MOLECULAR ARCHITECTURE OF THE KYPHAL WALL IN THE
WATER MOLD, ACELYA AMBISEXUALIS RARER
BY
JULIA BARTH REISKIND
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNI^/ERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1980
I would like to dedicate this dissertation to the memory of
John R, Raper, who initiated physiological studies of Aehlya by his
pioneering research on the hormonal aspects of mating, and who,
years later, stimulated my interest in fungal physiology and
genetics-.
ACKNOWLEDGEMENTS
I would like to thank Drs. Mildred M. Griffith, Chesley B. Hall,
Thomas E. Humphreys, and Paul H. Smith for serving as members of my
supervisory committee. I appreciate their availability and advice during
the course of this research.
I would also like to thank Drs. R. Michael Roberts and S. M.
Mahaboob Basha for the use of their facilities for the gas chromatographic
studies and for their advice on this aspect of the research. Appreciation
also goes to Dr. Arnold S. Bleiweis and Mr. Steven F. Hurst for their
assistance and facilities in the amino acid analyses, to Drs. Henry C.
Aldrich and Gregory W. Erdos for their help and equipment in the ultra-
structural studies, to Dr. Christine E. Carty for the techniques of
lipid extraction and analyses, and to Drs. Stephen G. Zam and Francis C.
Davis for their consultation throughout this study. Thanks also go to
Mr. Charles K. Cottingham for the preparation of cellulase from Aahlya
ambisexualis , to Dr. Michael LaBarbera for the use of the polarizing
light microscope, to Dr. Lewis Berner for aid in microphotography, and
to Dr. Jerome M. Aronson who did the x-ray diffraction analyses.
I would like especially to thank the chairman of my supervisory
committee. Dr. J. Thomas Mullins, for his support and advice throughout
this research. This study could not have been completed, much less
started, without his initial definition of the problem and subsequent
assistance.
Final thanks go to my husband, Jon, for his time, patience and
understanding during the long course of this task. I especially
appreciate his willingness to help in child care and other household
duties. I also thank my children, Alix and Michael, for their patience
during this study.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
INTRODUCTION 1
LITERATURE REVIEW 3
The Organism, Aahyla ambisearualis Raper 3
Higher Plant Cell'^Walls 5
Fungal Walls 9
Fungal Walls, Chemical Structure 12
Fungal Walls, Physical Structure 24
Fungal Walls, Morphology 25
Fungal Walls, Growth 28
MATERIALS AND METHODS 30
Organism and Culturing Techniques 30
Hyphal Wall Isolation and Purification 30
Chemical Fractionation of the Wall 31
Chemical Analyses of Wall Constituents 33
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase 41
Ultrastructural Studies 42
RESULTS 45
Criteria for Wall Purity 45
Chemical Fractionation of the Wall 45
Chemical Analyses of Wall Constituents 50
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase 78
Ultrastructural Studies 78
DISCUSSION 100
The Preparation of Wall Samples 100
Chemical Fractionation of the Wall 100
Chemical Analyses of Wall Constituents 107
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase Ill
Ultrastructural Studies 112
CONCLUSION 120
APPENDICES 122
A TECHNIQUES 123
Buffer-Water Washing of Isolated Walls 123
Chitin Isolation 123
Cellulose I Isolation 124
Preparation of Acid Swollen Cellulose 125
Enzyme Purification 125
Enzymatic Hydrolysis of Laminarin 126
Hydrolysis of the Unfractionated Wall with H2SO4 127
Description of Analyses Used for the Detection of
Neutral Sugars 127
Solubility Analysis of the Hexosamine Component of the Wall . 134
Uronic Acid Analysis 135
Lipid Extraction and Analysis 135
Phosphorus Analysis 136
Ultrastructural Studies - Thin Section 136
B RECIPES 137
Growth Media for A. amb-isexualis 137
Cadoxen Reagent 137
Schweitzer's Reagent , . 138
Anthrone Reagent 133
Glucostat Test 139
Cellulase Viscometric Assay . 139
DMAB Assay 140
Folin Test 141
BioRad Protein Assay 141
Lipase Assay , . . . . 141
