," which contains pantothenic acid. Concentrates of
coenzyme A have now been shown to act as the prosthetic
group of several enzyme preparations from bacteria and yeast
which catalyse reactions involving acetate or acetyl phosphate.
Thus a preparation which catalyses the condensation of
acetyl phosphate and oxalacetate to yield citrate is activated
by coenzyme A.' Coenzyme A has not yet been obtained in a

104 growth: synthesis of bacterial protoplasm

pure state but there is little doubt tbat it is a compound con-
taining pantothenic acid as part of its structure. The position
of biotin is very similar : deficient culture studies have shown
that biotin-deficiency results in impairment of oxalacetate
formation from pyruvic acid and COg, and in decreased activity
of aspartic acid and serine deaminases (see p. 163). Cell-free
preparations of aspartic acid deaminase can be activated by
biotin and adenylic acid; both factors are necessary and it is
fairly certain that the preparations contain a second enzyme
which brings about the synthesis of a co-aspartase from them.
A combined form of biotin has been isolated from yeast
extracts and this material will act as co-aspartase. Its
structure has not yet been determined.

In most of these cases we find that the biologically active
structure (= prosthetic group) is a more complex molecule
than the growth factor. This can be explained by saying that
the synthesis of the growth factor is more difi&cult for the
organism to accomplish than that of the rest of the prosthetic
group but, on the mutational explanation of variation, it is
probably more correct to say that mutations involving loss of
synthetic ability towards the growth factor give the organism
a greater energetic advantage (or " selection pressure ") in a
complex medium than those involving loss of ability to
synthesise the rest of the molecule. Whatever the explanation,
it does not follow that all strains of an organism which is
nutritionally exacting towards, say, pantothenic acid will
display a disability towards the whole molecule. Some
organisms can synthesise pantothenic acid if they are supplied
with jS-alanine. Others, which apparently require biotin, can
synthesise this factor if supplied with the pimelic acid part
thereof. In these cases, the lost enzymes are concerned with
the synthesis of the j8-alanine portion of pantothenic acid or
the pimelic acid portion of biotin respectively.

Many growth factors have been discovered by analysis of
complex "satisfactory" media, but one, j9-amino-benzoic acid ,
was first discovered as an antagonist to the drug sulphanila-
mide (seep. 113) and then shown to be a growth factor for, first,

FOLIC acid; strepogenin 105

CI. acetobutylicum. Its metabolic function is still not clear.
It lias been shown to form part of the structure of folic acid, a
growth factor for several Lactobacteriaceae, but it may have
functions other than those involved in the production of folic
acid. FoHc acid itself is presumably a prosthetic group but
no enzymes requiring it have yet been discovered. The
deficient-culture technique does not seem to have yielded
results, but the discovery that folic acid can be replaced in
growth media by mixtures of thymine and other purines and
pyrimidines has led to the suggestion that the fohc-activated
enzymes are concerned with purine and pyrimidine synthesis.
Similar evidence suggests that it may also be concerned in
methionine synthesis while ^-amino-benzoic acid, apart from
folic acid, may be involved in the synthesis of other amino-
acids, such as lysine.

A growth factor of unknown constitution and function is
Strepogenin. This is a substance necessary for the growth
of some Lactobacteriaceae and has the properties of a
peptide. It can be obtained from partial hydrolysates or
enzymatic digests of crystalline proteins such as insulin and
so must represent a part of the structure of these proteins.
It is thought that it is a specific peptide structure synthesised
by a specific enzyme which can be lost by mutation.

Haemophilus influenzae is exacting towards h.aematin, but
can in some cases dispense with this if growth takes place
under anaerobic conditions. It would appear that haematin
is necessary for some oxidative process, and since catalase is
a haematin-enzyme, it has been suggested that the haematin
is required for the synthesis of catalase which protects the
organism from hydrogen peroxide formed during aerobic
existence. This suggestion is supported by the fact that
haematin can be replaced as a growth factor by cysteine,
which decomposes HgOg by reduction.

In the same way that it is possible to select mutants of
Eherthella typhosa which can synthesise their own tryptophan,
it is also possible in some cases to select from cultures of
organisms exacting towards growth factors, mutants which

106 growth: synthesis of bacterial protoplasm

will be able to synthesise these factors. Thus it is possible to
"train" Staph, aureus to synthesise thiamine by serial sub-
cultivation in media containing progressively less of this
factor.

(e) Organisms exacting towards both amino-acids and
GROWTH factors: It has been suggested that an organism
becomes exacting when it grows for many generations in a
medium in which all growth requirements are provided ready
made. If the rate of growth is regulated by the rate of
synthesis of some factor, and that factor is provided ready
made in the environment, then the organism will be able to
grow more rapidly if it utilises the preformed factor than
if it is dependent upon the synthetic process. Likewise, a
mutant which has lost the ability to synthesise that factor
will grow as well as, if not better than, the synthetically
competent cell in the rich medium. Rich media would there-
fore be expected to select nutritionally-exacting mutants and,
in general, the richer the medium, the poorer would become the
synthetic abihties of the organisms using it as a natural
habitat. Soil organisms can find few or no complex growth
factors in their habitat, but organisms that have assumed a
parasitic existence in animal tissues are living in an environ-
ment rich in all those substances that go to make up proto-
plasm. In general, we find that the more parasitic an
organism, the more exacting are its growth requirements.
Some organisms such as S. haemolyticus are exacting towards
several amino-acids and several growth factors. The omission
of any one of these amino-acids or factors from the medium
renders it sterile towards this organism. The organism is
highly parasitic because only in the presence of tissues and
tissue products will such an array of factors be found naturally.
Parasitism leads to exacting growth requirements, and the
exacting nature of the growth requirements makes parasitism
obligatory. It does not follow that exacting organisms are
all parasitic on man, or that they are pathogenic, as patho-
genicity depends upon factors other than parasitism (see
Chap. XI). Organisms such as the Lactobacilli from milk or

CARBON DIOXIDE EEQUIREMENT 107

CI. acetobutylicum from molasses, are highly exacting as a
result of constant growth in these complex but inanimate
media.

Table X outlines the growth requirements of various
selected species that are mentioned in this book.

Carbon dioxide requirement

A distinction has been drawn between the autotrophic and
heterotrophic bacteria partly on the grounds that the former
utilise carbon dioxide as sole source of carbon, while the latter
utilise organic material as carbon source. It is not true,
however, that the heterotrophic bacteria are unable to utilise
carbon dioxide, for carbon dioxide is actually essential for the
growth of many, if not all, heterotrophic species. Whereas
the autotrophes utilise carbon dioxide as sole source of carbon,
the heterotrophes require traces only as a source of certain
essential carbon compounds. The requirement of carbon
dioxide was demonstrated by Gladstone and others, who
showed that if simple media are rendered COg-free, then many
heterotrophic organisms cannot grow or their growth is
greatly delayed, e.g. neither Esch. coli nor Eberthella typhosa
will grow in the absence of carbon dioxide, while the growth
of Staph, aureus is greatly delayed, presumably until the
metabolic activities of the inoculum have produced a threshold
concentration. The full function of carbon dioxide has not
yet been elucidated, but it has been shown in the case of the
fermentation of many organisms such as Esch. coli, the
Propionibacteria, etc., that carbon dioxide assimilation is
involved in the formation of succinic and other 4-carbon
dicarboxylic acids (see Chap. YII).

KNOWLEDGE OF SYNTHETIC PROCESSES FROM GROWTH
REQUIREMENTS

By studying the growth of bacteria in mixtures of pure
chemicals, we are able to divide organisms up into nutritional
groups along the lines indicated above. We start, on the one

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Proteus vulgaris

Proteus morganii

H. parainfluenzae

CI. acetobutylicum

Corynebact. diphtheriae

CI. sporogenes

Staph, aureus

8. haemolyticus

Lactob. casei

108

SYNTHETIC ABILITIES 109

hand, with a chemically defined medium, and from that we
grow a certain amount of bacterial protoplasm; in most
cases we have no knowledge of the intermediate chemistry
and metabolism. Exacting nutritional requirements show
us what certain organisms cannot do, and we make the
assumption that these synthetic disabilities of some organisms
are synthetic abilities in others. To what extent is this
assumption justified ?

In the first place, we are able to trace a paralleHsm between
exactingness and parasitic existence which suggests that the
former arises from constant growth in the presence of complex
essential substances of difficult synthesis ; in some cases we are
able to demonstrate a recovery of synthetic ability, such as
that towards tryptophan of exacting Eher. typhosa, serially
subcultivated in media containing progressively less trypto-
phan, or that towards thiamin of Staph, aureus subcultivated
similarly in media containing progressively less thiamin.

Secondly, in the case of certain growth factors such as
nicotinic acid, thiamin, pantothenic acid, pyridoxal, etc., we
are able to show a metabolic impairment of deficient cells
which is not found in non-exacting organisms, and in such
cases the metabolic impairment is related to those enzymes
which, as we know from studies in other tissues, have a
structure involving the growth factor as prosthetic group or
coenzyme.

Thirdly, by surveying the parts of a complex growth
factor which are required by various exacting organisms,
we can often show that some organisms can synthesise
certain portions of the molecule but not others, e.g. some
strains of the diphtheria organism require ^-alanine as growth
factor, while others require pantothenic acid ; pantothenic acid
will replace ^-alanine for the former organisms, but ^-alanine
cannot replace pantothenic for the latter; consequently the
former organisms cannot synthesise ^-alanine, but, given that,
can synthesise pantothenic acid, whereas the latter strains
have a wider disability in that they cannot accomplish this
further step but require the complete pantothenic acid molecule.

110 growth: synthesis of bacterial protoplasm

Fourthly, we can demonstrate the presence of the growth
factors in the constitution of non-exacting organisms by
the technique of microbiological assay (see below). For
example, if we make up a salt-ammonia-glucose medium and
carefully free it from nicotinic acid before inoculating with
Pr. vulgaris then no growth will occur. Consequently we
can use the appearance of growth as a test for the presence
of nicotinic acid and, over a certain range, the growth is
proportional to the amount of nicotinic acid added. In this
way we can show the presence of nicotinic acid in the proto-
plasm of non-exacting bacteria by boiling the cells and adding
the sterile extract to the basal medium. The test is highly
specific and, in an analogous manner, we can show the presence
of all these growth substances in the protoplasm of non-
exacting organisms (e.g. autotrophes).

So we get knowledge concerning the synthetic abilities of
nutritionally non-exacting organisms from studies of the
disabilities of exacting types. Autotrophic bacteria must be
able to synthesise all their amino-acids from ammonia and
carbon dioxide; the amino-acids are then condensed in various
stereochemical combinations to form proteins; some of these
proteins require the synthesis of complex prosthetic groups
before becoming active as enzymes; some enzymes are not
complete without carrier systems of coenzyme I nature; the
enzyme systems break down energy-yielding substances in
the environment with the production of acid, alkaline, and
toxic end-products, and further enzymes are synthesised to
neutralise or detoxicate such products. At each stage of
synthesis some organism finds the task too difficult, so we get
differentiation into autotrophic, heterotrophic, anaerobic,
exacting, parasitic, pathogenic, etc., organisms, and in each
case the differentiating property is a reflection of the synthetic
abilities of the organisms concerned.

Microbiological assay

In this chapter organisms have been mentioned which require
the presence of certain growth factors in the medium before

MICROBIOLOGICAL ASSAY

111

growth can occur. Many, if not all, of these growth factors
are of importance in mammalian nutrition, but are difficult to
estimate by chemical means owing to their complex structure
and the very small amounts in which they occur naturally.
If an organism is exacting towards a growth factor F, and is
inoculated under standardised conditions into a series of
media containing amounts of F varying from nil to sufficient
to give complete growth, then the growth is found to vary
with the amount of F in the medium as shown in Fig. 9.

Over a certain range of concentrations of F there is a linear,
or approximately linear, relation between the concentration

GROWTH

(turbidity,
dry weight

CELL-N,

ACID FORMED

ETC.)

CONCENTRATION OF F
Fig. 9.

and the amount of growth . If this relation can be standardised
consistently, then the growth can be used as a measure of the
amount of F in a given solution. This method of growth
factor or vitamin determination is known as microbiological
assay. For example, the growth of Pr. vulgaris can be used
for the assay of nicotinic acid. The method is open to many
sources of error: the basal medium must be completely and
easily freed from the factor to be assayed; the growth curve
must be accurately reproducible; and the growth must not
be affected by any other variable factor in the growth medium
or in the preparation of material added for assay. The
method can often be made to work with reasonable accuracy

112 growth: synthesis of bacterial protoplasm

for solutions of the assay factor, but difl&culties arise when
attempts are made to assay the factor in biological materials,
food-stuffs, etc., as, unless it is possible to extract the factor
easily and quantitatively, addition of the preparation con-
taining the factor is certain to involve the addition of salts,
amino-acids, and other growth factors which may affect the
growth and so vitiate the assay. The problem is to obtain a
satisfactory basal medium such that the addition of the assay
factor alone has any effect on growth. G-rowth may be
estimated turbidimetrically, but in the case of the ^omolactic
fermenters such as Lactobacilli or S. lactis, it is possible to
obtain a measure of the growth by titration of the acid formed
in the medium. Many workers prefer the titration method
to the turbidity measurement. Microbiological assay is
used for the estimation of nicotinic acid, pantothenic acid,
bio tin, pyridoxin, riboflavin, folic acid, thiamin, and certain
amino-acids.

NUTRITIONAL ANTAGONISM

We have seen that many pathogenic organisms have become
nutritionally exacting as a consequence of parasitic existence.
Such organisms are unable to grow in media which do not
contain certain growth factors, or in fully nutrient media if
the utilisation of the growth factors is prevented. The possi-
bility of preventing growth by interference with growth factor
utilisation was brought into prominence by the work of Woods
on sulphanilamide action.

Sulphanilamide, or ^-amino-benzene-sulphonamide, in low
concentrations prevents the growth of certain bacteria,
particularly the Gram-positive cocci. It can be shown that
sulphanilamide does not immediately kill bacteria in these
low concentrations, but prevents their division. Organisms
whose growth has been checked by sulphanilamide can
proceed to grow and multiply normally after removal of the
sulphanilamide. This can be demonstrated by inoculating a
sensitive organism into medium containing just sufficient
sulphanilamide to prevent its growth ; if the static culture is

SULPHANILAMIDE-^-AMINOBENZOATE COMPETITION 113

now subcultured into fresh sulphanilamide-free medium,
growth will occur as usual. Sulphanilamide is consequently
said to be "bacteriostatic," in that it prevents multiplication
without necessarily killing the organisms. The quantity of
sulphanilamide which is bacteriostatic for any given organism
varies greatly with the constitution of the medium in which
the test is carried out. For instance, media which contain
peptone or yeast extract can support growth in the presence
of much higher concentrations of sulphanilamide than simple
synthetic media. This is explained in the case of yeast
extract by the presence in the extract of a substance which is
antagonistic to sulphanilamide in that it prevents its bacterio-
static action on the bacteria. Woods investigated the
properties of this anti-sulphanilamide substance, and showed
that it has the properties both of a weak organic acid and of a
diazotisable aromatic amine. He then tested the anti-
sulphanilamide activity of jo-amino-benzoic acid, and found
that 1 molecule can neutralise the bacteriostatic action of
5000-25,000 molecules of sulphanilamide. No other substance
of this nature that was tested has such marked anti-sul-
phanilamide activity and, shortly after, other workers were
able to isolate jt?-amino-benzoic acid itself from yeast extracts.
In the extracts p-amino-benzoic acid exists in the free state
and also combined as a peptide with glutamic acid, and the
amount of (free -\- combined) substance is sufficient to account
for the total anti-sulphanilamide activity shown by the
extract. At the time of the demonstration of its anti-sulphani-
lamide action, no function had been attributed to ^-amino-
benzoic acid in bacteria, but within a few months two
Australian workers, Eubbo and Gillespie, were able to show
that it acts as a growth factor for CI. acetohutylicum. The
list of organisms which require ^-amino-benzoic acid as a
growth factor has now been exteruded to include certain
Acetobacter, Lactobacilli, and a mutant strain of Neurospora.
Eecently j9-amino-benzoic acid has been shown to form part
of the molecule of folic acid (Table VIII) which is required
by many of the Lactobacteriaceae.

CHEM. A. B. S

114 growth: synthesis of bacterial protoplasm

Fildes and Woods proposed that ^-amino-benzoic acid is
an " essential metabolite " for bacteria, and that if its meta-
bolism is in any way prevented, then growth ceases. Since
sulphanilamide and ;p-amino-benzoic acid have similar
chemical structures, it was suggested that sulphanilamide
acts as a competitive inhibitor of the enzyme carrying out the
essential metabolism of jo-amino-benzoic acid.

H^N-^-^-SO^NH^ H2N-:^~\-C00H
Sulphanilamide ^-amino-benzoic acid

The fact that folic acid contains jo-amino-benzoic acid as
part of its structure, indicates that the latter substance must
undergo some metabolism within the organism in order to
become incorporated in the larger molecule.

According to this theory, ^-amino-benzoic acid is an
" essential metabolite," and if the organism has no power to
synthesise this metabolite, then it becomes a growth factor
for that organism. Sulphanilamide acts by preventing the
utilisation of p-amino-benzoic acid. If this is the case, then
it should be possible to inhibit the growth of other exacting
organisms by presenting them with substances of structure
similar to that of their specific growth factors (" metabolite
analogues ") which will compete with the growth factor for
an enzyme surface, will block the metabolism of the growth
factor, and, consequently, prevent growth. For example,
Pr. vulgaris is exacting towards nicotinic acid; if we add
pyridine-3-sulphonic acid to the medium, we find that growth
is prevented by competition between the growth factor, nicotinic
acid, and its antagonistic analogue, pyridine-3-sulphonic acid.

_COOH ^SOgH

Nicotinic acid Pyridine-3-sulphonic acid

In this case it is interesting that pyridine-3-sulphonic acid
acts as an antagonist towards nicotinic acid, but not towards
nicotinic amide, so presumably the antagonist prevents the

METABOLITE ANALOGUES 115

synthesis of the amide from the acid. Many examples of
this growth factor antagonism have now been worked out and
some are given in Table XI. There is one difference between
these examples and that of sulphanilamide, in that whereas the
latter is effective against many organisms whether these are
nutritionally exacting towards ;p-amino-benzoic acid or not,
the nutritional antagonists are effective as growth inhibitors
only in those cases where the organism tested is nutritionally
exacting towards the factor concerned. Whether this is a
difference of principle or degree remains to be seen.

The main interest of this type of work was that it gave
promise of the rational development of chemotherapeutic
agents. Many nutritionally exacting organisms are patho-
genic, and if it were possible to prevent their growth
in vivo by nutritional antagonism, then the growth factor
analogues might form a valuable source of chemotherapeutic
agents. Pantoyl-taurine is effective as a bacteriostatic agent
against streptococcal infections in the rat and is antagonised
by pantothenic acid. So far, however, no marked advances
in the chemotherapeutic field have come from this research
for three main reasons : most of the antagonists so far prepared
are simple molecules, and are excreted too rapidly to be
effective m vivo ; the internal environment of the host contains
such quantities of the .natural growth factor that the com-
petitive amounts of analogue that must be injected 9,re
unreasonably large; and in some cases the effect of the
analogue is to deprive the host of the metabolism associated
with the growth factor as well as the organism (e.g. the adminis-
tration of pyrithiamin to rats gives rise to the symptoms of
vitamin Bj deficiency).

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118 growth: synthesis of bacterial protoplasm

for further reading

" The Physiology and Biochemistry of the Sulphur
Bacteria," Bunker, H. J., D.S.I.E. Special Eeport Series.

" The Transamination Eeaction," Herbst, R. M., Advances
in Enzymology, 1944, 6, 75.

"Growth Factors in Microbiology," Knight, B. C. J. G.,
Advances in Vitamins and Hormones, 1945, 3, 108.

" Bacterial Nutrition," Knight, B. C. J. G., M.R.C. Special
Report Series, H.M. Stationery Office.

Bacterial Chemistry and Physiology, Porter, J. R. (Wiley).

"Metabolite Antagonists," Roblin, R. 0., Chem. Rev., 1946,
38, 255.

Bacterial Metabolism, Stephenson, M. (Longmans).

" Heterotrophic Assimilation of Carbon Dioxide," Werkman,
C. H., and Wood, H. G., Advances in Enzymology, 1942, 2, 135.

" Biochemical Problems of the Chemo- Autotrophic Bacteria,"-
van Niel, C. B., Physiological Rev., 1943, 23, 338.

" The Relation of jo-aminobenzoic Acid to the Mechanism
of the Action of Sulphanilamide," Woods, D. D., Brit. J.
exp. Path., 1940, 21, 74.

Basis of Chemotherapy, Work, T. S., and Work, E. (Oliver
and Boyd).

CHAPTER VI

BACTERIAL POLYSACCHARIDES

The polysaccharides synthesised by bacteria have received
detailed attention from carbohydrate chemists, as not only
do they present a wide range of new structures, but the poly-
saccharide found in the capsules of some organisms determines
their immunological specificity. Polysaccharides are formed
by bacteria in capsules, extracellular gums and slimes, and
probably as stores of energy.

Gums and slimes

One of the earliest studies of Pasteur was concerned with
the " viscous fermentation " occurring in sugar solutions, and
he showed that the slime formation was due to infection of
the material with certain organisms. We know now of several
species of bacteria which are able to synthesise hydrophilic
polysaccharide gels which are composed mainly of either
glucosan (" dextran ") or fructosan (" levan "). Organisms
such as B. mesentericus and B. subtilis, when growing in the
presence of sucrose, give rise to a fructosan in which the
fructofuranose residues are linked as follows: —

H 0H>| IVh OH^

H 1 r CH^OH H ^ f CH^OH

OH H OH H

The organisms attack sucrose to form fructosan, but cannot
produce the gum from glucose or fructose alone, a mixture of
glucose and fructose, or from invert sugar. This suggests
that energy is required to link the fructofuranose residues, and
this energy is derived from the hydrolysis of the sucrose mole-
cule. A cell-free enzyme has been obtained from B. subtilis
which will carry out the synthesis of fructosan from sucrose :

sucrose > fructosan -f glucose.