Carbazole Test 142
Fiske-Subbarow Assay 143
C PERIPHERAL STUDIES 144
Dry Weight Determination of Washed Mycelium 144
GLC Analyses of H2SO4 Hydrolysates of Unfractionated Walls. . 144
Congo Red Stain of the Wall and its Fractions 144
Wall Width as Measured from Thin Section Micrographs 146
Observations of Enzymatically Treated Material by
Phase Microscopy 146
Observations of Hyphal Branching by Polarizing Light
Microscopy 149
REFERENCES , . 150
BIOGRAPHICAL SKETCH 163
vi
LIST OF TABLES
Table
1 Conditions of enzyme hydrolysis 35
2 Carbohydrate fractions of A. amb-isexualis wall 49
3 The separation of enzyme hydrolysates of wall fractions
of A. ambisexualis by paper chromatography 51
4 The separation of acid hydrolysates of wall fractions of
A. ambisexualis by paper chromatography 52
5 Periodate consumption and formate liberation of
A. ambisexualis ■wall fractions 66
6 Periodate consumption and formate liberation of
known polysaccharides 67
7 X-ray diffraction analysis of Schweitzer's and cadoxen
reagent-soluble fractions of A. ambisexualis wall 68
8 Analysis of solubility of glucosamine from
unfractionated walls of A. ambiseo::ualis 72
9 Amino acid profile of the total wall of A. ambisexualis
after chemical or buffer-water cleaning 73
10 Comparison of amino acid profiles of samples of the
total wall of A. ambisexualis during various stages
of chemical cleaning 75
11 Chemical constituents of the buffer-water washed
walls of A. ambisexualis 79
12 Microfibrillar diameter of various preparations from
A. ambisexualis 99
LIST OF FIGURES
Figure
1 Phase contrast photographs of cleaned walls 47
2 Decrease in total protein as a measure of wall purity ... 48
3 GLC of the IMS derivatives of the monosaccharides released
by hydrolysis of the wall fractions or the total wall by
unpurified A. nigev cellulase 55
4 GLC of the TMS derivatives of the mono- and disaccharides
released by acid hydrolysis of wall fractions and total
wall 58
5 GLC of the TMS derivatives of the mono- and disaccharides
released by hydrolysis of the wall fractions or the total
wall by laminarinase 61
6 GLC of the TMS derivatives of the monosaccharides released
by hydrolysis of the total wall with laminarinase and
unpurified A. nigev cellulase 64
7 X-ray diffraction patterns of cellulose II isolated from
A., amb-isexualis walls 70
8 Comparison of amino acid profiles of samples of the total
wall of A. amb-isexual-is during various stages of chemical
cleaning 77
9 Increase in reducing sugars of isolated wall after
hydrolysis by A., amhisexualis cellulase (uncorrected
data) 81
10 Increase in reducing sugars of isolated wall after
hydrolysis by A. ambisexualis cellulase 83
11 Surface replicas of isolated walls treated chemically ... 85
12 Surface replicas of wall fractions 88
13 Surface replicas of live hyphae after chemical treatment. . 90
14 Surface replicas of live hyphae after single enzyme
treatment 93
15 Surface replicas of live hyphae after sequential
enzyme treatment 95
16 Surface replicas of live hyphae after sequential
enzyme treatment 97
17 Molecular model of the carbohydrate portion of the
hyphal wall 106
18 Scheme for explaining the apparent increase in micro-
fibrillar width as a result of enzymatic or chemical
treatment 117
19 Diagrammatical representation of the hyphal wall based
on ultrastructural evidence 119
20 Periodate and iodate oxidation blanks 131
21 Periodate consumption of the wall fractions and the
total wall 132
22 Formate liberation of the wall fractions and the total
wall 133
23 GLC of the TMS derivatives of the monosaccharides
released by H2SO4 hydrolysis of the unf ractionated
wall 145
24 Apical and subapical sections of an k. omb-isesnMztis
hypha 148
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
MOLECULAR ARCHITECTURE OF THE HYPHAL WALL IN THE
WATER MOLD, ACELIA AMBISEXUALIS RAPER
By
Julia Barth Reiskind
June 1980
Chairman: J, Thomas Mullins
Major Department: Botany
In order to elucidate the molecular architecture of the hyphal wall