120

BACTERIAL POLYSACCHAEIDES

In other cases the extracellular gum or slime is a glucosan.
Organisms such as Leuconostoc dextranicus, Betacoccus arabi-
nosus, etc., will synthesise glucosan when grown in the presence
of sucrose. The glucosan has a long chain structure in which
the repeating unit is

Bacterial cellulose

Acetobacter xylinum (see p. 149) produces a slimy envelope
when growth takes place in the presence of sucrose or glycerol.
The envelope is composed of a polysaccharide which has the
same structure as that of vegetable cellulose, i.e. a chain
structure consisting of cellobiose as the repeating unit:

CH,OH

Capsule polysaccharides

Many organisms produce capsules and these are often,
though not invariably, composed largely of polysaccharides.
These are of considerable interest since they appear to confer
specificity upon the immunological response. For example,
the genus Pneumococcus can be divided into 32 types by
serological methods. If a serum to Type I is prepared by
injection of the intact organism into an animal, then that
serum reacts with Type I pneumococcus only. However, if
a serum is prepared against the proteins of the Type I pneu-
mococcus— in the absence of the polysaccharide — then the

TYPE SPECIFIC POLYSACCHARIDES 121

serum will react with any pneumococcus irrespective of type.
Further, the serum prepared against the intact cell will
precipitate the Type I polysaccharide, but not polysaccharides
prepared from other types, while the serum prepared against
the protein fraction will not precipitate any of the poly-
saccharides. It seems, then, that the antigen is the protein
part of the cell, but the presence of the polysaccharide
confers specificity upon the antibody response. This suggests
that the 32 pneumococcal types differ in the nature of the
polysaccharides composing the capsules. The structures of
several of these substances have been worked out; the Type III
polysaccharide consists of glucose and glucuronic acid residues
linked into chain formation as follows: —

COOH OH

CH2OH J 0, ^ A k . 9"

Y— f^ OH CHgOH r""0

OH COOH

Energy stores

In mammalian tissues, energy is stored in the glycogen
deposits while in plant tissues storage occurs mainly in the
form of starch. Bacteria are known to accumulate reserves
of polysaccharide material within the cell, but few studies
have been made as yet on the nature of these stores. For
example, when Esch. coli is allowed to metabolise glucose in
excess, then polysaccharide formation occurs within the cell,
but this material is itself metabolised as soon as the external
glucose is exhausted.

FOR FURTHER READING

Introduction to Carbohydrate Biochemistry, Bell, D. J.

(Univ. Tut. Press).

Bacterial Metabolism, Stephenson, M. (Longmans).

CHAPTER VII

PKOVISION OF ENEKGY: FERMENTATION

In the last chapter we considered the materials which are
essential for the growth of various bacterial species. The
growth process involves assimilation of these materials and
their elaboration into the constituents of the living cell. These
cellular constituents are often far more complex than the
nutrient materials; for example, the autotrophic bacteria
synthesise protein molecules from ammonia and carbon
dioxide. In other words, the energy content of the cell
constituents is higher than that of the raw materials and, con-
sequently, energy has to be suppHed before cell synthesis and
growth can occur. The gain in energy of the cell constituents
is obtained by degradation of other energy-rich materials in
the environment. The bacterial cell often obtains its energy
by the degradation of carbohydrates in the environment, and
this degradation can be accompUshed anaerobically, in which
case the process is called " fermentation," or aerobically by
oxidation processes. In this chapter we shall consider the
fermentation process.

The products of bacterial fermentation are many and varied,
and it has been shown in Chap. II that bacteria can often be
'^: separated and differentiated on the basis of their fermentation
reactions considered with respect to the sugars fermented and
the products formed from those sugars. It is undesirable to
deal here with the whole range of bacterial fermentations and,
indeed, many have not yet been worked out in detail. In
some cases bacterial fermentation provides an easily controlled
method for the production of a commercially valuable sub-
stance such as butyl alcohol, and in others the production of
an easily identified and specific product can be used as a
characterisation test. In these cases detailed investigations
have been carried out with the intention of elucidating the
metabolism involved. The problem is also of interest to the
biochemist interested in carbohydrate metabolism, and the
fermentation of Esch. coli and Aerobacter aerogenes has received

122

FERMENTATION PRODUCTS 123

such detailed attention that our knowledge of the intermediate
stages is now almost as great as that of the related glycolysis
cycles in yeast and other cells. The key substance in many
fermentations is pyruvic acid, CHg . CO . COOH, which is formed
by the breakdown of the carbohydrate molecule and is then
attacked in various ways by different organisms to give a
variety of products. In this chapter we shall first trace the
course of the formation of pyruvic acid and then show how
various organisms produce their varied fermentation products
by further elaboration of this key substance.

FERMENTATION OF GLUCOSE
Glucose is the carbohydrate whose fermentation has been
studied in greatest detail. Some organisms, including many
of the Streptococci and Lactobacilli, carry out a simple
fermentation of glucose with the production of lactic acid
in almost theoretical yield; such organisms are called homo-
lactic fermenters. The majority of heterotrophic organisms
produce a variety of products amongst which can be listed
the following: —

CO2 Pyruvic acid. Acetone,

CH3.CO.COOH CH3.CO.CH3

Hj Butyric acid, iso-Propyl alcohol,

CH3. CHg. CHg. COOH CH3.CHOH.CH3

Formic acid, Ethyl alcohol. Succinic acid,

H . COOH CH3 . CH2OH HOOC . CH2 . CH2 . COOH

Acetic acid, n-Propyl alcohol, Acetylmethylcarbinol,

CH3.COOH CH3.CH2.CH2OH CH3.CO.CHOH.CH3

Propionic acid. Butyl alcohol, 2.3.Butylene glycol,

CH3. CHg. COOH CH3.CH2.CH2.CH2OH CH3.CHOH.CHOH.CH3

Lactic acid, Diacetyl,

CHj.CHOH.COOH CH3.CO.CO.CH3

Fermentation releases energy. From this point of view it
is a less efficient form of metabolism than oxidation, for the
complete oxidation of glucose yields considerably more energy
than any fermentation can do, as Hhe latter involves the
production of partially reduced substances :

CgHigOe + 6O2 > 6CO2 + 6H2O + 674 Cals.

CgHiaOe > 2CO2 + 2C2H5OH + 22 Cals.

124 PROVISION OF energy: fermentation

A further consequence of obtaining energy by fermentation
is the accumulation of large quantities of waste products
which may be toxic to the organism in high concentration.
Consequently this form of metabolism is necessarily restricted
to small organisms living in a liquid medium, in which the
waste products are quickly removed by diffusion from the
immediate environment.

The anaerobic breakdown of glucose has been studied in
considerable detail of recent years, and great advances have
been made in our knowledge of the processes that take place
during this breakdown ("glycolysis") in muscle and yeast
cells. In the case of these cells it is comparatively easy to
make cell-free extracts of the cells and, from these, to make
preparations of the various enzymes present. In this way it
has been possible to disentangle the various steps in the
series of reactions and to isolate the enzymes catalysing these
steps. Our knowledge of bacterial glycolysis has lagged
behind that for yeast and muscle cells, as it is only of recent
years that efficient methods have been discovered whereby
bacterial cells can be disrupted and their enzymes liberated
in an active state. Consequently there are still gaps in our
knowledge of bacterial fermentation processes, and much
of the work has been concerned so far with investigating
whether the stages of breakdown of glucose by bacteria are
the same as those occurring in yeast. As far as the processes
concerned in the formation of pyruvic acid are concerned,
the answer appears to be that these processes are essentially
the same in bacteria as in yeasts and in various other tissues
that have been investigated.

BREAKDOWN OF GLUCOSE BY YEAST

Table XII outlines the steps and enzymes involved in the
breakdown of glucose to pyruvic acid by yeast cells. The
first step consists of a phosphorylation of glucose to glucose-
6-phosphate by the enzyme hexokinase, which catalyses the
transfer of the phosphate group from adenosine-tri-phosphate

GLYCOLYSIS CYCLE

125

126 PROVISION OF energy: fermentation

(ATP) to the 6-position in the glucose molecule. Glucose-6-
phosphate is then altered to fructose-6-phosphate by the
action of the enzyme phosphohexose-isomerase (sometimes
called oxoisomerase) and a second phosphate enters the
molecule in the 1 -position, the second phosphate again being
transferred from ATP, but under the action of the enzyme
phosphohexokinase in this case. This series of reactions
results in the formation of hexosediphosphate from glucose, two
molecules of phosphate being taken up from two molecules of
ATP with the formation of adenosine-di-phosphate in each case.

Hexosediphosphate then splits into an equilibrium mixture
of triosephosphates under the action of the enzyme zymo-
hexase (also called aldolase). The two triosephosphates are
glyceraldehyde-phosphate and dihydroxyacetone-phosphate,
and their interconversion is catalysed by the enzyme isomerase.
We are mainly concerned with the breakdown of glyceralde-
hyde-phosphate in fermentation reactions and, as this is
removed, dihydroxyacetone-phosphate isomerises to form
more glyceraldehyde-phosphate, so that eventually the whole
of the hexosediphosphate that is broken down by zymohexase
can pass through the series of reactions starting with glycer-
aldehyde-phosphate.

Glyceraldehyde-phosphate is oxidised by the enzyme
triosephosphate dehydrogenase (or glyceraldehyde-phosphate
dehydrogenase). This enzyme catalyses the transfer of
hydrogen from its substrate to coenzyme I, and is only active
in the presence of inorganic phosphate. The immediate
product of the oxidation is l.S.diphosphoglyceric acid:

CHO COOPO3H2

I Triosephosphate I

CHOH -f- HgO + coenzyme I + phosphate > CHOH + reduced

I dehydrogenase I coenzyme I

CH2OPO3H2 CH2OPO3H2

and the reduced coenzyme I is available as H-donator for
other reactions. The diphosphogly eerie acid can give up
the second phosphate to either adenyhc acid or adenosine-di-
phosphate resynthesising ATP in the presence of the necessary

TRIOSE PHOSPHATE OXIDATION 127

enzyme. Consequently this particular step in the breakdown
process has three results:

1. The oxidation of glyceraldehyde-phosphate to phospho-
glyceric acid.

2. The formation of reduced coenzyme I as H-donator.

3. The uptake of inorganic phosphate and its synthesis
into ATP.

Phosphoglyceromutase now catalyses the transfer of the
phosphate group from the 3-position in glyceric acid to the
2-position, and water is removed from 2-phosphoglyceric
acid under the action of enolase. Phospho-enol-pyruvic acid
is produced which can lose its phosphate by transfer to
adenylic acid or adenosine-di-phosphate with the formation of
pyruvic acid and the regeneration of ATP. The phosphoryla-
tion of glucose to hexosediphosphate involves the dephosphory-
lation of two molecules of ATP, while the further breakdown of
each molecule of glyceraldehyde-phosphate regenerates two
molecules of ATP.

In muscle, pyruvic acid is reduced to lactic acid by lactic
dehydrogenase working in reverse and utilising the reduced
coenzyme I as H-donator. The oxidation of glyceraldehyde-
phosphate and the reduction of pyruvic acid are thus linked by
coenzyme I acting as H-carrier between the two enzyme systems :

CHO ■ COOH

I Triosephosphate | ■RpdnopH

CHOH -f Coenzyme I > CHOH + -^^^^^^^

I ^ Dehydrogenase | coenzyme I

CH2OPO3H2 CH2OPO3H2

CH3 ^ . CH3 -\^

' Rpflnrpd Lactic |

*^0 +ccZ™eI ^ CHOH + Coenzyme I

I -^ Dehj^drogenase I

COOH COOH

128 PROVISION OF energy: fermentation

In yeast cells pyruvic acid is decarboxylated by the enzyme
carboxylase, and the acetaldehyde so formed is reduced to
ethyl alcohol by alcohol dehydrogenase acting in reverse in a
manner analogous to the lactic dehydrogenase of muscle,

Carboxylase

CH3.CO.COOH ^ > CIIaCHO + CO2

PTT nuf\ _L Reduced ___°1_^_^ CH3.CH2OH +
CH3 .CHO + ^^^^^^^ T T^ehydrogenar Coenzyme I

THE FORMATION OF PYRUVIC ACID FROM GLUCOSE
BY BACTERIA

In the scheme described above for the breakdown of glucose
by yeast, the initial stages consist of a phosphorylation of the
glucose molecule to form hexosediphosphate. The majority
of bacteria are unable to ferment glucose in the absence of
phosphate. This can be demonstrated very easily in some
cases by centrifuging the organisms out of culture, washing
them very thoroughly, and then incubating the washed
suspension of organisms with glucose in the presence and
absence of phosphate. If the washing has been successful in
removing phosphate from the organisms, then fermentation
will often not occur in the absence of phosphate, though it
proceeds normally in its presence. This constitutes a priori
evidence that phosphate is involved in the fermentation
processes, but the existence of the enzymes catalysing the
intermediate steps involved in the conversion of glucose to
hexosediphosphate has yet to be proved in bacteria. It is
highly probable that the same reactions occur, as Esch. coli,
for example, will ferment fructosediphosphate to the same
fermentation products as those obtained from glucose. Also
when glucose is being fermented by the cells, there is an uptake
of inorganic phosphate from the medium, and if sodium
fluoride (which inhibits enolase) is added to the fermentation
mixture, phosphoglyceric acid can be isolated as the chief

LIBEEATION OF ENERGY 129

product. This suggests that the same cycle of reactions is
occurring in Esch. coli as previously demonstrated in yeast.

If we assume that the initial steps in the breakdown are
those shown in Table XII, then the rest of the chain of
reactions has been proved. Using various methods for the
disruption of cells (see Chap. Ill), it has been possible to
obtain cell-free preparations of zymohexase (aldolase),
isomerase, glyceraldehyde-phosphate dehydrogenase, phos-
phoglyceromutase, enolase, lactic dehydrogenase, coenzyme I,
and adenosine-tri-phosphate from Esch. coli, Aerobacter
aerogenes, etc. There is little doubt then but that the main
glycolysis cycle, as set out in Table XII, can occur in these
bacteria. In general, the enzymes that have been isolated
from bacteria are essentially similar in properties to their
counterparts in other cells.

Liberation of energy

The formation of pyruvic acid from glucose via the cycle
described above, results in the liberation of energy which is
available to the organism for growth purposes. This energy
is made available by the building up and subsequent rupture
of the various phosphate bonds in the cycle. The energy
content of phosphate bonds differs with the nature of the bond.
Thus phosphate ester bonds of the type we get in hexose-
diphosphate ( — CHgOPOgHg) liberate comparatively little
energy when they are broken, but enol-phosphate bonds
( — COPO3II2) of the type we find in phospho-enol-pyruvic
acid are energy-rich, their rupture releasing about 12,000 cals.
per gram-molecule compared with 3,000 cals, liberated by
the rupture of an ester-bond. Glucose is thus phosphorylated
at a low energy level to hexosediphosphate, and this is built
up by the cycle of reactions to give the energy-rich bond in
phospho-enol-pyruvic acid. Energy cannot, of course, be
created and phosphoglyceric acid and phospho-enol-pyruvic
acid have approximately the same energy content, but whereas
the energy of phosphoglyceric acid is distributed over the

CHEM. A. B. 9

130 PEOVisiON OF energy: fermentation

whole molecule, in phospho-enol-pyruvic acid the energy is
concentrated in the phosphate bond.

A second energy-rich phosphate bond is built up during the
oxidation of glyceraldehyde-phosphate with the uptake of
inorganic phosphate to yield l.S.diphosphoglyceric acid. In
the presence of ADP, the diphosphoglyceric acid yields
S.phosphoglyceric acid and ATP. If the complete cycle is
now inspected from the point of view of phosphate bond
formation (Fig. 10) it can be seen that the initial phosphoryla-
tion of glucose to hexosediphosphate involves the utilisation of
two molecules of ATP, and that the hexosediphosphate then
splits to yield two molecules of triosephosphate each of which
is eventually converted to pyruvic acid, liberating two molecules
of ATP in the course of the metabolism. Thus two mole-
cules of ATP are required to start a cycle which yields four
molecules of ATP, a net gain of two molecules of ATP. The
pyrophosphate bond ( — P — 0 — P — ) of ATP is energy-rich and
this appears to be the form in which the cell stores its energy
until it is required. The synthetic mechanisms of the cell
require energy which is obtained by reactions involving phos-
phorylated intermediates obtained, in turn, by interaction with
the extra ATP formed by the glycolysis cycle. The cycle can
thus be looked upon as a machine for taking in glucose and
phosphate at a low energy level, winding the energy up into
specific bonds, and then transferring that energy, in the form
of such bonds, to a suitable store while discarding the waste
product as, in this case, fermentation products.

OH OH OH

CH,0 — P-O— P — 0 — P-OH

I II II II

0 0 0

HO HO
Adenosine-tri-phosphate

Bacteria differ from other tissues mainly in the way in which
they dispose of the pyruvic acid thrown out as a waste product
from the energy machine and so, in considering the further

ENERGY PRODUCTION

131

details of bacterial fermentation, we are concerned largely with
the methods utilised by the various organisms for the disposal
of their waste material — although, in some cases, the organisms
dispose of this material in such a way that more energy is made
available during the disposal process.

CHjOH

CH,OP

2ATP

Fig. 10.
THE FERMENTATION OF PYRUVIC ACID BY ESCH. COLI
Acetic and formic acid formation

Under aerobic conditions, Esch. coli oxidises pyruvic acid
directly to acetic acid.

CH3 . CO . COOH + 0 > CH3 . COOH + CO2.

132 PROVISION OF energy: fermentation

Under anaerobic conditions the first breakdown of pyruvic
acid is to acetic and formic acids by what was originally called
the " hydroclastic split ":

CH3.CO.COOH + H2O— >CH3.C00H + H.COOH (1)

However, it was found that when cell-free enzyme preparations
were used, this reaction would not take place unless phosphate
was present. This suggests that the formation of acetyl
phosphate occurs as an intermediate step in the breakdown,

CH3.CO.COOH + H3PO4 ^^ CH3.COOPO3H2 + H.COOH

(2)

If the enzyme preparation is freed from adenylic acid or
adenosine-di-phosphate (ADP), then acetyl phosphate is
actually found to accumulate, but in the presence of either
adenylic acid or ADP, the acetyl phosphate breaks down
to acetic acid iand the phosphate group is transferred to the
adenine compound with the synthesis of ATP:

CH3.COOPO3H2 + ADP > CH3.COOH + ATP . .(3)

In the intact organism the over-all result of reactions 2 and 3
is reaction 1, with the additional result that inorganic phos-
phate is taken up during the course of the reaction and
ATP synthesised.

Hydrogen and carbon dioxide formation

The phosphoclastic split described above (reaction 2),
releases formic acid as a product of pyruvic acid breakdown.
Formic acid is further broken down to hydrogen and carbon
dioxide by an enzyme called formic hydrogenlyase,

Formic
H.COOH ^Ha + COg (4)

hydrogenlyase

The hydrogen formed during fermentation is all produced
as a result of this reaction, though, as we shall see later, the
phosphoclastic splitting of pyruvic acid is not the sole source

LACTIC ACID FORMATION 133

of formic acid as precursor of hydrogen. Formic hydrogen-
lyase is a reversible enzyme, so that synthesis of formic acid
will occur in the presence of hydrogen and carbon dioxide.
In the absence of any side-reactions, the final result of formic
hydrogenlyase action is to produce an equilibrium mixture
of formic acid, hydrogen, and carbon dioxide. Formic
hydrogenlyase is an iron-activated enzyme, and if the
organism is grown in an iron-deficient medium, the formation
of the enzyme is prevented and, as a result, no gas is liberated
during the fermentation of glucose, formic acid accumulating
instead (see p. 83).

Lactic acid formation

Esch. coli possesses the same series of enzymes as those
involved in the production of lactic acid by muscle. It
possesses lactic dehydrogenase which will bring about the
reduction of pyruvic acid to lactic acid in the presence of a
H-donator, which is itself supplied by the presence of reduced
coenzyme I. The series of reactions involved in the fermenta-
tion of glucose includes the oxidation of glyceraldehyde-
phosphate to phosphoglyceric acid by triose-phosphate
dehydrogenase, the oxidation involving the reduction of
coenzyme I. If, however, the fermentation substrate is
pyruvic acid instead of glucose, then triose-phosphate
dehydrogenase is not involved and coenzyme I is not reduced.
This is probably linked with the facts that whereas lactic
acid forms approximately 50 per cent, of the products of glucose
fermentation, it forms only about 5 per cent, of the products
when pyruvic acid is the fermentation substrate. The main
reaction giving rise to lactic acid is presumably therefore :

CHO CHg COOH CH3

CHOH -h H2O + CO ^ <CHOH + CHOH (5)

I III

CH2OPO3H2 COOH CH2OPO3H2 COOH

the oxidation of glyceraldehyde-phosphate being linked to the
reduction of pyruvic acid by coenzyme I acting as H-carrier

134 PROVISION OF energy: fermentation

(see p. 127). However, lactic acid is still formed to some extent
from pyruvic acid, and it has been suggested that formic acid
will react with pyruvic acid to give lactic acid according to
the equation:

CH3 . CO . COOH + H . COOH > CH3 . CHOH . COOH + COg

(6)

It is found experimentally that a molecule of COg is formed
for every molecule of lactic acid produced. As a result of
this, the fermentation gases contain more carbon dioxide
than hydrogen, the amount of carbon dioxide in excess of
the hydrogen being equivalent to the lactic acid formed. The
experimental evidence in support of reaction 6 is not, however,
convincing at present. It has also been shown that the amount
of lactic acid produced is increased by increasing the carbon
dioxide present during the fermentation. Fermentation
studies carried out in the presence of carbon dioxide containing
isotopic 0^3 (" heavy carbon ") have shown that C^^Og is
fixed during the fermentation, and that some of this fixed-COg
appears in the ■ — COOH group of lactic acid.

In the case of the Gonococcus and S. faecalis it has been
shown that lactic acid can be formed from pyruvic acid by a
hydrolytic reaction (or dismutation) :

2CH3 . CO . COOH + H2O = CH3 . CHOH . COOH +

CH3.COOH + CO2.