in Aahlya ambisexualis Raper both chemical and morphological analyses
were done. Isolated cleaned walls were fractionated chemically. Acid
and enzyme hydrolysates of the resulting polymers or of the unfractionated
wall were analyzed for their neutral sugar content and for their pattern
of linkage and branching. Glucose was the only monosaccharide found, but
three disaccharides were detected, laminaribiose, gentiobiose, and cello-
biose, indicating the presence of 31,3; 31,6; and 31,4 linkages. Close
to 40% of the wall was found to consist of acid-soluble glucans of 31,3
linkages with single 31,6 linked glucose units every fifth monomer. A
much lower percentage (7%) of the wall was soluble in alkali following
the acid treatment. The structure of this fraction was determined to be
a linear polymer of 31,3 and 31,4 linkages with occasional 31,6 side
chains. About 20% of the wall was solubilized by known cellulose solvents
and was considered to be cellulose II based on x-ray diffraction analysis.
Nearly 6% carbohydrate remained after these treatments. This insoluble
residuum was found to have a linkage pattern similar to the alkali-soluble
fraction. Almost 3% of the wall was found to be glucosamine, most of
which was in an insoluble form. After additional analysis it was
concluded that this component was a weakly acetylated chitin. A 10%
protein component was found in the wall, and amino acid analysis revealed
the total spectrum of amino acids including hydroxyproline. Very small
amounts of uronic acids and phosphorus were found, but virtually no
lipid was detected.
Ultrastructural analyses of carbon-platinum surface replicas of
hyphae treated either chemically or enzymatically , of isolated walls
treated chemically, and of various wall fractions were performed. Both
laminarinase, laminar inase plus protease, and acid plus alkali treatments
removed the acid- and alkali-soluble glucans and revealed the underlying
microfibrils of cellulose. The addition of cellulase to the laminarinase
plus protease mixture resulted in virtual dissolution of the hyphae.
Cadoxen, following acid and alkali treatments, resulted in almost total
removal of the microfibrillar pattern. Observations of the surfaces of
the various wall fractions indicated that the acid-soluble phase was
amorphous, the alkali-soluble and insoluble residuum both faintly micro-
fibrillar, and the cellulose II strongly microfibrillar. The cellulose I
and chitin fractions were both uniformly microfibrillar. Morphologically,
the hyphal wall of A. cmbisexualis is similar to that of other Phycomycetes.
Basically, the wall consists of an outer amorphous portion of Bl,3 and
31,6 glucans which covers an inner microfibrillar component.
From these studies two models of the wall were designed. One is
a molecular model which attempts to describe the molecular architecture
of the wall. The other model is a diagram of the various wall components
based upon both chemical and ultrastructural studies.
INTRODUCTION
Hyphal walls in fungi, as in other organisms, provide an invalu-
able function. In addition to protecting the protoplast from environ-
mental damage, they also, by their rigid nature, aid the organism in
maintenance of its characteristic morphology. Although rigid, walls are
also pliant allowing the necessary morphological changes which occur with
growth. They also play a role in cellular recognition between different
organisms, as in host-parasite interactions and immune responses. Walls
are considered to be an integral part of the living system (Preston 1979),
perhaps as a single macromolecular entity (Keegstra et al. 1973). A
number of studies have been made in an attempt to elucidate the molecular
structure of the cell walls of various organisms. The common pattern
which emerges is that of a rigid fibrillar structure embedded in and
covered by an amorphous matrix. Pores, perhaps proteinaceous in nature
(Wrathall and latum 1973), are thought to exist in the wall and to allow
the passage of macromolecules (Farkas 1979).
In this study, the hyphal wall of the water mold Achlya ambisexualis
Raper was analyzed both chemically and ultrastructurally, and a model of
its molecular structure is proposed. Previous research on this organism
has indicated a correlation between lateral branching and increased
production and secretion of the enzyme cellulase (Thomas and Mullins
1969) . It has been theorized that localized hydrolytic action by this
enzyme "softens" or restructures the wall allowing branch initiation to
occur (Thomas and Mullins 1969). A logical step in the study of the role
1
of this enzyme in branching and growth is an in-depth analysis of the
"substrate," i.e., the hyphal wall.
One question which this study attempted to answer is "What are the
bonds of interest in the hyphal wall?" What aspect or aspects of the
wall are responsible for its integrity? Early observations that lysis
of A. ambisexualis hyphae occurred within two hours following the
application of a cellulase led to the suggestion that the structural
integrity of the hyphal wall resides directly in the cellulosic
component or indirectly between the cellulosic and another component
(Mullins 1979). This is in sharp contrast to Preston (1974a) in his
analyses of higher plant walls, who states that the matrix portion of
the wall is the key to its integrity. Another feeling, however, is that
both components are necessary for the integrity of fungal walls (Bartnicki-
Garcia and Lippman 1967; Sietsma et al. 1968, 1969; Hunsley and Burnett
1970; Tokunaga and Bartnicki-Garcia 1971). Whatever the case, the two
functions of the wall, maintenance of hyphal morphology and plasticity
to allow growth, must be borne in mind in the consideration of its
molecular structure. One further point is the observation of Hunsley
and Burnett (1970) of a more loosely arranged fibrillar structure at
growing hyphal apices than in more distal or nongrowing regions. As
branching occurs subapically, the conformation of the wall must be
changed in order for this to occur and it may be here that cellulase or
related hydrolytic enzymes play a key role.
LITERATURE REVIEW
The Organism, Aohlya ambisexualis Raper
Aahlya ambisexualis Raper belongs to the Class Phycomycetes,
Series Bif lagellate, Order Saprolengniales, Family Saprolegniaceae
(Alexopoulos 1952). An alteimate classification divides the Phycomycetes
into two subclasses, one of which is the Oomycetes which have differen-
tiated gametangia and to which Achlya belongs (Alexopoulos 1952). One
of the distinguishing characteristics of the Saprolegniales is the
possession of cellulosic cell walls, a situation not commonly found in
the fungi. Fungi of the genus Achlya inhabit fresh water and generally
form colonies around pieces of decaying plant and animal material. A.ahlya
ambisexualis is filamentous and is made up of coenocytic hyphae surrounded
by a rigid wall. A mass of hyphae is termed a mycelium. Septa, complete
plates in this fungus, are formed only at the base of the reproductive
structures or are sometimes found in aging mycelia. Vegetative growth
occurs at the hyphal apices, also the area of sporangial formation.
Sporangial spores are motile and biflagellate, one flagellum of the
whiplash type and the other of the tinsel type. Sexual reproduction is
by gametangial contact in which there is tranfer of male gametes produced
in an antheridium to female gametes or oospheres produced in a oogonium
via fertilization tubes (Alexopoulos 1952),
Sexual reproduction is initiated and sequentially controlled by a
series of diffusible hormones. Raper (1939a, b; 1940) first described
3
this hormonal mechanism in Aahyla. The sequence is described as follows:
(1) vegetative female hyphae secrete hormone A into the growth medium;
(2) this hormone is taken up by the male hyphae and the result is the
production of numerous lateral branches, termed antheridial hyphae; (3)
the induced male now secretes hormone B, (4) which is taken up by the
female and causes the induction of oogonial initials; (5) two additional
hormones, C and D, were postulated to be involved in the attraction and
appression of the antheridia to and onto the oogonium; (6) this is followed
by septal delimitation at the antheridial tip and at the base of the
oogonium to form the functional sexual organs. These two cells are the
site of meiosis and gametogenesis. Fertilization takes place with the
formation and inward growth of fertilization tubes from the appressed
antheridia into the oogonium. The species name, A. ambisexwilis, was
chosen by Raper (1939a) to emphasize the presence of a wide range of
sexual capacities among the various isolates, i.e., pure female or pure
male, or either male and female depending upon its mating partner.