Succinic acid formation

Succinic acid is formed to a variable extent during the
fermentation of either glucose or pyruvic acid by Esch. coli,
Aerobacter aerogenes, Propionibacteria, and other organisms.
For many years its formation constituted a puzzle, as it was
by no means obvious how a substance containing 4 carbon
atoms could be derived from a 6-carbon sugar or a 3-carbon
triose or pyruvic acid. Elsden of the Cambridge School
showed that succinic acid formation depends upon the presence
of carbon dioxide during fermentation, so that increasing the
CO2 tension results in increased succinic acid production or.

SUCCINIC ACID FORMATION 135

alternatively, rigid removal of carbon dioxide during fermen-
tation results in suppression of succinic acid production. This
suggests that carbon dioxide is assimilated during the fermenta-
tion and that the ultimate fate of the assimilated carbon
dioxide is to form succinic acid. This suggestion has been
investigated in detail by Prof. Werkman and his colleagues
at Iowa State College. They showed that if pyruvic acid
is fermented in the presence of carbon dioxide containing
isotopic C^^, then some of the heavy-carbon is assimilated
and appears in one of the — COOH groups of succinic acid.
They suggest the following scheme for the reaction: —

C*02 C*OOH C*OOH C*OOH C*OOH

+ 11 II

CH3 CH2 CH2 CH Succinic CH2

I I > I > II > 1 (7)

CO Oxalacetic CO Malic CHOH Fumarase CH dehydro- CHg

I decar- 1 dehydro- 1 1 genase i

I boxylase I genase I I I

COOH COOH COOH COOH COOH

Oxalacetic Malic Fumaric Succinic

acid acid acid acid

(C* = C13 isotope)

CO2 is first fixed by combination with pyruvic acid to
form oxalacetic acid. The enzyme involved in this fixation
process is oxalacetic acid decarboxylase working in reverse.
Oxalacetic acid is then reduced to malic acid by malic dehydro-
genase in reverse. Malic acid is converted to fumaric acid
by the action of fumarase and, finally, fumaric acid is reduced
to succinic acid by succinic dehydrogenase acting in reverse.
All these enzymes have been demonstrated in the bacteria
concerned, and it is probable that the " Wood- Werkman
scheme," as this is usually called, is responsible for part, at
least, of the succinic acid formation in these organisms.

This scheme accounts for succinic acid formation by the
fixation of carbon dioxide, but studies involving isotopic CO2
show that, although some of the succinic acid arises in this
fashion, the total formation of succinic acid cannot be
accounted for in this way. Werkman and his colleagues have
now demonstrated that there is a second method whereby

136 PROVISION OF energy: fermentation

succinic acid is formed in bacterial fermentation and that is
by debydrogenation of acetic acid,

CH3.COOH CH2.COOH

+ ^^1 +2H (8)

CH3.COOH CH2.COOH

In some cases, but not all, this reaction is easily reversed, and
acetic acid itself can be formed by reductive breakdown of
succinic acid. These reactions have again all been demon-
strated by application of techniques involving heavy-carbon.

THE FERMENTATION OF PYRUVIC ACID BY
AEROBACTER AERO GENES

Formation of acetylmethylcarbinol, etc.

Aerohacter aerogenes is an organism very similar in many
properties to Esch. coli, but systematically differentiated from
the latter by a positive " Voges-Proskauer test " (Table II).
This test consists of adding strong alkali to a 24 hours old
culture of the organism in glucose-peptone, and a positive
reaction is shown by the development of a pink colour near
the surface of the medium after 24-48 hours. The colour
starts to develop at the surface of the medium and slowly
spreads down into the liquid. The chemistry of the colour
reaction is complex and is due to a reaction between diacetyl,
CHg . CO . CO . CH3, and substances in the medium containing
a guanidino-group. To speed up the test it is usual nowadays
to add a trace of creatinine to the treated medium when,
if positive, the colour develops within a short time. Diacetyl
is produced by atmospheric oxidation of acetylmethylcarbinol
(acetoin), CH3.CO.CIIOH.CH3, which is a fermentation
product formed from glucose by this organism.

Glucose is fermented by the organism to pyruvic acid, as
usual, and Aerohacter aerogenes then attacks pyruvic acid in
two ways.

(a) By the phosphoclastic split to acetic and formic acids
in exactly the same way as Esch. coli.

ACETYLMETHYLCAEBINOL FORMATION 137

(6) By decarboxylation and condensation of two molecules
of pyruvic acid to form acetylmethylcarbinol,

2CH3 . CO . COOH > CH3 . CO . CHOH . CH3 + 2C0o .... (9)

Which of the reactions predominates depends upon the pK of
the growth medium. Acetylmethylcarbinol is formed only
when growth occurs at an acid ^H and the pH of optimum
activity of the enzyme involved lies between 4-0 and 5-5.
The enzyme has been obtained in a cell-free state, and is
developed within the cell only when growth occurs at an acid
pB.; if the ^H is maintained at an alkaline value by the
addition of alkali throughout growth, so that the fermentation
acidity is neutralised, then the acetylmethylcarbinol enzyme
is not formed and the fermentation of the organism is con-
sequently essentially similar to that of Esch. coli. It is not
possible to convert Aerobacter aerogenes into Esch. coli by
continued growth in alkaline media, as immediately growth
is resumed in acid conditions acetylmethylcarbinol formation
again takes place.

Reaction 9 thus occurs under acid environmental conditions.
Since it involves the conversion of two molecules of acid into
one molecule of a neutral substance, it acts as a neutralisation
mechanism (see p. 71), coming into action when the growth
environment becomes acid. This fact is further utiHsed as a
method of differentiating between Aerobacter and Escherichia.
If the two organisms are cultivated in a medium containing
a small amount of glucose, then both will ferment the glucose
with the formation of acid. However, the acetylmethyl-
carbinol formation by Aerobacter will result in the neutralisa-
tion of some of this acid and, as long as the glucose is not
present in excess, the final pH will be lower in the Escherichia
culture than in the Aerobacter culture. Consequently, if we put
up our cultures in peptone containing 0-2 per cent, glucose and
methyl red as indicator, we find that Esch. coli will turn the
indicator red in the course of its growth, while Aerobacter
will not; the Esch. coli is therefore said to be " methyl red
positive."

138 PROVISION OF energy: fermentation

Some strains of Aerobacter can effect a reduction of acetyl-
methylcarbinol to 2.3.butylene glycol:

CH3.CO.CHOH.CH3+2H^— CH3.CHOH.CHOH.CH3 (10)

This fermentation product is of considerable commercial
interest as it is a comparatively simple matter to convert it
by chemical means to butadiene, CH2=CH — CH^CHg,
which is one of the materials used to produce synthetic rubber
by polymerisation. Acetylmethylcarbinol also undergoes
oxidation by oxygen to diacetyl CH3.CO.CO.CH3, which is
the substance imparting the "buttery" smell to butter.

Acetylmethylcarbinol is produced by species of Pseudomonas
and Bacillus as well as by Aerobacter.

THE FERMENTATION OF PYRUVIC ACID BY
PR OPIONI BACTERIA

Certain bacteria found in Gruyere and Emmentaler cheeses
produce propionic acid amongst their fermentation products.
These Propionibacteria ferment pyruvic acid to form propionic
acid, and since they also reduce lactic acid to this same
product, it has been suggested in the past that lactic acid
forms an intermediate stage in the reduction of pyruvic acid
to propionic acid. However, Barker and Lipmann have
found that the decomposition of lactate is inhibited by sodium
fluoride at a concentration considerably less than that required
to prevent the formation of propionic acid from pyruvic acid.
This indicates that lactic acid cannot act as an intermediate
in the latter reaction, but that the following scheme is involved.

2H 2H
CH3.CO.COOH > X > CH3.CH2.COOH

\x t

^ I NaF

^CH^.CHOH.COOH

where the nature of X is unknown.

The Propionibacteria form succinic acid from pyruvic acid
by the fixation of carbon dioxide in the same way as that

PROPIONIC ACID FORMATION

139

described for Esch. coli. Werkman and his colleagues have
shown that if CO2 containing C^^ is used then the heavy-
carbon is fixed by the organism and appears in the — -COOH
group of succinic acid and also of propionic acid. This
suggests that propionic acid arises from the same initial
reaction, involving CO2 fixation, as succinic acid. It is now
known that the organism possesses a succinic decarboxylase
which removes COg from succinic acid, to form propionic acid.

CO2

CH,

COOH COOH
I

CH,

CH.

COOH
I

COOH

I
CH„

COOH

CH,

COOH

1 ^^ 1 ^=^

CO CHOH

II ^-=^
CH

1 -- >I
CH2 CH

COOH COOH

COOH

1 +
COOH CO,

(11)

Some strains of these organisms carry out a further reduction
of propionic acid to w-propyl alcohol,

CH3.CH2.COOH + 2H2 — >CH3.CH2.CH20H + H20 ...(12)

and when C^^02 is used in such a fermentation, C^^ appears
in the — COOH groups of succinic and propionic acids and
also in the — CH2OH of propyl alcohol.

CH3.CO.CO.CH3

CH3. CHOH. COOH

CH3.CO.CHOH.CH3

CH3.CHOH.CHOH.CH3

HOOC.CH2.CO.COOH

6 n®

H00C.CH=CH.COOH

n®

H00c.cH2.cH2.cooH

/©
H00c.cH2.cH3

/©
HOH2C.cH2.cH3

The fermentation of pyruvic acid by Esch. coli, Aerobact. aerogenes,
and Propionibacteria. The numbers in rings in the above scheme
refer to the reactions similarly numbered in this chapter.

140 PROVISION OF energy: fermentation

MISCELLANEOUS FERMENTATION REACTIONS
In tlie preceding sections we have traced the formation and
fate of pyruvic acid in various fermentations. It is not
possible, however, to derive all fermentation products from
pyruvic acid, and we must now consider the production of
some of these substances. The point is experimentally tested
by comparing the fermentation products of an organism
when, first, glucose and, second, pyruvic acid is used as
fermentation substrate. Thus the products of the fermenta-
tion of glucose by Esch. coli are Ho, COg, ethyl alcohol, formic
acid, acetic acid, lactic acid, and succinic acid, but if pyruvic
acid is the substrate then no ethyl alcohol is formed and very
much less lactic acid.

Ethyl alcohol formation

In the alcoholic fermentation of yeast (Saccharomyces
cerevisiae), alcohol is derived from pyruvic acid by the action of
carboxylase which decarboxylates pyruvic acid to acetaldehyde :

Carboxylase
CH3.CO.COOH > CH3.CHO + CO2,

and acetaldehyde is then reduced to alcohol by alcohol
dehydrogenase working in reverse. The coliform organisms
do not, however, possess carboxylase, and the presence of
this enzyme in bacteria has yet to be demonstrated. Ethyl
alcohol does not arise from pyruvic acid in these organisms.
If we return to the fermentation of glucose, as described
on p. 128, we find that the formation of pyruvic acid was
traced from hexosediphosphate through glyceraldehyde-
phosphate and phosphoglyceric acid. The oxidation of
glyceraldehy de-phosphate to phosphoglyceric acid forms half of
an oxido-reduction reaction, which is completed, for the other
half, by the reduction of dihydroxyacetone-phosphate to
a-glycerophosphate. If fluoride is added to the fermentation
system, then the reaction is stopped at this point and a
mixture of phosphoglyceric acid and a-glycerophosphate
remain as the products.

ETHYL ALCOHOL FORMATION 141

CH2OPO3H2 /'CHj.CHzOH

' ) +

CHOH — -^ < H.COOH

t I +

CH2OH u^ V Phosphate

H2O3 POHi C /^ \CH2OPO3H2

OH
HO H CH2OPO3H

CH3.CO.COOH

In the absence of fluoride, phosphoglyceric acid is fermented
to pyruvic acid, as described, and a-glycerophospbate is
fermented with the formation of ethyl alcohol and formic
acid. Dihydroxyacetone-phosphate and glyceraldehyde-
phosphate form an equilibrium mixture; consequently the
proportions of ethyl alcohol to pyruvic acid formed will
depend upon the reaction velocities of the various inter-
mediate reactions involved.

It will be noted that the oxidation of glyceraldehyde-
phosphate can be coupled either with a reduction of dihydroxy-
acetone-phosphate or with the reduction of pyruvic acid to
lactic acid. Since pyruvic acid will not accumulate until
the glycolysis cycle has proceeded through all the intermediate
steps, it follows that the reduction of dihydroxyacetone-
phosphate will occur predominantly during the starting-up
of the cycle and so can be looked upon as a " starter reaction "
which enables the cycle to get under way and consequently
to produce the main H-acceptor, pyruvic acid.

Investigations using isotopic-carbon have indicated that,
during the fermentation of glucose, ethyl alcohol can also be
formed by the reduction of acetic acid. Ethyl alcohol is not
formed during the fermentation of pyruvic acid but it is
possible that the reduction of acetic acid requires reduced
coenzyme I as H-donor, and this, in turn, requires the triose-
phosphate dehydrogenase system, as in the case of lactic acid
formation. This second method of alcohol formation has
not yet been confirmed by other techniques.

142

PROVISION OF energy: fermentation

It is now possible to outline the intermediate reactions
involved in the formation of all the fermentation products of
the coli-aerogenes group of organisms:
GLUCOSE

Hexose-di-phosphate

Dihydroxyacetonephosphote ^ Glyceroldehyde-phosphate

<»< -glycerophosphate Phosphoglyceric acid

ETHYL ALCOHOL Formic acid

Hi CO2

Phosphopyruvic acid

Pyruvic acid

Formic acid

ACETIC LACTIC SUCCINIC
ACID ACID ACID

CO-

ACETYLM ETHYL
CARBINOL

2.3. BUTYLENE
GLYCOL

Acetone-butanol fermentation

The fermentation of maize meal or molasses by CI. aceto-
hutylicum became of importance in the war of 1914-18 as it
was then the most satisfactory method of making acetone on
a commercial scale. Since that time the synthesis of acetone
by a cheaper chemical method has been worked out and the
fermentation method is no longer of such industrial importance.
A further product of the fermentation is butyl alcohol (butanol)
which is now required on a large scale as a paint and lacquer
solvent. As a result of the commercial value of the products,
the fermentation has received considerable attention which
has not, as yet, succeeded in unravelling all the intermediate
reactions. The research was hampered by the fact that, until
recently, it has not been possible to prepare washed suspensions
of the organism in an active state, but this difficulty has now
been largely overcome by the use of concentrated suspensions,
rather than washed suspensions, and a certain amount of
knowledge of the course of the fermentation has been obtained.

ACETONE-BUTANOL FERMENTATION

143

In the commercial process the raw fermentation substrate
is usually maize meal, and the organism attacks the starch of
maize meal by the production of two extracellular enzymes,
one of which is an amylase which breaks the starch down to
maltose, and the other is a maltase which hydrolyses the
maltose to glucose. The fermentation of glucose by CI.
acetobutylicum gives rise to the formation of hydrogen, carbon
dioxide, acetic and butyric acids, ethyl and butyl alcohols,
and acetone; the fermentation of pyruvic acid gives rise
mainly to hydrogen, carbon dioxide, and acetic acid, with traces
of butyric acid, ethyl alcohol, and acetone. The proportions
in the two cases are given in Table XIII, where the results are
expressed as molecules of product per mol. glucose or two
mols. pyruvic acid (equivalent to 1 mol. glucose) fermented.

TABLE XIII
Products of CI. acetobutylicum Fermentation

Product

Substrate-glucose
mols. /mol.

Substrate-pyruvate
mols./2 mols.

Hydrogen gas

1-87

1-88

Carbon dioxide

2-46

2-08

Butyl alcohol

0-6

001

Ethyl alcohol

0006

005

Acetone

014

0-08

Acetic acid

0125

1-52

Butyric acid

0-04

Investigations of this fermentation have been concerned
mainly with the production of the commercially valuable
substances, acetone and butyl alcohol. If the formation of
the various products is followed at intervals during growth in
a glucose-containing medium, then we find that the appearance
of these substances varies with the time as shown in Fig. 11.

In the early stages of growth, while the ^H is falling rapidly,
acetic and butyric acids are formed together with hydrogen
and carbon dioxide, but no acetone or butyl alcohol. Later in

144

PROVISION OF energy: fermentation

the age of the culture, when the pR has fallen to about 4-5,
acetone and butyl alcohol begin to appear; their appearance
is associated with a corresponding disappearance of acetic
and butyric acids and a small rise in pH. It would seem that
the formation of acetone and butyl alcohol, involving the
formation of neutral substances from acids, is a neutralisa-
tion mechanism which is brought into play when the environ-
mental pH becomes strongly acid. Their formation can thus
be regarded as a mechanism in the same class as that of

BUTYL ALCOHOL

AGE OF CULTURE

Fig. 11. Courseof fermentation of glucose by CZ. aceto^Mf^ZicMm
[after Davies and Stephenson, Biochem. J., 1941, 35, 1323].

acetylmethylcarbinol formation in Aerobacter aerogenes, and of
the production of amines from amino-acids by some strains
of Esch. coli. It would appear from Fig. 11 that the precursors
of acetone and butyl alcohol in the medium are acetic and
butyric acids respectively, and it has been shown that the
addition of acetate to the fermentation mixture results in a
marked increase in the production of acetone. At first it was
thought that a direct reduction of the acids by fermentation
hydrogen might take place, but it is fairly certain now that
this is not the case and that acetic and butyric acids undergo

ACETONE-BUTANOL FERMENTATION 145

further metabolic changes before giving rise to acetone and
butyl alcohol.

Acetone is produced by the decarboxylation of acetoacetic
acid:

Acetoacetic

CH3.CO.CH2.COOH -> CH3.CO.CH3 + CO2,

decarboxylase

and the acetoacetic decarboxylase, which has been obtained
in a cell-free condition from the organism, is formed within
the cells only when the environmental pH has fallen to a low
value — the growth conditions stimulating its formation being
similar to those stimulating the formation of the amino-acid
decarboxylases in other bacteria. Accumulation of acetoacetic
acid in the medium can be demonstrated by means of a colour
reaction, and the presence of the acid can be shown towards
the end of the growth of the culture when acetone formation
is taking place. There is little doubt but that acetic acid is
reduced to acetone through acetoacetic acid, but it is not
known how acetoacetic acid is formed froni acetic acid. Recent
investigations, using isotopic-C compounds, suggest that acetic
acid is also the precursor of butyric acid and butyl alcohol.

Iso-propyl alcohol, CH3 . CHOH . CH3, is formed by the
fermentation of CI. butylicum, an organism closely related to
CI. acetohutylicum. Isotopic-C studies again indicate that the
iso-propyl alcohol is formed by reduction of acetone, which is
formed by the organism in the same way as by CI. aceto-
hutylicum, but the acetone in this case is reduced to the
corresponding alcohol so rapidly that it does not accumulate
and appear as a fermentation end-product.

Fatty Acid Sjmthesis

The synthesis of butyric acid has been investigated in detail
by Barker and his colleagues for the case of CI. Muyverii. This
is a strict anaerobe, isolated from mud, which cannot utilise
glucose but requires ethyl alcohol and a fatty acid such as
acetate for growth. It obtains its energy by metabolism of
these substances resulting in the synthesis of higher fatty acids ;

146 PBOvisiON OF energy: fermentation

when growing on alcohol and acetate it synthesises butyric and
caproic acids with the Hberation of some hydrogen. If acetic
acid, isotopically labelled (C*) in the position CHgC^OOH, is
added to the culture, the resulting acids are labelled in the
positions CH3.C*H2.CH2.C*OOH (butyric) and CHg.C^Hg.CHa.
C*H2.CH2.C*OOH (caproic) indicating that they are formed by
condensation of acetate molecules. Cell-free enzyme prepara-
tions have been made which will accomplish the synthesis of
butyric and caproic acids from ethyl alcohol and acetate under
anaerobic conditions. Analysis of the reactions involved shows
that the alcohol is first oxidised to acetaldehyde and acetyl-
phosphate with the liberation of hydrogen, and this hydrogen
is then utilised to reduce (acetylphosphate -f acetate) to
butjrric acid. The over-all reactions can be represented: —

1. 2CH3.CH2OH + HsPO^^CHg.CHO -f CH3.COOPO3H2 +3H2

2. CH3.COOPO3H2 + CH3.COOH + 2H,->CH3.CH2.CH2.COOH -)-

H3PO4 + H2O

The initial step involved in the condensation of acetylphosphate
and acetate has not yet been clarified. The work on acetone
production, outlined above, suggests that acetoacetic acid
might be concerned but if this substance is added to the enzyme
preparation it is either split irreversibly to acetylphosphate and
acetate or, in the presence of hydrogen, reduced to ^-hydroxy-
butyric acid and does not give rise to butyric acid under any
condition tested. A number of other possible intermediate
substances have now been tested and the only one which will
give rise to butyric acid is vinyl acetate, CH2 = CH.CH2.COOH,
but isotope studies indicate that it is not, in fact, involved in
the production of butyric acid from acetylphosphate and
acetate. The present situation concerning the synthesis of
butyric acid by CI. kluyveri can be summarised diagram-
matically as follows: —

CH3.CH2OH CH2 : CH.CH2.COOH

I j 4H

CH3.COOH -f CH3.COOPO3H2 >? >CH3.CH2.CH2.COOH -f H3PO4

t 2H

CH,.CO.CHo.COOH-

FATTY ACID SYNTHESIS 147

Caproic acid may be formed by a further condensation of
acetylpliosphate with butyric acid: —

CH3.CH2.CH2.COOH + CH3.COOPO3H2 + 4H->

CH3.CH2.CH2.CH2.CH2.COOH + H3PO4 + H2O.

FOR FURTHER READING

" Bacterial Metabolism," Barker, H. A., andDoudoroff, M.,
A7in. Rev. Biochem., 1946, 15, 475.

" Studies on the Acetone-Butanol Fermentation," Davies, K.,
Biochem. Journal, 1941-3.

" Metabolic Generation and Utilisation of Phosphate Bond
Energy," Lipmann, F., Advances in Enzymology, 1941, 1, 99.

" Non-oxidative Enzymes," Mann, T., and Lutwak-Mann, C,
Ann. Rev. Biochem., 1944, 13, 25.

" Cellulose Decomposition by Micro-organisms," Norman,
A. G., and Fuller, W. H., Advances in Enzymology, 1942, 2, 239.

Bacterial Metabolism, Stephenson, M. (Longmans).

" Pyruvate Metabolism," Stotz, E., Advances in Enzymology,
1945, 5, 129.

"Fatty Acid Synthesis by Clostridium kluyverii,'' Stadtman,
E. E., and Barker, H. A., J. Biol. Chem., 1949, 180, 1085,
1095, 1117, 1169; 181, 221.

CHAPTER VIII

PROVISION OF ENERGY: OXIDATION

In the last chapter we dealt in detail with the anaerobic
decomposition of carbohydrates. In this chapter we shall
proceed to study the liberation of energy by oxidative pro-
cesses. As explained in Chap. Ill, oxidation does not
necessarily involve molecular oxygen since any substance
AHg can be oxidised by the general oxido-reduction reaction :

AH2 + B ^ A + BH2.

Oxygen can take the place of the hydrogen-acceptor B.
Oxidation reactions thus do not depend upon the presence of
atmospheric oxygen and can be carried out by anaerobic as
well as aerobic organisms. However, where the organism is
living an aerobic existence and a substrate AHg can be oxidised
directly, or indirectly through a chain of reactions, by oxygen,
the accumulation of reduced products is avoided and the
provision of energy by such complete oxidation is obviously
greater than by a fermentation process. Consequently a
facultative anaerobe grows more efficiently in air than
anaerobically.

The oxidation of carbon substances is too wide and complex
a subject to deal with in detail here, and we shall restrict the
discussion to the mechanisms employed by typical examples of
aerobic, facultatively anaerobic, and anaerobic bacteria to
carry out key oxidations centring on alcohol as substrate.

OXIDATIONS IN ACETOBAGTER

The commercial production of acetic acid as vinegar has
been based for many years on the power of certain strictly
aerobic- bacteria, Acetohacter, to oxidise ethyl alcohol. The
process has been commonly called " acetic fermentation,"

148

OXIDATION OF ALCOHOLS

149

though it cannot strictly be called a fermentation if we adhere
to the usual definition of fermentation as an anaerobic process.
The biological nature of the process was demonstrated by
Pasteur in 1862-4, and since then many organisms of this
genus have been isolated from vinegar vats, etc., and have the
property of oxidising alcohols to acid. Acetobacter are highly
aerobic and carry out three main types of oxidation:

1. Oxidation of primary alcohols to the corresponding acids:

CH3.CH2OH + 02= CH3.COOH + H2O.

2. Oxidation of aldehydes and aldohexoses to the corre-
sponding acids:

CHiOH

nA—\

H OH
GLUCOSE

CH2OH

COOH
HO

H OH

GLUCONIC ACID

3. Oxidation of certain secondary alcohols to the correspond-
ing ketones. The nature of the secondary alcohols was
studied in detail by Bertrand in a classical work on Acetobacter
xylinum. By studying a large number of secondary alcohols,
he showed that only those possessing a specific stereochemical
configuration are attacked. Using the old type of straight
chain formula, this specific group is

H H

HOCHo—C — C— which is oxidised to HOCH,— C— C-

OH OH

0 OH

and the — OH group oxidised must be in the j8-position and
adjacent to another — OH group. Thus an alcohol with the
structure

150 PROVISION OF energy: oxidation

H OH

I I

HOCH2 — C — C — is not attacked. Glycerol is oxidised to

I I
OH H

H H H

I I I
dihydroxyacetone HOCHg— C — CH > HOCH2— C— CH

II II I
OH OH 0 OH

A strain of Acetobacter is used to oxidise sorbitol to sorbose
as one of the steps in the commercial synthesis of vitamin C.

H OH H H H OH H

I I I I III

HOCHj-C-C-C-C-CHpOH >- H0CH2-C-C-C-C-CH,0H

I I I I I I I II

OH H OH OH OH H OH 0 . H

SORBITOL ^-SORBOSE ^ ^i/J OvCHzOH

HO ^ [^ OH

H OH

OXIDATION OF ALCOHOL BY ESCH. COLI

In the case of the oxidation of ethyl alcohol by Esch. coli
the enzyme system has been analysed and the various
components identified. The alcohol is oxidised to acetalde-
hyde in the first place:

CH3.CH2OH + 0 > CH3.CHO + H2O . . . . (1)

The enzyme concerned is alcohol dehydrogenase which
transfers hydrogen from alcohol to coenzyme I,

Alcohol

CH3 . CHgOH + coenzyme I >

dehydrogenase

CH3.CHO + reduced co. I (2)

Keduced coenzyme then reacts with the cytochrome system

ACETIC ACID FORMATION 151

of the organism, the transfer of the hydrogen being catalysed
by diaphorase (see substrate Type S3, Table III).

Diaphorase

Reduced coenzyme I + Cytochrome > Coenzyme

-f- Reduced cytochrome (3)

and the final link with atmospheric oxygen is made by cyto-
chrome oxidase catalysing the transfer of hydrogen from
reduced cytochrome to combine with atmospheric oxygen,
forming water. The complete series of reactions have the
over-all results of Reaction I. Acetaldehyde is further
oxidised, presumably to acetic acid in the first place, although
this does not seem to have been proved :

CH3.CHO + O ^CHg.COOH (4)

Mammalian tissues carry out a similar oxidation through
the action of aldehyde oxidase which is a flavoprotein and
catalyses the oxidation of its substrates by atmospheric
oxygen without the intermediary action of other carriers
(substrate Type S^, Table III). The oxidation has not been
studied in detail in Esch. coli. Acetic acid is also formed from
pyruvic acid and the enzyme concerned, pyruvic oxidase, has
been studied in extracts of L. delbreucHi. If the enzyme
preparation is purified and dialysed, it will attack pyruvic acid
only in the presence of thiamindiphosphate and inorganic
phosphate, and the products of the reaction are acetyl phos-
phate and carbon dioxide:

0 + CH3 . CO . COOH + HgPO^^

CH3.COOPO3H2 + CO2 + H2O (5)

If adenosine-di-phosphate (ADP) is added as phosphate-
acceptor, then the acetyl phosphate gives up its phosphate to
form adenosine-tri-phosphate and acetic acid:

CH3 . COOPO3H2 + ADP = €H3 . COOH + ATP .... (6)

The over-all reaction therefore involves an oxidation of
pyruvic acid to acetic acid and the synthesis of ATP from
inorganic phosphate. This reaction is therefore called an

152

PEOvisioN OF energy: oxidation

ACETIC ACID OXIDATION 153

" oxidative phosphorylation " and it is clear that it has
resulted in the production of energy-rich ATP. This reaction
demonstrates how oxidation energy is made available for
synthetic purposes by the cell; the energy released by the
oxidation accumulates in ATP just as it did in the case of
the oxidation of glyceraldehyde phosphate in the fermentation
cycle. Both fermentation and oxidation therefore have the
same end result in the synthesis of energy-rich ATP bonds
within the cell.

Under aerobic conditions, acetic acid itself is oxidised by
Esch. coli:

CH3.COOH + 2O2 = 2CO2 + 2H2O (7)

The enzymic processes involved in this reaction in bacteria are
not yet clear. In animal tissues a similar type of oxidation
takes place through a complex cycle known as the " Krebs "
or " citric acid cycle." This cycle involves a complex of many
enzymes intervening between acetate (or acetyl phosphate) and
the cytochrome system. It has two main results: (1) it splits
up the liberation of oxidative energy into small steps instead of
a single large outburst, and (2) since a number of steps involve
uptake of inorganic phosphate (not shown in Fig. 12) followed
by synthesis of ATP, it makes energy available to the cell in
the form of energy-rich phosphate bonds. The steps and
enzymes involved in the cycle are set out in Fig. 12. Although
this cycle has now been well estabUshed for animal tissues,
there is considerable doubt whether it exists in bacteria. That
part of the cycle connecting pyruvic acid through oxalacetic
acid to succinic acid is the same as that which has been
discussed in Chap. VII in the section on CO2 fixation; it has
recently been shown that acetyl phosphate will condense with
oxalacetate in the presence of coenzyme A to yield citric acid
in Esch. coli. On the other hand, Esch. coli is differentiated
from Aerobacter in that it cannot attack citric acid, while in
Azotobacter it is found that the rate of oxidation of acetate is
greater than that of any of the intermediate substances in
the postulated cycle. For the present it must suffice to

154 PROVISION OF energy: oxidation

record the cycle as known in other tissues and to point out that,
although there is evidence accumulating that some such
system does exist in bacteria, its nature and occurrence in any
bacterium has yet to be proved.

The oxidation process makes ATP available and this, in
turn, makes energy available for synthetic purposes. If we
follow the oxygen uptake during the oxidation of acetate by
Esch. coli, we find that the amount of gas taken up corresponds
to 60-75 per cent, of that required by the above equation for
complete oxidation. If the residual substrate is estimated,
we find that all the acetic acid has disappeared, although the
oxygen consumption does not correspond to 100 per cent,
oxidation. The portion of the acetic acid which has not been
oxidised is assimilated and incorporated in the cells by what is
called a process of ''oxidative assimilation." If we assume
that the material assimilated by the cells is of the nature
(HCOH), then the true equation for the oxidation is :

2CH3.COOH + 3O2 > (HCOH) + 3CO2 + 3H2O.

If the oxidation is carried out in the presence of sodium azide
or dinitrophenol the oxidative assimilation is prevented and
the oxygen consumption then corresponds to quantitative
oxidation according to Equation 7.

OXIDATION BY STRICT ANAEROBES

The methane that arises from stagnant and putrescent pools
is produced by bacterial action. The organisms responsible
belong to the genus Methanobacter, and their activities have
been studied in detail by Barker and his colleagues. Methano-
bacter omeliansJdi, like other organisms of this group, is a
strict anaerobe, and obtains energy by the oxidation of
alcohols. As it is a strict anaerobe it cannot utilise oxygen for
the oxidation process, but carries out an oxidation-reduction
process in which the H-acceptor is carbon dioxide, which is
reduced to methane according to the equation:

2CH3.CH2OH + CO2 > 2CH3.COOH + CH4.

METHANE FORMATION 155

The organism \vill not attack methyl alcohol but will grow on

ethyl alcohol, when the rate of growth bears a linear relation

to the acetic acid production, and the greater part of the

carbon of the organism is derived from the acetate so formed.

Proof that methane arises from carbon dioxide was obtained

by carrying out the oxidation of alcohol in the presence of

carbon dioxide enriched with isotopic-C, when it was found

that the carbon dioxide was converted to methane and the

bulk of the isotopic-carbon appeared in the methane produced,

although small amounts were assimilated by the organism.

The strictly anaerobic Clostridia obtain their energy in

many cases by oxido-reduction reactions. In some cases

the hydrogen donator and acceptor are both amino-acids,

so that a reaction occurs in which one amino-acid is oxidised

to the corresponding keto-acid, while the other is reduced

to the corresponding fatty acid, both amino-acids becoming

deaminated :

R X R X

I III

CHNH2 + H2O + CHNH3— ^ CO + 2NH3 + CHo

I I I I "

COOH COOH COOH COOH

This reaction is called the Stickland reaction and is discussed
in further detail in Chap. IX.

Alcohol is a hydrogen donator in the case of CI. kluyverii
studied by Barker and his co-workers (pp. 145-7). This organism
cannot attack glucose or pyruvic acid but obtains its energy by
the metabolism of ethanol and acetate. The growth requires
CO2, and isotopic studies have shown that the amount of CO2
assimilated is proportional to the amount of acetate meta-
bolised, but that 70 per cent, of the carbon of the CO2 appears
in the cellular material and none in the other products. The
main products of the metabolism of the ethanol and acetate are
butyric and caproic acids, but gaseous hydrogen is also formed
and this arises from oxidation of ethanol :

CH3CH2OH + HgO-^CHg.COOH + 2H2.

156 PEOVisioN OF energy: oxidation

In this case COg is utilised for cell synthesis, the energy-
being provided by fatty acid metabolism. However, in other
cases amongst the Clostridia, the CO2 can act as hydrogen
acceptor as well as a source of cell-carbon, e.g. Wieringa
isolated an organism from mud which reduces CO2 to acetic
acid in the presence of gaseous hydrogen as H-source :

2CO2 + 4H2 -> CH3 . COOH + 2H2O.

FOR FURTHER READING

" Microbial Assimilations," Chfton, C. E., Advances in
Enzymology, 1946, 6, 269.

" The Intermediate Stages in the Biological Oxidation of
Carbohydrate," Krebs, H. A., Advances in Enzymology,
1943, 3, 191.

Papers :

1. Bertrand, G., Ann. Chim. et Phys., 8, 181 (Acetobacter).

2. Still, L., Biochem. J., 1940, 34, 1177 {Esch. coli).

3. Barker, H. A., J. Biol. Chem., 1941, 137, 153 {Methano-
hacter).

CHAPTER IX
BREAKDOWN OF NITROGENOUS MATERIAL

The synthesis of protein is one of the main reactions involved
in the growth of the bacterial cell. In Chap. V we were
largely concerned with the nature of the bricks from which the
cell builds its protein and with the activation of the enzymes
concerned in the building process. The building process often
takes place at the expense of complex substances existing in
the environment. It is as though we wished to build a
laboratory on the site of an apartment; the one structure must
be demolished to its constituent units before the new one can
be constructed in its place. Consequently the growth of new
cells in an environment already utilised by previous growth
involves the degradation of the complex proteins, etc., left
by the earlier inhabitants, to assimilable material such as
amino-acids, ammonia, or even nitrogen, and simple carbon
substances. In this chapter we shall be concerned with these
breakdown reactions.

Proteolysis

Under this heading we group those reactions involved in
the hydrolysis of protein to amino-acids. The series of
enzymes involved in such breakdown has been studied with
great success in animals, and the proteolytic enzymes of the
mammalian intestinal tract have been divided into pepsin,
trypsin, " erepsin," polypeptidases, peptidases, etc., but
comparatively few studies in detail have been made of the
corresponding enzymes formed by bacteria. The native
protein molecule is too large to enter the bacterial cell, and
consequently if the organism is to utilise such molecules it
must first excrete extracellular enzymes to start the
hydrolysis. The power to excrete such enzymes in quantity
is restricted to comparatively few species. Some of the
Clostridia, such as CI. histolyticum and CI. sporogenes, excrete

157

158 BREAKDOWN OF NITROGENOUS MATERIAL

highly active proteases into their environment. This can
be demonstrated by filtering such organisms from culture,
when it will be found that the cell-free filtrate contains an
active proteolytic enzyme which can be concentrated and
precipitated by suitable protein precipitants. The lique-
faction of tissues around a wound is due to the proteolytic
activities of contaminants of this type. Other genera, such
as Proteus and Pseudomonas, have less marked proteolytic
activities, while Streptococci are sometimes feebly proteolytic.
Even proteolytic organisms will fail to grow when inoculated
into a medium containing native protein as sole source of
nitrogen, as they require some utilisable source of nitrogen
from which to synthesise the extracellular protease necessary
to initiate the hydrolysis of the protein.

Once native protein has been hydrolysed to peptones, the
majority of the heterotrophic organisms are able to utilise
these peptones as sources of nitrogen and/or energy. Thus
media in common use in the laboratory for general growth
purposes are prepared with a basis of peptone. It is highly
probable that genera and species differ widely in the proteolytic
enzymes which they produce, but there have not as yet been
sufficient studies of this aspect of the subject to make any
generalisations possible. The end-products of the breakdown
of proteins by bacterial proteases are amino-acids, and dis-
cussion of nitrogen metabolism must at present hinge mainly
on amino-acid metabolism, as it is here that the widest variety
of further breakdown products occur.

AMINO-ACID BREAKDOWN

If we consider the general formula of an amino-acid:

R.CHNH2.COOH

we find that there is no theoretically possible mode of attack
which is not employed by some organism or other under
some condition or other. The molecule can be degraded in
three main ways: (1) by removal of the — NHg group, or
deamination; (2) by removal of the — COOH group, or

AMINO-ACID BREAKDOWN 159

decarboxylation; or (3) by splitting or hydrolysis of the
molecule in some other position. In some cases we find that a
single organism may attack an amino-acid by both deamination
and decarboxylation, but the two processes do not take place
together as the enzymes involved are not formed under the
same conditions of growth. When growth takes place in an
alkaline medium, the carboxyl group of the amino-acid is
ionised, R.CHNHg.COO', and if the particular amino-acid
can be attacked by that organism, the specific deaminase
is produced and the organism will attack the amino-acid by
removal of the unionised — -NHg group. Conversely, if growth
takes place in an acid medium, then the amino-group of the
amino-acid is ionised, R . CHNH3+ . COOH, and the specific
decarboxylase will be produced so that the organism will
attack the amino-acid by removal of the unionised — COOH
group. For example, Esch. coli, grown in an alkaline medium,
will attack L-glutamic acid with the formation of a-keto-
glutaric acid and ammonia:

HOOC.CH2.CH2.CHNH2.COOH -f 0

> HOOC . CH2 . CH2 . CO . COOH -f NH3,

but the same organism grown in an acid medium can no
longer attack the molecule by deamination but does so,
if at all, by decarboxylation

HOOC . CH2 . CH2 . CHNH2 . COOH

> HOOC . CH2 . CH2 . CH2NH2 -f CO2.

The products of amino-acid breakdown may thus be pro-
foundly influenced, not only by the particular organism, but
by the ^:>H of the medium in which that organism is grown
(see Chap. IV).

DEAMINATION

Removal of the — NHg group from an amino-acid may be
accomplished by different bacteria in different ways, such as by
oxidation, reduction, desaturation, hydrolysis, etc., of the
substrate molecule.

160 BREAKDOWN OF NITROGENOUS MATERIAL

Oxidative deamination

Oxidative removal of the — NHg group is accomplished
according to the equation

R.CHNH2.COOH + 0 > K.CO.COOH + NH3,

with the production of the a-keto-acid corresponding to the
amino-acid attacked. This is the type of breakdown found
in mammalian kidney cells, but whereas most amino-acids
are attacked by oxidative deamination in these tissues, in
bacteria this method of attack is restricted to certain organisms
and specific amino-acids. Esch. coli is known to deaminate
glycine, L-alanine and l -glutamic acid in this way with the
formation of glyoxylic, pyruvic, and a-ketoglutaric acids
respectively.

H2NCH2 . COOH -f 0 > CHO . COOH + NH3

CH3 . CHNH2 . COOH -f 0 > CH3 . CO . COOH + NH3

HOOC.CH2.CH2.CHNH2.COOH + 0

^^ HOOC . CH2 . CH2 . CO . COOH + NH3.

In the case of glutamic acid the reaction is really accomplished
in two stages, in which the first stage consists of a dehydro-
genation to imino-glutaric acid, which is then spontaneously
hydrolysed to ketoglutaric acid.

COOH COOH COOH

I I I .

CH2 CH2 CH2

I Glutamic I I

CH2 - 2H ^— ^ CH2 -f H2O ^^ CH2 + NH3
I dehydrogenase I I

CHNH2 C-NH C = 0

I I I

COOH COOH COOH

This deamination process is essentially similar to that
which occurs in mammalian tissues, with the difference that
whereas liver L-glutamic acid dehydrogenase requires
coenzyme I, the enzyme of Esch. coli requires coenzyme II.

OXIDATIVE DEAMINATION 161

Both stages of the reaction are reversible so that L-glutamic
acid can be synthesised from ammonia and a-ketoglutaric acid.
This may represent the main path of glutamic acid synthesis
in bacteria. In mammalian tissues, the glutamic acid dehydro-
genase may act as a carrier system for the deamination (or, in
reverse, the synthesis) of other amino-acids, as some workers
claim that transamination occurs between either a-keto-
glutaric acid and any other amino-acid or, alternatively,
between glutamic acid and any other a-keto-acid. This may
not be the case in bacteria, as so far it has not been possible
to demonstrate transamination other than in a very restricted
sense in these organisms (see p. 94), and there is definite
evidence, on the other hand, that the deamination of some
amino-acids passes through reactions quite different from those
involved in the postulated glutamic acid carrier system of
mammalian tissues.

Esch. coll would seem to attack the three amino-acids
mentioned by oxidative deamination, but no others. Stumpf
and Green have, however, recently found an L-amino-acid
oxidase in Pr. vulgaris, Ps. pyocyanea, and Aerobacter aerogenes,
which attacks 11 amino-acids: the laevo-isomeis of phenyl-
alanine, tyrosine, leucine, iso-leucine, methionine, tryptophan,
histidine, norleucine, norvaline, amino-butyric acid, and
arginine. In each case the corresponding ke to-acid is formed
according to the equation

R . CHNH2 . COOH + 0 > E . CO . COOH -f NH3.

The enzyme has been isolated in a cell-free state by disin-
tegrating a thick suspension of the bacteria wdth supersonic
vibrations. Other common naturally-occurring amino-acids
are not attacked in the presence of the enzyme, although the
intact organism is capable of a wider range of deaminating
activities and must consequently possess other enzymes
affecting the deamination of these other amino-acids. The
enzyme cannot be obtained from Esch. coli, S. haemolyticus,
B. suhtilis, or Staph, aureus. The suggestion is made that
there are several bacterial amino-acid oxidases, and that the
specificity of these enzymes varies with their source.

CHEM. A. B. 11

162 BREAKDOWN OF NITROGENOUS MATERIAL

Reductive deamination

In this case hydrogen is added to the substrate with the
production of a saturated fatty acid:

R . CHNHg . COOH + 2H -> R . CH2 . COOH + NH3.

This type of deamination has been demonstrated with certain
strict aerobes (e.g. Mycoh. phlei) in the case of aspartic acid,
which is reduced to succinic acid with the liberation of ammonia :

HOOC.CH2.CHNH2.COOH + 2H

> HOOC . CH2 . CH2 . COOH + NH3.

Desaturation deamination

In this case NH3 is removed from the amino-acid molecule,
leaving an unsaturated fatty acid:

R.CH2.CHNH2.COOH ^R.CH= CH.COOH + NH3.

When intact cells of Esch. coli deaminate aspartic acid, the
final product is succinic acid, but if the deamination takes
place in the presence of certain inhibitors such as toluene,
then the end-product is not succinic acid but fumaric acid,
and the deamination takes place according to the equation :

HOOC . CH2 . CHNH2 . COOH

> HOOC . CH = CH . COOH + NH3.