Hormone A was chemically characterized by McMorris and Barksdale
(1967) and renamed antheridiol. Several structures were proposed
(Arsenault et at. 1968) and two isomers of one of the proposed structures
were synthesized, one of which resembled natural antheridiol in its
physical and biological properties (Edwards et al . 1969). Hormone B
was similarly chemically characterized and renamed oogoniol by McMorris
et at. (1975) . Both antheridiol and oogoniol are steroids and are the
best characterized sex hormones of this structure found in the plant
kingdom.
Strain E 87, a pure male, was used in this research. The response
by this strain to antheridiol has been examined biochemically. Both
antheridial branching and an increase in the secretion of cellulase into
the wall and its subsequent release into the medium have been found as a
result of antheridiol treatment. The role of cellulase has been postulated
to cause localized wall softening allowing antheridial branching to occur
(Thomas and Mullins 1969). Observations of lateral wall thinning at the
sites of branch initiation gave support to the wall softening theory
(Mullins and Ellis 1974) . RNA and protein synthesis have been found to
be required for both branching and cellulase production (Kane et at.
1973; Horowitz and Russell 1974; Timberlake 1976).
Higher Plant Cell Walls
Although the principal subject of this dissertation is the study of
a fungal wall, some discussion of higher plant cell wall structure and
growth is relevant. Albersheim and co-workers (Albersheim et al. 1973;
Bauer et al. 1973; Keegstra et al. 1973; Talmadge et al. 1973; Albersheim
1974) have done the most recent and major work in this area and this has
generated considerable interest (Monro et al. 1974, 1976). In terms of
carbohydrate polymers, the higher plant cell wall is more complex than
that found in fungi. The technical approach, however, for studying both
the chemical and physical aspects of walls is similar.
In general, more detailed wall analyses have been done on primary
walls rather than secondary. Primary walls are less differentiated and
occur in plant cells which are still growing. The basic cell wall
structure of a number of dicotyledonous plants has been analyzed and
found to be basically similar (Albersheim et al. 1973). Where differences
in structure occur, they are usually in the linkage, number, and types
of attached residues which act as side chains. Such differences, for
example in the hemicellulose or pectic portion of the wall, result in
changes in physical properties and thus biological function (Aspinall
1973) . Pectic substances and hemicelluloses together with cellulose
form the bulk of the primary cell wall of higher plants. The carbohydrate
polymers of the plant cell wall are: (1) cellulose; (2) hemicellulose
(xylans and glucamannans) ; (3) pectic substances [galacturonans, arabinans,
galactans and/or arabinogalactans, and rhamno galacturonans (Talmadge et
al. 1973)]; (4) glycoproteins (Aspinall 1973).
Most of these studies employed cultured cells and involved isolating
and cleaning the cell walls, followed by fractionation either by the use
of purified hydrolytic enzymes, or alkali, urea, and mild acid (Albersheim
et al. 1973; Bauer et al. 1973; Talmadge et al. 1973; Albersheim 1974).
Monosaccharides, fractionated polysaccharides, and total wall poly-
saccharides were analyzed for their monomeric structure, type of glycosidic
linkage, and anomeric configuration by a combination of gas chromatography-
mass spectrometry methylation analysis.
Analyses of an endopolygalacturonase digest indicated that the
pectic polysaccharide portion consists of a rhamnogalacturonan main
chain with side chains of arabinans and galactans. The galactan has been
postulated to serve as a bridge to the hemicellulosic portions of the
wall (Talmadge et al. 1973). The hemicellulose portion of the wall is
basically a xyloglucan poljnner. This component consists of two fragments,
one, a seven-unit sugar, and the other, a nine-unit one. In addition to
xylose and glucose, small smounts of galactose and fucose are present in
the larger fragment (Bauer et al. 1973; Albersheim 1974). It has been
speculated that covalent linkages exist between the pectic polysaccharides
and the hemicellulosic portion of the wall, while non-covalent linkages
link the cellulosic and hemicellulosic wall components (Bauer et at.
1973). The amount of xyloglucan is sufficient to cover, via the formation
of hydrogen bonds, all of the cellulose fibrils (Bauer et at. 1973).
Lamport and Northcote (1960) reported the existence of a specific
protein occurring in plant walls which contains an imino acid, hydroxy-
proline, which is usually found only in trace amounts in cytoplasmic
protein. It was suggested that this protein might be responsible for
cross linking various wall components and that wall extension might be
caused by the enzymatic reduction of disulphide bridges (Lamport 1965)
or at least by the lability of certain covalent linkages in this glyco-
protein (Lamport 1970). Structural studies of hydroxyproline-rich
glycopeptides have indicated a polypeptide backbone with oligoarabinose
side chains (Lamport 1967, 1969). Subsequently a hydroxyproline-rich
glycopeptide was isolated which contained galactose bound by the hydroxyl
group of serine, and a tentative structure was devised consisting of a
serine with an attached galactose and four hydroxyprolines each with
four arabinose molecules (Lamport 1973). There are many questions concern-
ing cell wall proteins; for example, what sugar is covalently bound, is
there more than one structural protein in the wall, and to which
component of the wall is the protein attached (Preston 1979)?
The only molecular model which will be considered in detail is that
designed by Keegstra et at. (1973) based on the chemical analyses of
sycamore cell walls. Briefly, the matrix of the cell wall includes the
pectic substances, the proteinaceous component, and the hemicellulosic
materials while the cellulosic portion makes up the microfibrillar region.
Covalent cross linkages are postulated to hold the matrix together, while
hydrogen bonds are responsible for cementing the cellulose portion and
for binding the cellulosic molecules to the xyloglucan component of the
matrix. The hydrogen bonding is so extensive that it is considered to
have strength comparable to the covalent linkages. In the model the
glucose moiety of the hemicellulose component lies parallel to the axis
of the cellulose fiber and is bonded by hydrogen bonds. Arabinogalactan
chains lying perpendicular to the cellulose fibrils bind the hemicellulose
to the pectic substances by glycosidic linkages and may also play a role
in binding the hydroxyproline-rich protein (Albersheim et at. 1973).
Taking all the bonds into account a rigid matrix is formed (Albersheim
1974).
Cell elongation, as envisioned in the model, occurs by the ability
of the cellulose molecules to slide past each other, suggesting that
certain bonds are labile. It is postulated that for nonenzymatic
creep — the slow yielding of the cell wall under constant stress
thought to be responsible for cell growth (Preston 1974^ — to occur, only
four consecutive hydrogen bonds need to be broken and it is thought that
these bonds exist between the xyloglucan chains and the cellulose
microfibrils. The rate of creep can be enhanced by lowering the pH or
raising the temperature. Thus auxin may act because of its ability to
stimulate growth via the activation of a hydrogen ion pump (Keegstra
et at. 1973). An alternative suggestion (Albersheim 1974) is that bond
breakage and reformation is mediated enzAnnatically with the involvement
of a hydrolase and a synthetase.
This model has been criticized on several points (Preston 1979;
Monro et at. 1976). In addition to criticisms of the techniques used,
Preston (1979) felt the binding of xyloglucan and cellulose is unlikely
because of the highly branched nature of the hemicellulose. Stronger
criticism of the model comes from the work of Monro et at. (1974, 1976).
The model, or working hypothesis, devised by these researchers differs
from Albersheim's in several respects. A small amount of hemicellulose
(30% or less) is thought to serve as a covalent bridge between the
protein and the microfibrils. In addition, a fraction of the wall
protein itself or in conjunction with a polysaccharide is felt to be
covalently bound to the microfibrils. More hydrogen bonds are implied
in this model, especially in the matrix. Monro et at. (1974, 1976)
have suggested that the bonds controlling creep should be at right
angles to the direction of elongation, which is not the case in the
Albersheim model. The Albersheim model is an explanation for cell
expansion rather than for cell elongation (Monro 1976). Longitudinal
growth in the Monro model occurs in the hydrogen bonded matrix region
by the shearing of these bonds and is independent of microfibrillar
orientation. These authors state also that studies of the roles of
synthetic and hydrolytic enzymes must be done in order to reach a
better understanding of what occurs in cell enlargement.