In the absence of inhibitors fumaric acid is reduced to succinic
acid. The enzyme responsible for the desaturation deamina-
tion is called " aspartase," and has been isolated in a cell-free
state. The aspartase reaction is reversible so that aspartic
acid can be synthesised from ammonia and fumaric acid.
Since the reaction is reversible, the end-products of either
forward or back reactions form an equilibrium mixture of
ammonia, fumaric acid, and aspartic acid. When the intact
organism is used as source of the enzyme, the equilibrium
mixture is further complicated by the presence of another
reversible enzyme, fumarase, which catalyses the hydration
of fumaric acid to malic acid. In the intact organism the
deamination of aspartic acid may lead to the formation of

ASPARTASE 163

any or all of the following products : ammonia, fumaric acid,
succinic acid, malic acid.

HOOC.CH2.CH0.COOH

Succinic // dehydrofieiiase

Aspartase yf

HOOC.CH0.CHNH2.COOH F=^ HOOC.CH=CH.COOH + NH3

HOOC.CH^.rHOH.OOOH
Aspartase is found in many facultative anaerobes but is not,
apparently, involved in the reductive deamination of aspartic
acid to succinic acid by certain strict aerobes (as above).

When cell preparations of aspartase are left to stand on the
bench, especially if the j^H is adjusted to around 4-0, the
activity steadily declines. The lost activity can be restored
if biotin is added to the cell suspension, which suggests that a
biotin-containing coenzyme is involved in aspartase action.
However, adenylic acid can also restore the lost activity but
much larger concentrations are required. The cell-free
aspartase has now been resolved into specific protein and
coenzyme portions, and the protein can be activated by the
addition of both adenylic acid and biotin, neither being active
alone. It is probable that adenyUc acid and biotin combine,
in the presence of an enzyme contained in the preparation,
to form a complex active as co-aspartase.

A further example of desaturation deamination is the
breakdown of histidine by Esch. coli to give urocanic acid,
so called since it was first isolated from the urine of dogs.
HC=C— CH2.CHNH2.COOH HC=C— CH=CH.COOH

HN N HN N +NH3

\^ \^

CH CH

Hydrolytic deamination

A method of deamination that is theoretically possible
involves hydrolysis to the correspondiug hydroxy-acid :

R . CHNH2 . COOH + H2O > R . CHOH . COOH + NH3

164 BREAKDOWN OF NITROGENOUS MATERIAL

but, with one exception, such a reaction has not been demon-
strated in bacteria. A claim has been made that aspartic
acid is hydrolytically deaminated to malic acid by Ps,
fluorescens liquefaciens :
HOOC.CH2.CHNH2.COOH + H2O

> HOOC.CH2.CHOH.COOH + NH3.

The evidence for the reaction is not direct and requires con-
firmation with a cell-free enzyme system.

Aspartic acid

Since aspartic acid is oxidatively deaminated to oxalacetic
acid by Haemophilus influenzae, it provides an example of a
substrate which can be deaminated in the four ways so far
discussed :

HOOC . CH2 . CO . COOH HOOC . CH2 . CH2 . COOH

Oxidative \ deamination Reductivey^ deamination

{H. Influ\enzae) /(Strict aerobes)

HOOC . CH2 . CHNHo . COOH

Desaturation /^ deamination Hydrolytic -v deamination

1/ (Aspartase) {Ps. \ fluorescens)

HOOC . CH = CH . COOH HOOC . CH2 . CHOH . COOH

Dehydration deamination

There is one particular example of this type of deamination
and that is the breakdown of L-serine by Esch. coli; the
postulated course of the breakdown is as follows : —

CH3

^C=0 +NH3

COOH

The experimental fact is that serine is attacked anaerobically
to liberate pyruvic acid and ammonia. To explain this
reaction the above steps have been postulated, starting with a
dehydration of serine to the unsaturated amino-acid by an
enzyme called " serine dehydrase." Preparations of washed

CH20H
CHNH2

-H2O-

CH2 CH3

II 1
^ C NH2 ^=i C-NH + H2O

COOH

COOH COOH

STICKLAND KEACTION 165

cells lose their serine dehydrase activity under conditions
similar to those described for loss of aspartase activity and can
also be restored by the addition of biotin. This type of
deamination is only made possible by the unique structure of
the serine molecule. An analogous reaction occurs with
cysteine, when the first step is a removal of HgS by " cysteine
desulphurase," after which the course of the breakdown is
presumably the same as that postulated for serine :

I 11 - I I

CHNH2 - H2S > C— NH2 ^=^ C=NH + HoO ^=3^ C=0 + NH3

COOH COOH COOH COOH

The cysteine enzyme has also been obtained in a cell-free
condition from Esch. coli; it is inactivated by dialysis and
the activity restored by the addition of zinc, magnesium, or
manganese.

Deamination by the strict anaerobes

Some of the Clostridia employ specific methods for the
deamination of some amino-acids. CI. sporogenes was first
studied by Stickland, who found that washed suspensions of
this organism are unable to deaminate any amino-acid if this
is added by itself to the suspension. Using reducible dyes
as H-donators and acceptors, he found that some amino-
acids are deaminated in the presence of a H-acceptor and some
in the presence of a H-donator dye; in other words some
amino-acids act as H-donators and some as H-acceptors. If
two amino-acids, one from each group, are added together to
the suspension of organisms, then deamination of both occurs
according to the general equation:

R X R X

! I L I

CHNH2 H- H2O -f CHNH2 — > CO + 2NH3 -f CH2

I II I

COOH COOH COOH COOH

In this reaction the molecule R . CHNH2 . COOH undergoes

166 BREAKDOWN OF NITEOGENOUS MATERIAL

oxidative deamination, the H-acceptor being another amino-
acid molecule rather than a coenzyme as in the deamination of
glutamic acid by Esch. coli. It is possible that a coenzyme is
involved as carrier in the " Stickland " reaction, but the
enzyme kinetics of the reaction have not yet been studied in
detail. The amino-acids so far tested fall into the following
groups : —

H-acceptors H-donators

Glycine Alanine Leucine

Proline Valine Phenylalanine

Hydroxyproline Cysteine Serine

Ornithine Histidine Aspartic acid

Arginine Glutamic acid

Tryptophan
CI. sporogenes also possesses a very active hydrogenase
enzyme activating molecular hydrogen so that it can be
utilised by the H-acceptor group of amino-acids. The
products of reduction have been isolated and identified in
some cases. Proline undergoes reduction with opening of
the ring to give S-amino-valeric acid without animonia
formation

HoC — CH,

"l I -f2H ^CHaNH^.CHa.CHg.CHa.COOH

HgC CH.COOH

N ■■

H
Ornithine, on the other hand, is also reduced to S-amino-valeric
acid but with the liberation of one molecule of ammonia,

CH2NH2 . CH2 . CH2 . CHNH2 . COOH -f 2H

> CH2NH2.CH2.CH2.CH2.COOH -f NH3.

Glycine is reductively deaminated to acetic acid, and the
Stickland reaction between glycine and alanine takes place
as follows, presumably with the intermediate formation of
pyruvic acid from alanine,

STICKLAND REACTION 167

2CH2NH2.COOH + CH3.CHNH2.COOH + 2H2O

= 3CH3.COOH + 3NH3 + CO2.

This oxido -reduction reaction between two amino-acids,
usually called the " Stickland reaction," would seem to be
specific for certain Clostridia such as CI. sforogenes and CI.
botulinum, but is not carried out by all Clostridia. Other
members of the genus employ a different method of deamina-
tion in which single amino-acids are attacked with the
liberation of ammonia and gaseous hydrogen. The growth of
certain Clostridia on meat media results in the formation of
considerable volumes of gas, hence the name " gas gangrene "
given to the clinical condition following the infection of
wounds with certain pathogenic Clostridia. The greater part
of this gas is liberated during the deamination of certain
amino-acids. For example, CI. tetmiomorphum attacks tyro-
sine and histidine with the liberation of hydrogen and ammonia.
In most cases the products of the deamination have not been
fully identified and we do not know how the hydrogen is
formed during the deamination process, but it has been
suggested that we have here a form of oxidative deamination,
consisting of dehydrogenation followed by release of the
hydrogen as molecular hydrogen instead of combination
with a H-acceptor.

DECARBOXYLATION

The removal of the terminal — COOH group of an amino-
acid is carried out by specific amino-acid decarboxylases
with the formation of the corresponding amine :

R . CHNH2 . COOH > R . CH2NH2 -f CO2.

The decarboxylases are specific for the natural isomer of one
amino-acid. From studies of these enzymes in a cell-free
state, it seems that only such amino-acids are attacked as
have at least one chemically active (polar) group in the
molecule other than the terminal — COOH and the a-NHg
groups. Thus decarboxylases have been described for
arginine, lysine, ornithine, histidine, tyrosine, glutamic acid,

168 BREAKDOWN OF NITROGENOUS MATERIAL

aspartic acid, and possibly tryptophan, but for no monamino-
monocarboxylic acids. In each case a simple decarboxylation
occurs with the production of the corresponding amine or,
with the dicarboxylic acids, of the a>-amino-acid. Any
alteration in the structure of the amino-acid such as methyla-
tion of either — NHg group in the diamino-acids, or of substi-
tution of the — OH in tyrosine, or the S-COOH in glutamic
acid, results in inactivation, since combination between the
decarboxylase protein and the substrate is thus prevented
(see Chap. III). The addition of — OH to the substrate such as
occurs in hydroxy lysine, dihydroxy tyrosine, or hydroxy-
glutamic acid slows down, but does not prevent, decarboxyla-
tion by the corresponding decarboxylase.

H2N. /. H,N.

>C— NHCH2.CH0.CH2.CHNH2. COO H > >C-NHCH2.CH2.

HN^ " ' HN^ CH2.CH2NH2

L-Arginine Agmatine

H2N.CH2.CH2.CH2.CH2.CHNH2. ICOQIH > H2N.CH2.CH2.CH2.CH2.

CH2NH2

L-Lysine Cadaverine

H2N.CH2.CH2.CH2.CHNH2. |coo|h — > H2N.CH2.CH2.CH2.CH2NH2

L-Ornithine Putrescine

HC=CCH,.CHNH2. ICOOIH HC=CCH2.CH2NH2

I I ' > I 1

N NH N NH

Y Y

H L-Histidine H Histamine

H0<^~ VHo.CHNHo. ICOOIh > H0<;^~'^CH2.CH2NH,

L-Tyrosine Tyramine

__/

HOOC.CH2.CH2.CHNH2. |C00|H > HOOC.CH2.CH2.CH2NH2

li-Glutamic acid y-Amino-butyric acid

HOOC.CH2.CHNH2. |COO|H > HOOC.CH2.CH2NH2

L- Aspartic acid j8-alanine

PKOPEETIES OF AMINES 169

Many of the biologically produced amines have physiological
or pharmacological activities, and so their production in vivo
by bacteria might have important consequences. For
example, histamine, produced by the decarboxylation of
histidine, is known as a " depressor substance" in that injec-
tion of small quantities into an animal results in a rapid fall
in blood pressure. It also produces contraction of smooth
muscle and causes a general condition in the animal analogous
to " shock." Tyramine, on the other hand, is a " pressor
substance" in that injection causes a rise of blood pressure.
Its general properties are the opposite of those of histamine,
and its action on injection is similar to that of adrenaline,
but much less active weight for weight. Since the action of
tyramine resembles that of adrenaline which is secreted by
sympathetic nerve endings, tyramine is said to be a " sympa-
theticomimetic " drug, while histamine is " parasympathetico-
mimetic." The other amines are less active and, in general,
the diamines such as putrescine and cadaverine (from ornithine
and lysine respectively) have weak parasympatheticomimetic
activities, while the mon-amines have weak sympathetico-
mimetic activities. The guanidine nucleus in agmatine,
produced by decarboxylation of arginine, confers an insulin-
like activity upon this amine but it is not possible to use it
as an insulin substitute as its repeated administration gives
rise to liver damage. The products of decarboxylation of the
dicarboxylic acids have no known pharmacological properties,
although j8-alanine is a growth factor for some micro-organisms
and forms part of the pantothenic acid molecule.

The amino-acid decarboxylases are formed only when growth
takes place in an acid environment, and they have unusually
acid activity -^H optima varying from 2-5 for histidine decar-
boxylase {CI. welchii) to 5-5 for the ornithine decarboxylase
of CI. sejpticum. Six of the enzymes have been obtained in a
cell-free condition and five of them, the decarboxylases of
lysine, arginine, ornithine, tyrosine, and glutamic acid, have
pyridoxal phosphate as prosthetic group. The histidine
decarboxylase apparently does not require this prosthetic group.

170

BREAKDOWN OF NITROGENOUS MATERIAL

The distribution of the amino-acid decarboxylases amongst
bacteria appears to be haphazard (see Table XIV). Esch. coli
may have the decarboxylases specific for arginine, lysine,
ornithine, histidine, glutamic acid, and very occasionally
tyrosine, but wide strain variations are found as some strains
may have five of these enzymes while others may have two,
one, or none. Many Streptococci possess tyrosine decarboxy-
lase, but strains differ widely in the activity of the enzyme.
Clostridia again show wide differences; CI. welchii may
possess both histidine and glutamic acid decarboxylases, CI.
septicmn ornithine decarboxylase only, and many other
species have no decarboxylases. Aspartic acid decarboxylase
has been found in the symbiotic nitrogen-fixing organisms
Rhizohia.

TABLE XIV

DlSTRIBTTTION OF AmINO-ACID DECARBOXYLASES

Organism

Esch. coli ...

S. haemolyticus

Proteus vulgaris . . .

Gl. welchii ...

CI. septicum

CI. aerofoetidum ...

CI. sporogenes

Ehizobium legum-
inosarum

Decarboxylase Substrate :

ARGININE DIHYDROLASE 171

SPLITTING OF THE MOLECULE

Two examples of this type of amino-acid degradation will
be discussed.

Arginine dihydrolase

The Gram-positive Streptococci and Staphylococci are very
exacting in their amino-acid requirements, and this synthetic
disability is accompanied by very restricted catabolic activities.
The only amino-acid from which these organisms can liberate
ammonia rapidly is arginine. The breakdown of arginine is not
a simple decarboxylation or deamination, as analysis of the
products shows that these are ammonia, carbon dioxide, and
ornithine. In mammalian liver we get a somewhat similar
breakdown of arginine in which urea is first split from arginine
by arginase, and can then be decomposed to ammonia and
carbon dioxide by urease:

Urease 2NH3
HN^ .NH2 HgN^ /NH2 > +

CV C ^^^ CO,

^NHCHg li H2NCH2

I ^ .1

CH2 Arginase Urea ^ll2

I +H2O > I

CH2 CH2

CHNH, CHNH

COOH COOH

Arginine Ornithine

However, there is no evidence of the intermediate formation
of urea during the degradation of arginine by Streptococci,
and these organisms do not possess urease. The reaction is
presumably a direct hydrolysis as shown below, and the enzyme

172 BREAKDOWN OF NITROGENOUS MATERIAL

concerned has been called "arginine dihydrolase " to distin-
guish it from the arginase of liver cells.

^NHCHj H2NCH2

I I

^^2 Arginine ^Hg

I +2H2O > I +2NH3 + CO2

CHg dihydrolase CHg

I I

CHNH2 CHNH2

I I

COOH COOH

Arginine dihydrolase is possessed by most Streptococci to
varying extent. Its function is not clear, as arginine is an
essential amino-acid for the organisms which consequently
appear to attack one of their essential nutrients. It is possible
that these organisms, which carry out a simple Aomolactic
fermentation of glucose, depend upon arginine dihydrolase
action for the provision of the carbon dioxide which is essential
for their growth.

The production of indole from tryptophan

In Chap. II various biochemical tests were outlined for
the systematic characterisation of bacteria. One of these
tests is the formation of indole in protein-containing and
protein-digest media by Esch. coli. It has been known
from the early days of bacteriology that some organisms,
particularly those of the coli group, produce a strongly
smelling substance from protein digests, and that this sub-
stance reacts with j9-dimethyl-amino-benzaldehyde in alcoholic
HCl to produce a pink colour. The substance was known
as indole, and Ehrlich worked out a simple appHcation of the
colour reaction for testing the production of indole in cultures.
In 1901 Hopkins and Cole showed that the precursor of

INDOLE FORMATION 173

indole is tryptophan. The bacterial reaction occurs aerobically,
and the oxidation of one molecule of tryptophan to indole is
accompanied by the consumption of five atoms of oxygen.
We can make guesses at the nature of possible intermediate
substances, but if theoretically possible intermediates such
as indole-acetic, -propionic, -pyruvic, -acrylic, -lactic acids,
or the corresponding aldehydes, are added either to cultures
or to washed suspensions of active organism, they do
not give rise to indole and consequently cannot be indole
precursors. In an atmosphere of hydrogen Esch. coli produces
indole-propionic acid from tryptophan, but this must involve a
different metabolic path from that followed aerobically, as
indole-propionic acid is not decomposed further under aerobic
conditions. It seems that the first step in the aerobic decom-
position of tryptophan is not related to those steps discovered
for other amino-acids.

Recent studies with the bread mould, Neurospora crassa,
have shown that tryptophan is synthesised by this organism
from indole and L-serine and that the reaction is reversible:

+ CH2OH.CHNH0.COOH X \ CH2.CHNH2.

I III COOH

H H + ^2^

The same workers suggested that a similar reaction is involved
in the synthesis and breakdown of tryptophan by Esch. coli.
Further studies have not confirmed this suggestion, as a cell-
free preparation has been obtained from Esch. coli. and the
breakdown of tryptophan by the preparation results in the
liberation of indole, ammonia, and pyruvic acid.

/\ CH2.CHNH2.COOH /\

I I I -fHgO^ ^1 I I+NH3+CH3.CO.COOH

H H

The preparation will not attack either serine or alanine so
these cannot be intermediates in the reaction. The oxygen

+ +

I I

I I I

o. I

+3 i-j3 p t3

«^ W;£;^w

I I I

www

+ +

s s

§ a ^ a
w3w2

+
I I I I S I

W "IT

ow I

!^ ^ W pj'

+ + + ^

+■

^ >

■.a

WW. 2

+ + + I +

o "^

> t> o g ^3
g S S g o

f-, ^ O l-l r^-l

s a

w-5
J- s

^ § .a -a

Qj O) M t-i

J ft hj hij

-^3

:t^

bJD

<I1

1-5

.3 I
^ 2

174

AMINO- ACID METABOLISM 175

consumption accompanying indole formation must be involved
in the oxidation of pyruvic acid.

The amino-acid metabolism of Esch. coli, Streptococci, and
two typical Clostridia is summarised in Table XV.

FOR FURTHER READING

" Enzymes Involved in the Primary Utilisation of Amino-
acids by Bacteria," Gale, E. F., Bad. Rev., 1940, 4, 135.

" Bacterial Amino-acid Decarboxylases," Gale, E. F.,
Advances in Enzymology, 1946, 6, 1.

" Nitrogen Metabolism," Gale, E. F., Annual Rev. Microbiol,
1947, 1, 141.

CHAPTER X

THE NITROGEN CYCLE

Gaseous nitrogen is fixed by certain bacteria with the
formation of organic nitrogenous matter. This organic
material is decomposed by other organisms with the production
of ammonia. Ammonia is oxidised to nitrate by the nitrifying
autotrophes, and certain Chromobacteria have the property
of liberating gaseous nitrogen from nitrate. So nitrogen
completes a cycle: the steps in this cycle are indicated below
and will now be considered in greater detail.

PROTEIN

Azotobacier

ATMOSPHERIC

AMINO-ACIDS

< °'

NITROGEN

Rhizobia

1 AMINES

/'/

^ cl

'^Chromobacteria "

Pseudomoha daceae
1 /

NH3

NH2OH

NO2'

NO3'

c .

/Vitrosomonas
c

Nicro-
bacter

(a) Nitrogen-fixation

In Chap. V it was shown that certain organisms are able
to utilise atmospheric nitrogen as a source of nitrogen for
growth purposes. This fact has been used since very early
days of agriculture as a means of fertiHsing soil. The majority
of plants lead an autotrophic type of existence and draw upon
the inorganic nitrogen of the soil for their nitrogen require-
ment. Consequently the growth of a heavy crop of grain
results in the depletion of the soil-nitrogen, and cropping of
the same soil year after year results in a steadily decreasing
yield of grain until eventually such cultivation is no longer
economical. Since the times of Virgil it has been known
that this depletion can be countered in one of two ways:

176

NITROGEN FIXATION 177

either by leaving the soil fallow for a year or, alternatively,
by growing acrop of clover, vetches, alfalfa, or other leguminous
plant. Either of these measures results in a replenishment of
the soil-nitrogen, and it is possible to grow further successful
crops of grain. Both of these natural fertilisation measures
owe their ef&cacy to the action of bacteria in " fixing "
atmospheric nitrogen and so rendering it available in the soil
in a form which can be utilised by plants.

In the case of the field left fallow, the organism mainly
concerned is the strict aerobe, Azotobacter. Berthelot showed
in 1885 that if soil is left exposed to the air, then its nitrogen
content slowly increases and that this increase takes place
at the expense of atmospheric nitrogen. He further showed
that the responsible agent is biological, since the process
can be stopped by heat or by treating the soil with caustic
chemicals. It was some years before any organism was
isolated from soil which has the property of fixing nitrogen
and the first such organism isolated was CI. pastorianum,
a strict anaerobe which is of less importance than Azotobacter.
Azotobacter is able to grow rapidly in the presence of gaseous
nitrogen as sole source of nitrogen and of carbohydrate as
carbon and energy source. Growth is such that there is a
constant ratio between nitrogen fixed and carbohydrate
utilised. In soil the limiting factor is often the amount of
carbohydrate available, and this explains the practice of
some Indian farmers of enriching their soil by ploughing waste
molasses into it. Despite many studies on Azotobacter, we
are still without any definite knowledge of the chemistry of
the fixation process (see p. 90).