Fungal Walls
In general fungal walls appear to be simpler in structure than
those of higher plants, at least simpler in the types of sugar monomers
present. Hyphal walls are described as complex microfibrillar systems,
the microfibrils embedded in a matrix, generally made up of glucans,
mannans, and galactans (Northcote 1963; Aronson 1965; Rosenberger 1976).
The microfibrillar component is usually chitinous, but in a few cases is
cellulosic. A commonly used analogy of the combination of the matrix
10
and microfibrils is that of reinforced concrete (Rosenberger 1976) .
Fungal walls consist generally of 60 to 90% polysaccharide; other
components are uronic acids, protein, lipids, melanin (in some cases),
polyphosphates, and inorganic ions. Carbohydrate-protein complexes
are formed by ester, o-glycosidic, and glucosamine linkages (Sturgeon
1974) . A few detailed studies on yeasts and dermatophytes have been made
on wall glycoproteins and peptido-polysaccharides (Gander 1974) . In
general these compounds function as enzymes or recognition sites in
mating type or host-pathogen relationships (Gander 1974) and do not
appear to have a role in wall structure.
Ultrastructural studies depict the wall as existing in basically
two layers. The outer layer is the matrix and the inner, nearest the
plasma membrane, is the microfibrillar. The change in layers in the
wall is gradual rather than abrupt (Bartnicki-Garcia 1973) . In some
structures there is a third layer, melanin, which lies outside the matrix.
Basic wall form seems to be similar in the various taxonomic groupings
of fungi, even though the chemical composition differs (Bartnicki-Garcia
1973).
A correlation exists between the chemical structure of the wall
and the major taxonomic groups of fungi (Bartnicki-Garcia 1968). Eight
wall categories were created and the various taxonomic groups were
placed in the appropriate one. Members of the first two categories
contain cellulose as the microfibrillar portion, but one has glycogen
as the matrix and the other glucan. The Acrasiales belong to the
former group, while the Oomycetes belong to the latter. Organisms
of the third category have both cellulose and chitin microfibrils
11
and are represented by members of the Hyphochytridiomycetes. The
fourth is known as the chitosan-chitin category and includes the
Zygomycetes. The fifth and by far the largest group is the chitin-
glucan one which includes the Chytridiomycetes, the Ascomycetes, the
Basidiomycetes, and the Deuteromycetes. Most yeasts belong in the sixth
category, the mannan-glucan one. Yeasts with carotenoid pigments are
placed in the seventh category, the mannan-chitin one. The last
category consists of polygalactosamine-galactan, and are represented by
the Trichomycetes.
In terms of the various polymers found in fungal walls, a distri-
bution pattern can be made (Rosenberger 1976). R-glucans with 61,3 and
31,6 linkages are found in most groups except the Mucorales, while
S-glucans, al,3 linked, are limited to the Ascomycetes and the Basidiomycetes.
Cellulose is found in a few Phycomycetes, while chitin is more universal.
Chitosan and a polysaccharide of galactosamine are found in the
Mucorales, and the Ascomycetes and Hyphomycetes respectively. Poly-
uronides are known in the Mucorales. Protein, or at least the common
amino acids, is found universally. Hydroxyproline is reported in those
which have cellulose in their walls. A more recent study has noted the
presence of hydroxyproline in the basidiomycete Tvemella (Cameron and
Taylor 1976), where chitin occurs.