In the days before artificial fertilisers were available, the
alternative open to the farmer, instead of leaving his fields
fallow, was to cultivate on his fields a crop of one of the
leguminous plants, i.e. those plants having nodules on their
roots. The function of the root-nodules was first made
apparent in 1888 by Hellriegel and Wilfarth, who studied the
growth of peas and the formation of root-nodules on their

CHEM. A. B. 12

178

THE NITROGEN CYCLE

roots in soil and sand under controlled conditions. Their
results may be summarised as follows: —

Condition of
Soil

Presence of
Fixed-nitrogen

Growth

Formation of
Root-nodules

Normal

+ /-

+ + +

Sterile

—

Sterile

+ +

—

Non-sterile

+ + +

In normal soil, normal growth occurs and nodules are formed
on the roots ; analysis shows that an increase in the nitrogen-
content of the system (soil -f plant) has occurred during
growth. If the peas are sown into sterile sand from which
all fixed-nitrogen has been washed away, then no growth
can occur and no nitrogen-fixation takes place. If the
sterile washed sand is treated with fixed-nitrogen in the
form of nitrate, etc., then growth occurs, but no formation
of nodules takes place, and analysis shows that the nitrogen-
content of the system (soil + plant) has remained constant,
so that nitrogen-fixation has not occurred. On the other hand,
if the soil has been freed from fixed-nitrogen but not sterilised,
then normal growth takes place, nodules appear on the roots,
and the entire nitrogen-content of the plant is obtained by
fixation of atmospheric nitrogen. These experiments demon-
strate that (1) nitrogen-fixation occurs only in the presence
of root-nodules, and (2) nodules never form on the roots if
growth takes place in sterile soil.

The formation of nodules on the roots of these plants is
due to the action of bacteria called Rhizobia, which exist in
soil and are able to penetrate the root-hairs of certain plants
with which they come into contact. When penetration has
taken place the root-cells in the vicinity of the invading
bacteria are stimulated to division and the increased growth
so produced gives rise to the nodule. The Rhizobia in contact
with the plant within the nodule are now able to carry out

SYMBIOTIC NITROGEN FIXATION 179

nitrogen-fixation, but the process is essentially a symbiotic
one, in that neither the plant alone nor the free-living
Rhizobium can fix nitrogen. The plant, as shown above, can
grow in sterile soil as long as it is provided with a source of
fixed-nitrogen, but it cannot utilise atmospheric nitrogen
under these conditions. Rhizobium can lead a free-living
existence and can grow normally in a medium containing
fixed-nitrogen, but it cannot fix atmospheric nitrogen when
growing apart from a host-plant. There is an important
specificity between the particular Rhizobium and the plant
with which it can enter into a symbiotic relation, thus Rh.
trifolium can form nodules only on the roots of clover, and
can fix nitrogen only in symbiosis with that plant, while
Rh. leguminosarum can form nodules only on the roots of
the pea. Further than this, some strains of Rh. trifolium,
for instance, are better nitrogen-fixers in symbiosis with clover
than are others, and it often pays a farmer nowadays to obtain
a suitable strain with which to inoculate his soil before sowing
this type of crop.

The chemistry of the symbiotic nitrogen-fixation process
has been the subject of much work in Helsinki University and
also in American laboratories. Prof. Virtanen of Helsinki
has published a series of papers in which he claims to have
elucidated the chemical processes involved but, unfortunately,
attempts to confirm this work in other laboratories have not,
so far, met with success. The scheme put forward by Virtanen
was as follows: —

Nitrogen is fixed by Rhizobia with the formation, after
unknown intermediate stages, of hydroxylamine. At the
same time carbohydrate is broken down within the host-
plant with the formation of oxalacetic acid. Hydroxylamine
and oxalacetic acid combine spontaneously to form the
corresponding oximo-succinic acid which is then reduced by
the organism to aspartic acid. The aspartic acid, in turn,
acts as a source of other amino -acids in the plant through
transamination.

180 THE NITROGEN CYCLE

N, > ? > NHoOH

Rliizohium \

\ — ^ HOOC.CHo.C (NOH).COOH

/ I Ehiznhiii.m

c^Hj^Oe — > H00C.CH2.C0.C00H ;

^""'* HOOC.CHo.CHNHs.COOH

Rhizolnuyn ^--^^ Plant

HOOC.CH2.CH2NH2 ^^ R.CHNH2.COOH

The evidence put forward by Virtanen in support of this
scheme includes the following points: —

1. The demonstration that aspartic acid and its decarboxyla-
tion product, jS-alanine, are excreted into the soil around the
roots of young pea plants, and that this excretion occurs
only from the nodules.

2. The demonstration of oxalacetic acid in the sap of the
host-plant. *

3. The isolation of oximo-succinic acid from the soil around
the nodules.

4. The demonstration that Rhizobia can grow on a nitrogen-
free medium provided that oxalacetate is added to the medium
— when growth occurs with nitrogen-fixation.

5. That transamination between aspartic acid and pyruvic
acid will take place in the presence of extract of pea with
the formation of oxalacetic acid and alanine.

COOH CCOH

CH2 ^113 01x2 ^^z

I + I > I + I

CHNH2 CO CO CHNH2

II II

COOH COOH COOH COOH

This evidence is quite formidable, and would seem to provide
satisfactory confirmation of the fixation hypothesis, but

NITRIFICATION 181

several key-points, namely the proof of aspartic acid excretion
by the nodules and of the growth of Rhizobia in nitrogen-free
oxalacetate media, have not so far been confirmed in other
laboratories. Also the evidence of the nature of the oximo-
succinic acid isolated from soil ic not altogether convincing.
Wilson, in America, has carried out much careful and detailed
research into the symbiotic nitrogen fixation process, and has
not obtained evidence in confirmation of Virtanen's hypothesis.
An interesting point discovered by this group of workers is
that the fixation process is inhibited by the presence of
hydrogen.

The root-nodules contain a haematin-pigment resembling
haemoglobin, but the part played by this substance in the
fixation process has not yet been elucidated.

(6) Interchange of bacterial nitrogen and ammonia

The interchanges between bacterial protein, amino-acids,
and ammonia have been dealt with in detail in these last
two chapters. Degradation of amino-acids will take place
either by deamination or by decarboxylation, according to
the pH holding in the environment at the time. If
decarboxylation occurs, the nitrogenous product is an amine,
and if the environmental ^H subsequently returns to neutral
or alkaline values, then amines themselves are oxidised by
certain bacteria, particularly Pseudomonas, with the liberation
of ammonia.

The oxidation of ammonia to nitrate in the soil is called
" nitrification," and the biological nature of the process was
first demonstrated in 1877 by Schloesing and Muntz. They
poured sewage effluent through a long tube containing sand
and chalk, and showed that, after the fluid had been passing
through the tube for a few days, ammonia entering the tube
was removed during the passage of the effluent through the
tube, and replaced by nitrate in the fluid issuing from the tube.
They showed that treatment of the contents of the tube with

182 THE NITEOGEN CYCLE

heat or caustic chemicals stopped the nitrification process
so that ammonia passed through uncha'nged. If the passage
of the sewage effluent were maintained, then the culture was
slowly re-established and nitrification resumed after a few days.
It was thirteen years after this demonstration that the first
nitrifying organism was isolated in pure culture. In Chap. V
the nitrifying organisms Nitrosomonas and Nitrobacter aie
described as strict autotrophes whose growth is inhibited by
the presence of organic matter. Since it was the custom in
those days to attempt the isolation of organisms by growth
on the surface of solidified gelatine, it is understandable why
no successful isolation of nitrifying bacteria was achieved
for some time. It was not until Winogradsky invented the
purely inorganic solid medium consisting of inorganic salts
incorporated in a silicic acid gel that the first successful
isolation of a nitrifier was accomplished. It then became
obvious that two organisms are involved in the nitrifying
process, the first (Nitrosomonas) carrying out oxidation of
ammonia to nitrite, and the second (Nitrobacter) completing
the oxidation of nitrite to nitrate. The two processes are not
interchangeable, for while Nitrosomonas can grow on a
synthetic medium containing ammonia as source of nitrogen
and energy,

2NH3 + 3O2 > 2HNO2 + 2H2O + 79 Cals.

it cannot grow on a medium containing nitrite but no ammonia.
Similarly, Nitrobacter cannot grow in a nitrite-free medium, but
must obtain its energy from the oxidation of nitrite to nitrate :

HNO2 + 0 > HNO3 -f 21-6 Cals.

The process of nitrification is peculiar to these two organisms,
and can consequently only take place when conditions are
suitable for the growth of strictly autotrophic organisms.

(d) Reduction of nitrate to ammonia

This change is one that can be accomplished by several
organisms, including Esch. coli and CI. welchii. In the presence
of a hydrogen donator, Esch. coli can reduce nitrate to nitrite

NITRATE REDUCTION 183

by means of an enzyme called " nitratase." Hydrogen may
be supplied by a dehydrogenase such as, for example, formic
dehydrogenase, so that the organisms can oxidise formic acid
anaerobically in the presence of nitrate :

H.COOH + HNO3 > CO2 + H2O + HNO2.

Both Esch. coli and 01. welchii possess an active hydrogenase
(p. 48) and in the presence of hydrogen, we find that the
reduction of nitrate proceeds further than nitrite on to
ammonia, with the probable intermediate formation of
hydroxylamine :

H2^=^2H

2H + HNO3 ^ HNO2 + H2O

4H + HNO2 > NH2OH + H2O

2H + NH2OH > NH3 + H2O

or, the over-all reaction:

HNO3 + ^^2 > NH3 + 3H2O.

The interchange between ammonia and nitrate is thus rever-
sible, but whereas the forward reaction (c) can be carried out
by certain strict autotrophes only, the reduction process (d)
can be performed by a number of heterotrophic organisms,
both strictly and facultatively anaerobic.

(e) Denitrification

When certain species of Serratia, Chromobacteria, and
Pseudomonadaceae are grown in media containing either
nitrate or nitrite as source of nitrogen, there is a disappearance
of fiji:ed-nitrogen from the culture and bubbles of gaseous
nitrogen form in the culture fluid. The chemistry of this
denitrification process has not yet been worked out; it is
not reversible. The " Chromobacteria " thus provide the
final link in the cycle which starts with the fixation of atmo-
spheric nitrogen by Azotobacter, etc., passes through the
heterotrophic interchange of organic nitrogenous compounds
with final degradation to ammonia, through autotrophic

184 THE NITROGEN CYCLE

nitrification, and finally to denitrification and the liberation
of gaseous nitrogen again.

Conditions affecting the nitrogen cycle

Whenever we have a thoroughly mixed bacterial popula-
tion, we have the possibility that organisms and enzymes
for the entire nitrogen cycle will be present. Which particular
part of the cycle will predominate at any moment will depend
on conditions holding in the immediate environment. The
various reactions in the cycle will be conditioned as follows : — ■

ORGANIC N < a Ng

t
b e

d d I

NH3 ^ _^ NO2 < ^ NO3

c c

Reaction (a) can occur only in the absence of fijxed-nitrogen.

(6) Breakdown to ammonia will occur as long as there is
excess of organic-nitrogen ; if ammonia is the more abundant
and a source of carbon is present, then organic-nitrogen will
be synthesised at the expense of ammonia,

(d) Can occur only in the presence of organic matter or
hydrogen as H-donator.

(e) Occurs in presence of nitrate or nitrite as sole source
of nitrogen.

Sewage purification

The nitrogen cycle can function in whole or in part in the
soil and is put to use in sewage purification. Raw sewage
as it comes to the sewage farm contains much organic material
and is heavily inoculated with heterotrophic organisms. In
the settling tanks proteolytic organisms digest the solid
material forming utilisable organic-nitrogen, which is broken
down by heterotrophic action to ammonia. The fluid sewage

SEWAGE PURIFICATION 185

is then usually treated to some aeration process or trickled
over coke filter beds in which a high degree of aeration takes
place. In the upper layers of the beds the breakdown of
organic-nitrogen is completed and organic matter extensively
oxidised. In the lower layers conditions are suitable for the
growth of autotrophes and nitrification takes place. The
bacterial population of the filter bed is very mixed and a
certain amount of denitrification may also take place, the
composition of the final effluent depending upon the design
of the bed, the speed of flow of sewage, the time since the bed
was rested, etc. If the filter bed is kept in continual use
the coke becomes " choked " with bacterial growth and the
purification process slows down. Consequently the beds
have to be rested periodically by stopping the flow of sewage
for a few weeks, when the filters clear themselves by auto-
digestion, during which the cycle is repeated on a smaller
scale, having bacterial protein as starting point instead of
sewage.

FOR FURTHER READING

Cattle Fodder and Human Nutrition, Virtanen, A. I. (C.U.P.).

The Biochemistry of the Symbiotic Nitrogen Fixation Process,
Wilson, P. W. (Univ. Wisconsin Press, Madison).

CHAPTER XI

PATHOGENICITY; CHEMOTHERAPY

From a medical point of view, bacteria are divided into
" pathogens " or organisms capable of causing disease in a
host, and " saprophytes " or organisms which are harmless
to other creatures. Those organisms, such as the normal
bacterial flora of skin, mouth, and intestine, which live in
constant association with man without causing any disease
or lesion, are called " commensals." An organism may be
potentially pathogenic in one situation and a commensal in
another as, for example, CI. welchii which is a normal harmless
inhabitant of the intestine, but is pathogenic should it get
into a deep wound.

The majority of bacteria are saprophytic, and in this chapter
we intend to consider, as briefly as possible and from a bio-
chemical aspect, what it is that differentiates a pathogenic
from a saprophytic organism.

The healthy interior tissues of animals are sterile, and are
maintained so by the action of both fixed and wandering cells
which have the power to ingest and digest bacteria by the
process known as phagocytosis. In mammalian blood, for
example, certain of the white corpuscles have this phagocytic
property, and if a foreign particle or organism enters the
blood-stream, then these phagocytes are attracted towards
the foreign substance, surround it, and attempt to destroy or
engulf it. Other phagocytic cells are fixed to the capillary
walls of the liver, etc., and the whole complex of fixed and
wandering scavenging cells is known as the " reticulo-
endothelial system." When an organism enters the blood-
stream or other tissue, a race ensues between the capacity of
the organism to multiply on the one hand, and the capacity
of the phagocytes to destroy the invading cells on the other.
If the phagocytes win rapidly no disease symptoms appear,
but if the bacteria are able to paralyse or outgrow the
phagocytes, then there usually follows some disturbance of

186

VIRULENCE

187

the organisation or metabolism of the host, which becomes
apparent as cHnical disease.

Bacteria may enter the tissues in a variety of ways : through
the respiratory passages, through the tonsils, through the
intestinal wall or, above all, through any type of wound.
If saprophytic bacteria enter by any of these routes, they are
rapidly and effectively destroyed by the reticulo-endothelial
system, but a pathogenic organism is able to grow within the
host's tissues and its capacity to do so is a measure of its

Fig. 13.

" virulence." Fig. 13 shows the effect of injecting 1 million
streptococci into the blood-stream of a healthy animal. If
the organisms belong to an avirulent strain (Case 1), then
there is a steady decrease in the number of bacterial cells in
the blood-stream from the time of injection until eventual
disinfection. If the strain is highly virulent (Case 3), then,
after an initial decrease in numbers, the organism begins to
multiply rapidly and there is a steadily increasing number of
organisms in the blood-stream until eventually the host dies
as a result of their activities. In the intermediate case of a

188 pathogenicity; chemotherapy

moderately virulent organism (Case 2), there is the usual small
decrease, followed by increasing numbers — during which phase
clinical symptoms appear — then a period during which the
organisms are being countered by the defence mechanisms
of the host, and finally a clearing of the blood-stream as
phagocytosis is successful.

Virulent organisms differ from avirulent forms in two main
respects. In the first place, virulent bacteria often possess
a capsule which protects them against the action of the
phagocytes. Virulent pneumococci possess a capsule com-
posed of polysaccharide and if this capsule is removed by
enzymatic means, the organism, though viable, is no longer
virulent and is rapidly removed in vivo by the cells of the
reticulo-endothelial system. The protective capsule is not
always polysaccharide in nature as the capsule of B.
anthracis, for example, consists of a polypeptide of the
unnatural D -glutamic acid.

A second property which often distinguishes a virulent
organism from an avirulent one is the power of the former to
produce a " toxin." A toxin can be described as a substance
which is secreted specifically by an organism, which produces
general toxic reactions in the host and which gives rise to the
production of specific antibodies in the host's blood-stream.
When virulent organisms are injected, after an initial phase of
establishment, they proceed to multiply and produce toxin.
At the same time, the phagocytes mobilise around the site
of entry, and one of the actions of the toxin is often to
antagonise the phagocytic action and so impair the defence
mechanism. The circulation of toxin in the host's blood-
stream exerts the toxic action with the production of clinical
disease. A further effect of the toxin circulating in the blood
is to stimulate the cells of the bone-marrow to produce specific
antitoxin, the chief function of which is to render the toxin
ineffective. If the bone-marrow response is sufficiently rapid,
then antitoxin is poured into the blood, the toxin neutralised,
and the reticulo-endothelial cells once more enabled to attack

TOXIN PRODUCTION 189

and phagocytose the invading organisms. If the toxin
production is sufficiently powerful or the bone-marrow response
too slow, then the toxic action may cause the death of the
host as in Case 3. If the toxin production is moderate and
the bone-marrow response adequate, then the initial advantage
given to the organism by its toxin production is overcome and
the invading cells eventually removed (Case 2). If the
invading cells are avirulent or saprophytic and produce no
toxin, then the reticulo-endothelial system is able to sterilise
the host's tissues without difficulty (Case 1).

A pathogenic organism differs, then, from a non-pathogen
in that it possesses the power to produce a toxin, enabling it
in some way or other to gain an advantage over the defence
mechanisms of the host. This is not the sole property
necessary to produce a successful pathogen, as the property
of forming a toxin cannot be effective until the organism has
actually invaded the host's tissues. Consequently a second
factor of pathogenic importance is the degree of '' invasive-
ness," or capacity to penetrate the host's tissues.

" Toxin " and " invasiveness " are names of two properties
of a pathogenic organism that can be more correctly described
in chemical terms. This branch of bacterial chemistry is one
which is only now being developeci, and has not as yet reached
a stage where generalisations can safely be made. Conse-
quently we shall take a specific case of disease causation,
namely, the production of " gas gangrene " by the infection
of a wound with CI. welchii, and endeavour to explain the
pathogenicity on a chemical basis. This disease is chosen
as example as it has been very intensively studied in recent
years, so that we now understand more of the chemical nature
of the pathogenic action of CI. welchii than of any other
organism.

BIOCHEMISTRY OF GAS GANGRENE

Gas gangrene is the name given to the clinical condition
following the infection of tissue, usually through a wound,
with CI. welchii and certain related strict anaerobes. It is

190 pathogenicity; chemotherapy

characterised by liquefaction of the tissues around the wound
and the appearance of bubbles of gas within the muscular
tissue around the wound — hence the name " gas gangrene."
The gas may accumulate to such an extent that the infected
flesh crackles when handled. The local condition in the
wound is accompanied by pronounced shock, fever, and
collapse which, in the absence of therapeutic measures, is
usually fatal.

Gas gangrene became serious during the fighting in Flanders
in 1914-18, when it was, for a time, one of the major causes
of death following wounding. Bacteriological examination
of the wound and the wound exudate showed heavy infection
with certain species of strictly anaerobic bacteria. The main
organisms concerned are three Clostridia: CI. welchii, CI.
septicum, and CI. oedematiens, placed in order of importance.
Other Clostridia, particularly CI. sporogenes and CI.
histolyticum, were often found in association with these three;
these are not themselves pathogenic, but it has been observed
that gas gangrene infections where the pathogenic organism
is accompanied by one of these non-pathogenic species, are
considerably more dangerous and progress with greater
rapidity than those where a simple infection of CI. welchii,
etc., exists.

The Clostridia live a saprophytic existence in the intestinal
contents of many animals. Several distinct toxigenic types
of CI. welchii have been identified, of which some, types B, C,
and D, are associated with diseases of young farm animals.
CI. welchii, Type A, the causal organism in gas gangrene,
appears to lead a normal and harmless existence in the
intestinal contents of man and animals. The organisms are
voided with the faeces, and a certain proportion of them form
spores in the unsuitable environment of field and soil. The
spores can remain in the resting state on the soil for many
years, if necessary, until they eventually fall into an environ-
ment suitable for vegetative existence when they germinate
to form viable cells ready for multiplication. A suitable
environment for germination is provided by the tissues of a

GAS FORMATION IN GAS GANGRENE 191

wound. Consequently, whenever a wound becomes con-
taminated with soil, dirt, or dust containing manure, there is a
possibility of gas gangrene infection from Clostridium spores
in the manure particles. When soldiers fighting on cultivated
land become casualties, the probability of infection is high,
and gas gangrene becomes a major hazard of war.

CI. welchii is a moderately proteolytic, highly fermentative
organism. When the viable organism begins to multiply in
the tissues surrounding an infected wound, the following
changes take place: —

1. Liquefaction of the tissues

This is due to the action of extracellular proteases attacking
tissue proteins and breaking them down to their constituent
amino-acids with the consequence that the tissue loses its
structure and " dissolves." In particular, CI. welchii excretes
a coUagenase, a proteolytic enzyme which hydrolyses the
collagen of muscle-fibres, including the sarcolemma, with the
result that the fibre-bundles disintegrate.

2. Production of gas

There are two main sources of the gas which appears in the
infected tissue. First, the organism is able to ferment muscle
glvcogen with the production of hydrogen, carbon dioxide,
acetic and butyric acids, and other products. However, if
CI. welchii is grown in the presence of meat protein freed from
glycogen, gas is still produced in large quantities. This gas
is produced by the deamination of certain amino-acids,
especially serine, with the liberation of Hg, CO2, NH3, etc.
(see p. 167). The action of the proteases on tissue protein
assures a steady supply of free amino-acids to act as source
of gas in this way,

3. Production of histamine

CI. welchii, when growing in an acid medium, produces
histidine decarboxylase. Histidine is liberated from tissue
protein by the action of proteases, and the fermentation of

192 pathogenicity; chemotherapy

muscle glycogen leads to localised pockets of acid, consequently
conditions are suitable for the production of histamine within
the wound. The histamine content of the muscles of a cat
may increase by 100-250 per cent, when CI. welchii infection is
established, but since it is not possible to demonstrate any
increase in the blood histamine, it is doubtful whether this
histamine production has any generalised effect.

The three occurrences so far outlined are the result of the
simple metabolism of the organism and play their part in the
superficial characteristics of the disease and its clinical picture,
but are not seriously concerned in the lethal nature of the
infection. When infection with CI. welchii is accompanied
by contamination with either CI. sporogenes or CI. histolyticum
then these three factors so far discussed become exaggerated
as both these Clostridia are highly proteolytic. Consequently
their presence leads to a more rapid liquefaction of the tissues
with increased supply of amino-acids to the pathogenic
organism.

4. Invasion of the tissues

If a number of experimental infections of animals are made
with various strains of CI. welchii, it is found that whereas
some organisms establish themselves in the wound quickly,
penetrate the tissues, and set up a fulminating gas gangrene,
others, though of equal toxicity, are unable to establish
themselves or to penetrate the tissue. The strains are said
to vary in their invasiveness. Penetration of tissue by
bacteria is opposed by a barrier of highly viscous mucoprotein
between the tissue cells, and many organisms are unable to
penetrate such a barrier. Highly invasive organisms have the
power to decompose the polysaccharide portion of the complex
by the formation of an extracellular enzyme. The poly-
saccharide concerned in muscle tissue is called hyaluronic
acid and consists of equal parts of glucuronic acid and N-acetyl-
glucosamine. Some organisms excrete an enzyme called
" hyaluronidase," which is able to attack and decompose
hyaluronic acid. The chemistry of the breakdown is not yet

CL welchii toxins 193

known in detail, and it is probable that hyaluronidase is
actually a mixture of enzymes. The result of hyaluronidase
action on hyaluronic acid is a great reduction in viscosity
accompanied by hydrolysis and liberation of N-acetyl-
glucosamine. The barrier to penetration is thus overcome
by those organisms, including CI. ivelcMi, which can excrete
hyaluronidase.

5. The production of toxins

It is not the proteolysis, gas or histamine production, or
hyaluronidase action of CI. welchii that eventually kills the
host, but the production of toxins by the organism. It is
possible to grow the organism in a suitable medium, filter
off the organism, and kill a host animal by injecting the cell-
free medium containing toxins elaborated by the organisms
during growth. The medium can be concentrated and
fractionated, and the toxin extracted in a highly purified
state. It has all the properties of a protein and its toxic
action is destroyed by heat.

By fractional precipitation the " toxin " of CI. welchii,
Type A, can be divided into two:

[a) a-ToxiN: this substance has three biological actions:

1 . If it is mixed with a suspension of red blood corpuscles,
it brings about haemolysis or disintegration of the cells and
is consequently said to be " haemolytic."

2. In its presence, tissue cells disrupt and the toxin is said
to be " necrotic."

3. If minute amounts are injected into an experimental
animal, the animal dies within a few minutes, so that the toxin
is " lethal."

(6) ^-ToxiN : this is also haemolytic, but is neither necrotic
nor lethal, except in comparatively large doses. The lethal
properties of CI. welchii reside mainly in the a-toxin.

In the past there have been two main theories concerning
the possible nature of toxin action : (a) that toxins are enzymes

CHKM. A. B. J3

194 pathogenicity; chemotherapy

which interfere in some way with the essential metabolism
of the host ; (6) that they act in some other way by blocking
an essential activity in the host.

MacFarlane and Knight have shown that preparations of
the a-toxin possess the activity of a lecithinase, and that during
purification, increase of toxicity is paralleled by increase of
lecithinase activity so that the purest preparations of toxin
are also the most active lecithinase preparations.

Lecithinase hydrolyses lecithin with the liberation of
phosphocholine:

CHgO-E^

CH2O— Ri

CH0-R2
0

: II

CH2O : p -0-

Lecithinase
H2O

CHO— 112

+ Phosphocholine

-C2H,-N(CH3)3

("H2OH

Ri, R2 = fatty

acid

residues.

Lecithin is an essential component of the membrane of cell-
walls, so that if the lecithin is decomposed, then the cell-wall
disintegrates. If the lecithin of the cell-wall of a red blood
corpuscle is hydrolysed, then the cell-wall disrupts and the
red cell haemolyses. In the same way tissue cells disintegrate,
and it is only reasonable to suppose that such a reaction, taking
place generally throughout the body, would be lethal in its
final effect. It is highly probable that the a-toxin of CI.
ivelchii is actually a very active lecithinase. Both a-toxin
and lecithinase are inactive in the absence of calcium ions,
and the lecithinase activity of the preparations is inhibited
by the specific antitoxin.

The chemical action of the ^-toxin has not yet been dis-
covered. It also acts on some substance in the wall of the
red blood cell in a way that leads to disintegration but,
assuming that it is likewise an enzyme, its substrate has not

NATURE OF BACTEKIAL TOXINS l95

yet been identified. The ^-toxin is active only in a reduced
state and can be completely inactivated by oxidation; the
nature of effective oxidising and reducing agents suggests
that the group which is oxidised in the toxin molecule is
— SH, oxidation to — SS — ■ leading to inactivation.

We have attempted in this way to explain the toxicity
of CI. welchii by analysing the various factors which act
biochemically in a way that we might expect would explain
the toxic action. In the same way we endeavour to explain
the oxidations carried out by bacteria in terms of the activity
of isolated enzyme systems. When we come to integrate
our findings with isolated enzyme systems in terms of the
activities of the intact cells, we find that the interplay of
various environmental factors, etc., complicate the reactions
established in vitro. In the same way we find that we cannot
explain the complete pathological picture found in gas gangrene
in terms of the activities of the separate factors we have
discussed. The exact importance of the role played by toxins
in the clinical picture is not yet clear, and it has been suggested
that some part may be played by substances released from
muscle cells and necrotic tissues on disruption. There are
almost certainly some factors brought into play by the inter-
action of the infecting organism and the infected host which
have not yet been revealed by studies in vitro.

THE NATURE OF BACTERIAL TOXINS

The a-toxin of CI. welchii is probably an enzyme whose
substrate is an essential structural unit in the cells of the
host. The pathogenicity of this organism depends largely
upon its power to excrete certain enzymes which attack the
tissues of the host as substrate. It does not, of course, follow
that all toxins are necessarily enzymes, but such a hypothesis
fits in with what is known of the nature of many of them We
must now expect that work will be intensified with a view to
estabHshing the enzymatic nature of other toxins and the
identity of their substrates. The work is difficult as there is
often no clue as to the possible nature of the substrate ; with

196 pathogenicity; chemotherapy

CI. welchii a clue to the nature of the substrate existed in the
fact that when the organism is grown on serum-plates or in
egg-media, the medium becomes turbid and this turbidity is
due to the formation of minute fat droplets. We know now
that these arise from the hydrolysis of lecithin in such media.
Haemolysins, such as the ^-toxin of CI. welchii and the toxins
of S. haemolyticus, presumably act by degradation of a vital
constituent of the wall of the red blood cell, but there seem
to be no obvious clues to the chemical action of other toxins
such as that formed by CI. hotulinum which is probably the
most active of all exo- toxins.

The toxins of certain pathogens, such as CI. tetmium (tetanus)
or CI. botulinum (botulism), seem to be very much more active
than those of CI. welchii, and it may be that they act as
enzyme inhibitors rather than as enzymes themselves. It
has been suggested that the toxin of CI. tetmium is an inhibitor
of choline esterase, but proof is not yet available. These
two toxins of CI. tetmium and CI. botulinum have been
obtained in a crystalline state very recently, so we may expect
further developments in the near future.

THERAPY: THE COMBATING OF PATHOGENIC BACTERIA

At the beginning of this chapter it was shown that whether
or not disease follows the contamination of a host's tissues
with a bacterium depends upon the relative activities of the
bacterium and of the reticulo-endothelial system. The aim
of medical science is to prevent the organism gaining the
final advantage in any infection or, referring back to Fig. 13,
to reduce Case 3 to Case 2 and, if possible, to Case 1. The
most successful ways of accomplishing this are based on two
fundamental principles :

1. Immunological defence

A property of a toxin or any foreign protein in the blood-
stream is to stimulate the formation of an antitoxin or anti-
body by the bone-marrow. The chemistry of antigens and

IMMUNISATION 197

antibodies is a specialised branch of the subject outside the
scope of this book, but one obvious method of combating
infection is to assist the production of the antibody against
the infecting agent. This can be done in various ways,
which have varying effectiveness against different organisms.
In cases where the infection is already established and toxaemia
present, it is sometimes possible to inject the specific anti-
toxin itself. Antitoxin is made by injecting sub -lethal doses
of toxin into a large animal such as a horse, removing the
plasma after antitoxin formation has occurred, and using some
preparation of this plasma as a source of antitoxin. It is
more satisfactory to produce antitoxin in the blood of the
host itself and, if possible, to produce this prior to infection
so that accidental contamination with the pathogen will not
advance into virulent infection. Such " active immunisation "
€an often be accomplished by the injection of some harmless
preparation of the toxin or organism and so stimulating the
antibody response that the antibody concentration in the
blood-stream will remain effective for some considerable
time after immunisation. To stimulate such a response
three main types of preparation are used: (1) a " vaccine "
consisting of a suspension of organisms which have been
killed either by heat or by chemical treatment and are conse-
quently non-viable; (2) preparations of the toxin itself,
injected in minute doses at first and then in increasing doses
at intervals until a sufficient antitoxin response has been
built up; (3) toxoid preparations — if the toxins of some
organisms such as CI. ivelchii, CI. tetatium, Corynebacterium
diphtheriaef etc., are treated with a weak solution of formalde-
hyde, their chemical structure is altered in some way which
results in the destruction of their toxic nature but not of their
ability to stimulate antitoxin formation; consequently, in
these cases, it is possible to inject a^relatively large amount of
" toxoid " to stimulate a correspondingly large antibody
response without any toxic effect on the host. All of these
methods are used to combat specific infections and each has
its advantages in certain conditions, but all are dependent

CHEM. A. B. 13*

198 pathogenicity; chemotherapy

upon the antibody response being effective over a reasonably
long period, as it is undesirable to repeat the treatment
frequently.

2. Chemotherapeutic intervention

If the immunological method is ineffective or difficult, an
alternative method of therapy is to prevent the growth of
the organisms by chemical means. We must distinguish
between bactericidal agents, which actually kill the organisms,
and bacteriostatic agents which do not kill but prevent
multiplication of the bacterial cells and so allow the reticulo-
endothelial system to attack and remove the invaders. The
main chemotherapeutic agents in use to-day are:

Natural antibiotics: substances produced by micro-organisms
and which are naturally bactericidal or bacteriostatic.

1. Penicillin: a substance produced by various moulds,
particularly Penicillium notatum. It is bactericidal in very
high dilution against Gram-positive bacteria — Staph, aureus
being inhibited in vitro by 1 part penicillin in 3 x 10^ parts
water or medium. PenicilKn is exceptional in that it is non-
toxic to man so that large amounts can be injected to deal
with established or stubborn infections. One of its actions is
to prevent the assimilation of glutamic acid, and possibly
other amino-acids, needed by Gram-positive species for the
synthesis of bacterial protein (see p. 99). Sensitive organisms
continue to grow for a time after the addition of penicillin to
the medium, but the growth produces abnormally large and
distorted cells. After this short period of growth the cells
become non-viable and eventually undergo lysis. Penicillin has
no effect on the respiration of washed suspensions of Staph,
aureus. The chemistry of penicillin has received intensive
attention during recent years but there is still some doubt
about its structure. There are several substances produced
by moulds which have the properties of penicillin and
differ in the chemical structure of the group R in the
following formulae.

PENICILLIN AND STREPTOMYCIN 199

CH,

CH — COOH

S^ N
\ / \
HC C = 0
\ /
CH
I

NH
I

CO
I

R R = ^_J>-CH2- (Penicillin l)

= CH3CH2CH = CH-CH2- (Penicillinn)
Formula of Penicillin.

2. Streptomycin: a substance excreted by the mould
Streptomyces griseus and active against both Gram-positive and
Gram-negative bacteria. Its use in medicine is restricted since
many pathogenic organisms rapidly acquire resistance against
it. Streptomycin is the first antibiotic to be effective against the
tubercle organism in vivo and it has been of great use in com-
bating tuberculosis. Attempts are being made to extend its
cHnical usefulness by giving it in conjunction with some other
drug such as a sulphonamide or sulphone which, by preventing
multiplication of the organisms, will also prevent acquirement
of resistance to streptomycin. The formula is given below.
Streptomycin may act as a nutritional antagonist as its
structure contains the unnatural analogue of glucosamine and
a possible analogue of inositol. Its mode of action is not yet
clear; experiments with intact sensitive cells suggest that it
interferes with some stage in the oxidation of pyruvate. The
stage affected is concerned with a reaction involving pyruvate
and oxalacetate, possibly a condensation similar to that
occurring in the citric acid cycle (see p. 153). However, it is
not known whether this cycle functions in bacteria and no
demonstration of an action of streptomycin on a cell-free
bacterial system has yet been pubhshed.

200

pathogenicity; chemotherapy

Formula of Streptomycin

H MH — CZ

/^\

HOCH HCOH

I u

HOCH C NH

HO A O H

L/CH^OH \|

(Streptidine)

N- methyl-L-glucosamine

3. Toxic peptides secreted by bacteria: B. brevis is a
spore-bearing soil organism which secretes an antibacterial
substance, called tyrothricin. This is a mixture of peptides,
the most important being Tyrocidin and Gramicidin. Tyro-
cidin is a surface-active substance which kills both Gram-
negative and Gram-positive bacteria by dissolving lipoid
materials in their cell-walls. Gramicidin is much less toxic
and is bacteriostatic towards Gram-positive bacteria; it is
thought to act by interfering with the assimilation and meta-
boHsm of phosphate. Both tyrocidin and gramicidin are too
toxic for general clinical application, although purified grami-
cidin can be used locally in wounds. These two peptides
proved to be the forerunners of a series of similar substances
produced by bacteria, especially those of the genus Bacillus.
Of recent years, antibiotics called Polymyxin, Aerosporin,
Bacitracin, Subtilin, Bacillin, etc. have been described. They
all seem to be polypeptide in nature and contain some un-
natural D-amino-acid residues. Some are known to be cyclic
polypeptides. They are stable substances and vary consider-
ably in their antibacterial properties: bacitracin has a range
of activities very similar to that of penicillin, while polymyxin
is effective against organisms of the Gram-negative group
which are comparatively resistant to penicillin. The clinical

ANTIBIOTICS

201

application of many of these substances is still in doubt as
their use is often accompanied by damage to the kidney
tubules ; whether this is caused by toxic impurities or whether
it is a corollary of the excretion of peptide substances is not
yet known.

4. AuREOMYCiN, Chloromycetin: These substances are
organic bases formed by species of Streptomyces and have a
wide range of antibacterial activities. Their discovery is of
great importance for several reasons: first, they are more
stable than other antibiotics that can be used chemothera-
peutically; second, they are the first substances to be effective
against rickettsial and virus diseases ; and third, the structure
of Chloromycetin is relatively simple and the active substance
has been synthesised. The synthesis is not difficult and
Chloromycetin will probably be the first antibiotic which can
be produced more cheaply by chemical synthesis than by
biological production.

NO;

HCOH

HC NH-

CHCZ,

Chloromycetin

SuLPHONAMiDES : various derivatives of jo-amino-benzene-
sulphonamide are used with considerable success against
Gram-positive organisms, while some Gram-negative organ-
isms are susceptible to the action of the more active
derivatives such as sulphadiazine or sulphathiazole. Success-
ful derivatives are those in which the sulphonamido-N group
contains a substituent such as pyridine, pyrimidine, thiazole,
etc. ; the pyrimidine and thiazole derivatives have a relatively
high solubility in blood-plasma and can be used for disinfection

202 PATHOGENICrTY; CHEMOTHERAPY

of the blood-stream. SulphaguaDidine is comparatively
insoluble, is scarcely absorbed from the gut and consequently
finds use as an intestinal disinfectant. The sulphonamides
act by competing with ^-amino-benzoic acid in some essential
metabolic path in the organism (see Chap. V).

y Y s CH

HzN/ yS0^HH^ y ^ | ||

N / HzN/ ysOz — HH — C CH

SULPHANILAMiDE

SULPHATHIA20LE
/NH2
H,U{ >S02-N=C

^NH2

SULPHAGUANIDINE

Marfanil, etc. : Marfanil is ^-sulphonamido-benzylamine,
but its action appears to bear no relation to ^-amino-benzoic
acid metabolism, as it is not antagonised by this substance and
sulphonamide resistant organisms are sensitive to marfanil.
It is more effective than the sulphonamides in the presence of
pus, but is non-effective on injection, probably since it is
decomposed by the amine oxidase of body tissues. Derivatives

H^N— CH2— ^~^— SO2NH2

Marfanil

in which the amino- and amido-groups are substituted prove
effective against the Clostridia.

AcRiDiNE Derivatives: certain mono- and di-amino-
acridines, such as proflavin, acriflavin, etc., are active in high
dilution against Gram-positive bacteria and, to a less extent,
against Gram-negative organisms. They are relatively non-
toxic to man. Since acridines combine with nucleotides, it is
thought that these substances interfere with coenzyme
systems of bacteria and so block certain metabolic paths.
Their use, other than for superficial appUcation, has been

ANTIBACTERIAL AGENTS

203

largely discarded in favour of sulphonamides and penicillin.
!nHo HpNL i L iNHg

h^nI

CH3 CI

Acriflavin Proflavin

Triphenylmethane dyes : mixtures of tetra-, penta-, and
hexa-methyl-tri-amino-tri-plienyl-metliane dyes are effective
against some Gram-positive bacteria, but are also somewhat
toxic to the host. They are commonly used in burn dressings,
where their coagulant properties are of use.

,CH,

/^N'~CH3

HpN

o-^

HHCl

OxX^

^3\ /

CH,

CRYSTAL VIOLET
(CARBINOL BASE)

p-ROSANILINE

The relative effects of some of these antibacterial substances
when tested in vitro against a typically Gram-positive Staph,
aureus and Gram-negative Esch. coli are given in Table XVI.

TABLE XVI

Limiting Effective Molar Concentration of some
Antibacterial Agents

Concentrations expressed in fiM = MxlO~^

Staph, aureus

Esch. coli

fjM

Penicillin

0-03

300

Sulphathiazole

0-5

Crystal violet

0-3

Streptomycin

Aureomycin

100

Acriflavin

Sulphanilamide

All these values are subject to wide variations with the strain of the
test organism and with the nature of the growth medium.

204 PATHOGENICITY ; CHEMOTHERAPY

The effective dilution of any particular substance may vary
considerably with the nature of the medium and conditions
of test.

FOR FURTHER READING

" Microbiology," Dubos, R., Ann. Rev. Biochem., 1942,
11, 659.

" The Lecithinase Activity of CI. welchii Toxins,"
Macfarlane, M. G., and Knight, B. C. J. G., Biochem. J.,
1941, 35, 884.

" The Chemistry of Antigens and Antibodies," Marrack,
J. R., M.R.C., Special Report Series, H.M. Stationery Office.

INDEX

ACETALDEHYDE, 33, 39, 151 i
Acetic acid formation, 131-2, 136,
143, 145-7, 166

oxidation, 153

Acetoacetic acid, 145-6

— decarboxylase, 54, 79, 145
Acetobacter, 19, 113, 148-50

— xylinum, 19, 120, 149
Acetone, 29, 79, 145
Acetone-butanol fermentation,

142-5
Acetylase, 34, 103
Acetylmethylcarbinol, 19, 71, 136-8
Acetyl phosphate, 35, 55, 130, 146,

151
Acridines, 12, 202-3
Acriflavin, 202-3
Adaptive enzymes, 65-6
Adenine-nicotinamide-dinucleotide,

31
Adenine-riboflavin -dinucleotide,

32
Adenosine -tri-phosphate, 32, 55,

129-31, 151
AdenyHc acid, 32, 35, 129-31, 163
Adrenaline, 169
Aerobacter aerogenes, 20

acetvlmethylcarbinol, 136-8

differentiation, 20, 22, 137,

153

— — succinic acid, 134-5

— indologenes, 83
Aerobes, strict, 13
Aerosporin, 200
Age of culture, 75-81
Agglutination, 14
Agmatine, 168, 169
D- alanine, 94

Alanine deaminase, 67, 160
j3-alanine, 104, 109, 117, 168, 169
Alcohol dehydrogenase, 46, 73,
128, 150

— oxidation, 148-56
Aldehydes, 43, 53, 149
Aldohexose, 149
Aldolase, 125, 126, 129

Amine oxidase, 43, 181, 202
Amines, 54, 167-70
Amino-acids as H-acceptors, 166
Amino-acid assimilation, 69,99,198

— deaminases, 159-67

-decarboxylases, 33, 54, 71,

72, 79, 167-70

growth requirements, 93-100

metabolism, 155, 158-75

D-amino-acid oxidase, 39, 43
L-amino-acid oxidase, 43, 52, 161
p - amino -benzene - sulphonamide,

112
^-amino-benzoic acid, 38, 113-15
y-amino-butyric acid, 54, 168
Aminopolypeptidase, 51
8-amino -valeric acid, 166, 174
Amylase, 52, 143
Anaerobes, 13, 145
Aneurindiphosphate, 33
Antibiotics, 198-201
Antibody, 14, 121, 196
Antigen, 14, 196
Antitoxin, 188, 196
Arabinose, 64
Arginase, 171
Arginine breakdo^vn, 51, 172, 174

— decarboxylase, 54, 167-70

— dihydrolase^ 79, 172

— synthesis, 97
Asparagine, 84

Aspartase, 27, 35, 104, 162-3, 164
Aspartic acid, 26, 104, 162-3, 164, 180

— decarboxylase, 168, 170
Aspergillus, 195
Assimilation, amino-acids, 69, 99,

198

— oxidative, 154
Athiorhodaceae, 87
Aureomycin, 201
Autotrophes, chemosynthetic, 84-5,

181-3

— photosynthetic, 85-7
Autotrophic heterophants, 87
Azide, 154

Azotobacter, 16, 45, 54, 92, 95, 177

205

206

INDEX

BACILLACEAE, 17, 18
BacilHn, 200
Bacillus, 18, 138, 200
Bacillus anthracis, 21, 188

— brevis, 3, 200

— mesentericus, 119

— methanicus, 87

— phlei, 162

— subtilis, 21, 22, 119, 161
Bacitracin, 200

Bacteria, chemical agents, 1-9

— description, 10

— identification, 11-23

— size, 10
Bacterial cellulose, 120

— chlorophyll, 86

— enzymes, 24-56

— pigments, 45, 86

— polysaccharides, 119-21

— vitamins, 100
Bacteriostasis, 113, 115, 198
Barbituric acid, 117
Betacoccus arabinosaceus, 64, 120
Binary fission, 10, 75, 76, 82
Biochemical mutants, 97
Biocytin, 35, 163

Biotin, 35, 89, 104, 112, 163, 165
Butadiene, 138
Butvl alcohol, 71, 123, 142-5
Butylene glycol, 123, 138
Butyric acid, 71, 142-7, 155

CALCIUM, 83, 92
Caproic acid, 146-7, 155
Capsules, 119, 120-2, 188
Carbohydrate, growth effect, 67-8
Carbon dioxide, arginine dihydro-
lase, 172

— autotrophic requirements, 83-7,

— fixation, 104, 134-5, 139, 156

— heterotrophic requirements,

107, 155
Carbonic anhydrase, 34
Carboxylase, 33, 53, 140
Carboxy poly peptidase, 51
Casei factor, 89

Catalase, 34, 43, 46, 72, 83, 105
Cell-division, 75
Cell-size, 76

Cell-wall, 200

■ — permeabihty, 62, 99

Ceilobiose, 52, 120

Cellulase, 52

Cellulobacillus myxogenes, 52

CeUulose, 4, 52, 120

Cheese, 188

Chemosynthetic autotrophes, 83-5,

181-2
Chemotherapeutic agents, 115,

198-203
Chitin, 4

Chloromycetin, 201
ChlorophyU, 10, 86
ChoHne, 35, 103
Chromobacteria, 176, 183
Chromosome, 59
Citric acid, 35, 153
Citrulhne, 94
Classification, 15-23, 59
Clostridia, 18, 142-7, 155-6, 165-7,

169-70, 174
Clostridium acetobutylicum, 21

acetoacetic decarboxylase

53, 145

amylase, 52, 143

fermentation, 142-5

growth factors, 107, 108, 113

starch hydrolysis, 52

— aerofoetidutn, 170

— botulinum, 21, 165, 194

— butylicum, 145

— histolyticum, 157, 190, 192

— kluyverii, 145, 155

— oedematiens, 190

— pastor ianum, 92, 177

— septicum, 169, 170, 190

— sporogenes, 108, 157, 165-7, 174,

190, 192

— tetanomorphum, 167

— tetanum, 21, 22, 45, 196

— welchii, 21

amino-acid metabolism, 174

gas gangrene, 189-95

histidine decarboxylase, 169,

170, 191

hydrogenase, 182-3

life cycle, 186, 190

nitrate reduction, 182-3

starch breakdown, 52

toxins, 193

INDEX

207

Clostridium welchii, toxoid, 197
Coaspartase, 104, 163
Cobalt, 83
Cocarboxylase, 33
Coenzyme A, 34-5, 103, 153
Coenzyme I, formula, 3, 31

— synthesis, 3, 100

— systems, 42, 46-8
Coenzyme II, 32, 42, 46
Coenzymes, 31-5
Colony form, 12, 19
Commensal, 186
Compensatory enzyme formation,

72-3
Competitive inhibition, 37-8, 114
Constitutive enzymes, 65
Copper, 34, 83
Corynebacterium diphtheriae, 20,

197
Crystal violet, 12, 203
Cultural characteristics, 12
Cystathionine, 98
Cysteine, 49, 98, 105, 165, 174

— desulphurase, 165
Cytochrome, 44-6, 150-2

— reductase, 33, 41

DEAMINASE formation, aero-
biosis effect, 71

age of culture effect, 78

glucose effect, 68

|)H effect, 71-2

Deamination, 159-67

— anaerobes, 165-7

— dehydration, 164

— desaturation, 162

— effect of pH, 159

— hydrolytic, 163

— oxidative, 160

— reductive, 162
Decarboxylation, 6, 52-4, 79, 159,

167-70
Dehydration, 5, 49
Dehydrogenases, 39-43, 44, 46, 48
Denitrification, 183
Dephosphorylation, 6, 55-6
Depressor drugs, 169
Desoxyribonucleic acid, 61
Desulphovibrio desulphuricans, 20
Diacetyl, 123, 138

Diamines, 169
Dihydroxvacetone, 125-6, 140-1,

ISO"'
Dihydroxyphenylalanine, 168
Dinucleotides, 31-2
Diphosphoglyceric acid, 125-6,

130
Disintegration of cells, 29-30
Drug resistance, 63

EBERTHELLA typhosa, 21, 22,

62, 95, 99, 107, 108
Emmentaler cheese, 188
Energy, fermentation, 129-31

— Methanobacter, 154-5

— oxidation, 148-56

— stores, 121, 131
Energy-rich bonds, 32, 55, 129
Enolase, 125, 127, 128, 129
Enterobacteriaceae, 16-18
Environmental ^H effect, 71-5
Enzyme formation, 58-84

actual constitution, 63

adaptive, 64-6

age of culture effect, 76-80

constitutive, 65

glucose effect, 66-70

- — • — mutation, 60-3

— — pH effect, 67, 71-2

potential constitution, 59-64

precursor, 69

salt requirement, 83

suppression, 69

temperature effect, 74-5

Enzymes, 24-56

— cell-free, 28-30, 146

— effective activity, 72-4

— extracellular, 29, 50, 79, 157,

191, 195-6

— fermentation, 124-9

— nature of, 24, 30

— optimum pH, 71-3

— potential activity, 71-3

— specificity, 25-6, 36-7

-— substrate combination, 36

— toxins as, 194-6
Escherichia coli, 20, 38
acetic oxidation, 153

alanine deaminase, 67, 72,

160

208

INDEX

Escherichia coli, alcohol dehydro-
genase, 46, 73

■ amino-acid deaminases, 160,

165

decarboxylases, 71-2, 75,

170

aspartase, 162-3

carbon dioxide requirements,

107

citric acid formation, 35

cysteine desulphurase, 165

• cytochrome, 44-5

fermentation, 128-9, 131-6,

140-50

formic dehydrogenase, 44

glutamic acid dehydrogenase,

47, 52, 160

growth requirements, 69, 108

histidine breakdown, 163

• hydrogenase, 48, 74, 183

• indole formation, 172-4

iron requirements, 83

lactic acid formation, 133

— — ■ mahc dehydrogenase, 46

mutabile, 61-2

mutants, 94

nitratase, 48, 183

■ rate of division, 8, 82

serine dehydrase, 49, 71, 164

strain differences, 21-2

• transaminase, 95

-tryptophan metabolism,

172-4
Essential metabohtes, 82, 114
Ethyl alcohol, 39, 123, 140-1, 145,

155
Euhacteriales, 16, 17

FACULATIVE anaerobes, 13, 163
Families, 15
Fats, 4, 48

Fatty acids, 48, 87, 145-7, 156, 162
Fermentation, adaptive nature,
64-6

— Aerobacter, 136-8

— CI. acetobutylicum., 142-5

— Esch. coli, 131-6

— glucose, 123-45

— Propionibacteria, 138-9

— Schemes, 125, 139, 142

Fermentation, "viscous," 119
Fertilisation of soil, 87
Fission-fungi, 15
Flavine-adenine-dinucleotide, 32,

41
Fluoride, 128, 138, 141
Folic acid, 89, 103, 110, 112
Foot-and-mouth virus, 10
Formic acid, 28, 131-2

— — ■ dehydrogenase, 44, 72, 83,

183

— hydrogenlyase, 83, 132
Fructosan, 119
Fructose, 20, 87
Fructosediphosphate, 128
Fructose-6-phosphate, 126
Fumarase, 26, 135, 162-3
Fumaric acid, 26, 93, 135, 162-3

GALACTOSE, 64, 69
Galactozymase, 65, 69
Gas gangrene, 167, 189-95
Gel, polysaccharide, 119
Gelatinase, 50
Gelatine, 50, 84
Gene, 59, 96
Genera, 17-18
Genetic constitution, 59-63
Gluconic acid, 149
Glucosamine, 199
Glucosan, 120

Glucose, effect on enzyme forma-
tion, 66, 69

— fermentation, 123-45

— oxidation, 64

— protein-sparing action, 66
Glucose-6-phosphate, 126-9
Glucozymase, 68, 69, 74
Glucuronic acid, 121, 192
Glutamic acid, assimilation, 69,

99, 198

deaminase, 68, 160, 161

decarboxylase, 54, 160,

167-70
dehydrogenase, 47, 52, 93,

161

^polypeptide in capsules, 188

synthesis, 161

Glyceraldehyde-phosphate, 126-9,
133, 140, 153

INDEX

209

Glycerol, 150
Glycerophosphate, 140
Glycine, 160, 166, 174
Glycogen, 52, 87, 121, 191
Glyoxyhc acid, 160
Gonococcus, 20
Gramicidin, 200
Gram stain, 12, 22
Green sulphur bacteria, 86
Growth factors, 59, 100-6
analogues, 112-15

— phases, 75, 81
Guanidine, 136, 169
Gums, 119

HAEMATIN, 34, 43, 105, 181
Haemoglobin, 34, 181
Haemolysin, 20, 193
Haemophilus influenzae, 105, 164

— parainfluenza e, 70, 100, 108
Heavy-carbon, 134, 135, 139, 145,

146
Heterotrophic bacteria, 89-107
Hexokinase, 55, 124
Hexosediphosphate, 54, 124-6, 129,

141
Histamine, 168, 169, 173, 201
Histidine assimilation, 69

— decarboxylase, 168-70
Homocysteine, 98
Homolactic fermentation, 123, 172
Homoserine, 98

Hyaluronic acid, 192
Hyaluronidase, 192-3
Hydrocarbons, 4, 5, 87
Hydroclastic split, 132
Hydrogenase, 43, 64, 69, 71, 113
Hydrogen carrier, 31, 38-48

— formation, 132, 146, 155, 167

— oxidation, 4

— peroxide, 43, 45, 105

— sulphide, 20, 86, 165
Hydrolysis, 6, 106, 146-8
jS-hydroxy butyric acid, 146
Hydroxy lamine, 48, 87, 176,

179-80, 183
Hypoxanthine, 89

IDENTIFICATION, 11-14, 22,

58, 86

Immunisation, 196-8

Immunological specificity, 120-1

Indole, 19, 98, 172, 174

Indole-propionic acid, 173, 174

Inhibition, competitive, 37, 114

Initial stationary phase, 75

Inoculum, 76, 107

Inorganic salts, 83

Inositol, 199

Insulin, 105

Internal environment, 8, 9, 69, 72,

99
Intestinal flora, 17, 19
Invasiveness, 189, 191-3
Iron, 34, 83, 84, 92, 133
— bacteria, 84
Isoleucine, 97
Isomerase, 126, 129
Isopropyl alcohol, 123, 145
Isotopic-carbon, 134, 135, 139,

145, 146

KERATIN, 4

a-ketoglutaric acid, 93-5, 159, 160,

174
Krebs cycle, 153

LACTASE, 62

Lactic acid, 125, 127, 133

LactobaciUi, 15, 17, 94, 103, 107,

112, 113, 123
Lactobacillus acidophilus, 45

— casei, 20, 108

— delbreuckii, 55, 151

— plantarum, 103
Lactobacteriaceae, 15, 17, 105, 113
Lag phase, 75

Lancefield groups, 14
Lecithinase, 194
Leguminous plants, 177
Leptothrix ochracea, 84
Leucine, 50, 161, 166
Leuconostoc dextranicus, 120
Levan, 119
Light energy, 85
Linked oxido-reduction, 47, 85,

127, 133
Lipoids, 200
Liquefaction of tissues, 200, 201

210

INDEX

Lock and key analogy, 37
Logarithmic growth phase, 75
Lysine, 51, 97, 99, 168, 174
— decarboxylase, 36, 167-8

MAGNESIUM, 34, 83, 86, 165
Mahc dehydrogenase, 26, 46, 101
Malonic acid, 37, 38
Maltase, 52, 143
Maltose, 51, 64
Manganese, 165
Marfanil, 202

Maximum stationary phase, 75
Membrane equilibria, 83
Meningococcus, 20
Metabolites, essential, 82, 114
Methane formation, 48, 154-5

— oxidation, 87
Metkanobacter, 48, 154

— omeliansJci, 154
Methionine, 97, 98, 105, 161
Methyl alcohol, 155

— red test, 137
Microaerophilic bacteria, 13
Microbiological assay, 110-12
Micrococcus lysodeikticus, 30, 54

— pyogenes, 20
Milk, 20

Molasses, 105, 143, 177
Molybdenum, 83, 84, 92
Morphology, 11
Mud organisms, 86
Multiplication rate, 8, 75
Mustard gas, 61
Mutation, 23, 59-63, 104
Mycobacterium tuberculosis, 45

N-ACETYL-GLUCOSAMINE, 192
Natural antibiotics, 198-201
Nature of bacteria, 10-23
Neisseria gonorrhoeae, 20

— intracellularis, 20
Neurospora, 59, 95

— p-amino-benzoic-less mutant,

113

— genetic constitution, 59, 61

— mutants, 59, 61, 96-8

— tryptophan synthesis, 98

Neutrahsation mechanisms, 9,

71-2, 110, 137, 144
Nicotinamide, 58, 89, 100, 116
Nicotinamide-nucleoside, 100
Nicotinic acid, 100, 103, 110, 111,

113, 117
Nitratase, 48, 183
Nitrate reduction, 182-3
Nitrification, 84, 181, 185
Nitrobacter, 19, 84, 176, 182
Nitrogen, atmospheric, 89, 176-81
— fixation, 89, 176-81
Nitrogen-cycle, 176-85
Nitrophenol, 154
Nitrosomonas, 19, 86, 176, 182
Nodules, 177-81
Non-exacting heterotrophes, 92
Nucleic acid, 81
Nutrient salt mixture, 83
Nutrition, 36, 83-110
Nutritional antagonism, 112-15

OPTICAL specificity, 25
Optimum pYL, 24, 72

— temperature, 74
Orders, 15

Ornithine, 36, 97, 166, 171-3

— decarboxylase, 54, 168-70
Oxalacetic acid, 26, 27, 46, 94-6,

104, 135, 153, 179-80

— decarboxylase, 27, 54, 134-5
Oxidase systems, 42-3
Oxidation mechanisms, 5, 38-48,

58
Oxidative assimilation, 154

— deamination, 160

— energy, 55, 148, 156

— phosphorylation, 153
Oximo-succinic acid, 180
Oxygen requirement, 13

PANTOTHENIC acid, 35, 89, 103,

104, 108, 109, 112
Pantoyl-taurine, 97, 99, 115, 116
Pararosaniline, 12, 203
Parasitic existence, 74, 106, 109,

110
Parasympatheticomimetic drugs,

169

INDEX

211

Pathogenicity, 186-96

Pathogens, 20, 110, 115, 186

PeniciUin, 12, 198-9

Penicillium, 95, 97, 198

Pepsin, 51

Peptidase, 50

Peptidases, 50-1, 157

Peptide, ^-amino-benzoic acid, 113

PermeabiUty, 29, 62, 79

Peroxidase, 34

^H effect on enzymes, 71-4, 159,
163

• — growth limits, 71

Phagocytosis, 186

Phases, growth, 75

Phenylalanine, 51, 161, 166

Phosphatase, 34, 83

Phosphocholine, 194

Phosphoclastic split, 132

Phosphoglyceric acid, 39, 125-7,
128-31, 133, 141

Phosphoglyceromutase, 126-7

Phosphohexokinase, 125, 127

Phosphohexose-isomerase, 125, 126

Phosphopyruvic acid, 55, 128-9

Phosphorylation, 6, 32, 55-6,
129-31

Photosynthetic autotrophes, 85-7

Physico-chemical growth con-
ditions, 70-5

Pigments, 45, 86

Pimehc acid, 89, 104

Plating, 12

Pneumococcus, capsules, 60-1, 95,
120, 188

— hydrogen peroxide sensitivity,

— Type interchange, 61
Polar groups, 36, 37, 54, 167
Polymyxin, 200
Polyphenol oxidase, 34, 83
Polysaccharides, 49, 50, 60, 68,

119-21, 188
Positional isomerism, 2-3
Potential activity, 72-4
Pressor drugs, 169
Proflavin, 203
ProHne, 166, 174

Propionibacteria, 7, 107, 134, 138-9
Propionic acid, 7, 87, 138-9
Propyl alcohol, 123, 145

Prosthetic groups, 31-5
Proteases, 25, 49-51, 79, 157
Protective mechanisms, 72-3
Protein breakdown, 67, 157-8
Proteins, 3, 49, 82, 93, 99, 157-8,

181
Proteus fluorescens liquefacienSf 164

— morganii, 108

— vulgaris, 20, 195

amino-acid decarboxylases,

180

— oxidase, 43, 161

colony form, 13

— — growth requirements, 1 00,

108, 111

identification, 22

Pseudomonas, 95, 138, 158, 181

— pyocyanea, 19, 45, 161
Purines, 80, 105

Purple sulphur bacteria, 86
Putrescine, 168, 169, 174
Pyocyanine, 19, 45
Pyridine-3-sulphonic acid, 114, 116
Pyridoxal, 89, 101-3, 109

— phosphate, 33, 54, 95, 103, 169
Pyridoxamine phosphate, 33
Pyridoxin, 89, 94, 101-3, 112
Pyrithiamin, 115, 116
Pyrophosphatase, 83
Pyrophosphate bond, 130
Pyruvic acid, carboxylase, 53, 128,

140
fermentation by Aerobacter,

136-8

Esch. coli, 131-6

Propionibacteria, 138-9

formation from glucose,

124-9
metabohsm, general, 98, 101,

104, 123, 153

oxidase, 55, 151

transamination, 93-5

RACEMASE, 95

Rate of multiphcation, 8, 75, 106

Reaction chains, 26, 27, 97

Reduction, 38

— deamination, 162

Respiratory activities, 2

Reticulo-endothelial system, 186-9

212

INDEX

Rhizobaceae, 92
Rhizobium, 170, 176, 178-81

— leguminosarum, 170, 179

— trifolium, 179
Riboflavin, 32, 103, 112, 116
Riboflavin-phosphate, 33, 41
Rickettsia, 10

Root nodules, 177-81
Rough variation, 60-1
Rubber, sj'nthetic, 138

SA CCHA ROM YCE8 cerevisiae,

140
Salicin, 18, 22
Salmonella, 21
Saprophytes, 186
Scavenging action, 4, 186
Schizomycetes, 15
Serine, 49, 98, 164-5, 173, 174

— dehydrase, 49, 104, 164-5, 174
Serological characteristics, 14, 23
Sewage purification, 184-5
Shigella, 21, 95

Shock, 169

Sihcic acid gel, 84, 182

Shmes, 119

Soil fertihsation, 90-2, 176-81

— organisms, 19, 20, 75, 84-7, 106,

176
Solid media, 84, 182
Sorbitol, 150
Sorbose, 150

Species, 18-21, 58, 157, 158
Specificity, enzymes, 25-8, 36,

49-51, 161

— immunological, 119, 120-2
Spores, 11, 17, 22, 59, 190
Staining reactions, 12
Staphylococcus, 20, 30, 95, 99, 171

— aureus, 13, 20, 22, 45, 63, 101,

103, 107, 108, 109, 117
Starch, 143

Stationary growth phases, 75
Stickland reaction, 155, 165-7
Strains, 21
Strepogenin, 105
Streptidine, 200
Streptococci, 15, 17, 18, 79, 95, 99,

101, 123, 171, 174
Streptococcus faecalis, 20, 45, 95

Streptococcus haemolyticus, 14, 20,
22, 106, 108

— lactis, 20, 112
Streptomyces, 199, 201
Streptomycin, 63, 199
Subtilin, 200

Succinic acid, 25, 38, 134-6, 163

— dehydrogenase, 25, 74, 134-6,

153, 163
Sucrose, 64, 119-21
Sulphate, 20, 97
Sulphonamides, 34, 38, 63, 103,

105, 112-15, 199, 201-2
Sulphur, 4, 85, 86

— bacteria, 86-7
Sulphuric acid, 4, 85, 86
Supersonic vibration, 30
Sympatheticomimetic drugs, 169

TEMPERATURE, growth, 74

Therapv, 196-203

ThermolabiHty, 25

Thiamin, 101, 103, 112, 116

Thiamindiphosphate, 33, 53, 151

Thiobacillus thio-oxidans, 19, 85

Thiorhodaceae, 86

Thymine, 105

Toluene, 29

Toxins, description, 188

— CI. welchii, 189-91

— nature of, 195-6
Toxoid, 197

Transaminase, 26, 33, 93-5, 102, 180
Transforming principle, 60
Tribes, 17

Triosephosphate, 124-7, 133
Triphenylmethane dyes, 12, 203
Trypsin, 51

Tryptophan breakdown, 68, 166,
168

— synthesis, 62, 97, 98
Tubercle, 14, 199

Type specific polysaccharides, 120
Typhoid organisrn, 22, 99, 107, 108
Tvramine, 168-9, 174
Tyrocidin, 200
Tyrosine, 51

— decarboxylase, 54, 79, 102,

168-70
Tyrothricin, 3, 200

INDEX

213

ULTRA-VIOLET

graphy, 80
Uracil, 89, 117
Urea, 84

spectrophoto-

Urease,

171

Uric acid oxidase, 43
Urocanic acid, 163

VACCINE, 197

Valine, 97, 166

Vanadium, 83

Viable cells, 75

Vibration, 30

Vinegar vats, 149

Virulence, 188-9

Virus, 10

"Viscous fermentation," 119

Vitamin B^, 115

Vitamin C synthesis, 150

Vitamins, bacterial, 100

Voges-Proskauer test, 22, 136-7

WASHED suspension, 28, 29
Wood-Werkman scheme, 135
Wound infection, 167, 189-95

X-RAY mutants, 59, 61, 95, 96
Xylose, 19

YEAST, 59, 69

— amino-acid synthesis, 70

— carboxylase, 53, 128

— extracts, 35, 100, 113

— fermentation, 54, 114-17, 140

— oxidation mechanisms, 38-48

— size, 10

ZINC, 34, 83, 165

Zymohexase, 125, 126, 129

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