By altering the metabolism of the cell wall constituents, a
fungus can change its morphology (Dow and Rubery 1977). Such alterations
may involve changing from a mycelial to a yeast form or the reverse, or
changing to a reproductive, survival, or invasive mode. Studies with
Muoov roux-ii indicated that there are higher quantities of protein and
mannose in the cell walls of yeast forms as opposed to mycelial
12
(Bartnicki-Garcia and Nickerson 1962; Bartnicki-Garcia 1968; Dow and
Rubery 1977) . Additional differences include the presence of weakly
acidic polysaccharides in yeast walls and strongly acidic ones in
mycelial walls (Dow and Rubery 1977) . Quantitative differences in the
chemical composition have been noted in the walls of the different
structures, such as sporangial and hyphal, within a single organism
(Bartnicki-Garcia and Reyes 1964; Bartnicki-Garcia 1968; Cole et at.
1979; Mendoza et at. 1979).
Fungal Walls, Chemical Structure
Before describing the chemical structure of fungal walls certain
inherent shortcomings of studies of this nature will be discussed. The
first problem can be stated simply by the questions, "what is a clean
wall?" and "what is the method used for determining cleanness?" Walls
cleaned with hot alkali appeared pure microscopically, and chemical
analyses revealed that many covalently bound amino acids were released
(Cameron and Taylor 1976) . Are these components part of the wall
structure? Loosely bound wall constituents may protrude into periplasmic
space; are these inherent structural compounds? An isolated wall is
quite a different thing from a wall which is part of a living system
and this must also be borne in mind (Crook and Johnston 1962; Cameron
and Taylor 1976) . Enzyme degradation is a coiranonly used method for
studying wall composition but there are drawbacks to this technique, such
as the use of impure enzymes and rearrangements in wall architecture
caused by partial digestion (Farkas 1979) . The products of chemical
degradation must also be viewed with reservation due to the lability
of certain constituents (Talmadge et at. 1973). Bearing these thoughts
13
in mind, it becomes clear that the results of an analysis of wall
composition must be regarded with circumspection (Cameron and Taylor
1976) . This does not mean that tentative wall models cannot be drawn,
but that they must be considered in the light of the above restrictions.
Basidiomycete Walls
Detailed studies of isolated walls of Schizophyllimi aornmune have
been made over the last few years by Wessels and his group (deVries
and Wessels 1972, 1973a, b; Wessels et at. 1972; Sietsma and Wessels
1977, 1979). A lytic enzyme preparation from Triahoderma vivide grown
on isolated Schizo'phytlum walls was found to be active against the known
substrates chitin and R- and S-glucan, thus giving a clue to the identity
of the wall components (deVries and Wessels 1972, 1973a, b) . The most
external portion of the wall in S. aornmune is a water soluble layer of
mucilage. The basic structure of this component is a 61,3 glucan back-
bone with single glucose units linked through the sixth carbon atom of
every third glucose residue (Wessels et at. 1972; Sietsma and Wessels
1977). An alkali soluble al,3 linked chain, the S-glucan, lies adjacent
to the mucilage and next to this is the alkali insoluble R-glucan which
is: similar structurally to the mucilage except that it is more highly
branched (Sietsma and Wessels 1977) . The R-glucan is closely associated
with the chitinous portion, identified by x-ray diffraction studies, of
the wall. A tentative model of the R-glucan complex was postulated in
which covalent linkages were suggested between the chitin and the
R-glucan portions (Sietsma and Wessels 1979). Exo- Sl,3-glucanase
hydrolysis of the R-glucan followed by chitinase treatment yielded a
compound containing N-acetylglucosamine, glucose, lysine, citrulline.
14
glutamate, and glucosamine. The model drawn from these data describes
this portion of the wall as consisting of a linear chitinous chain
which is linked to an R-glucan oligomer by a bridge containing lysine,
citrulline, glutamic acid, glucose, and N-acetylglucosamine. In
summary, it was found that hyphal wall fragments consisted of 67.7%
glucose, 3.4% mannose, 0.2% xylose, 12.5% N-acetylglucosamine, 6.4%
amino acids, and 3.0% lipid. The mannose and xylose monomers are
associated with the S-glucan component.
Chemical analyses of the walls of Tremella mesenteviaa indicated
the presence of xylose, mannose, rhamnose, and fucose in addition to
glucose (Cameron and Taylor 1976) . The amino acid content of these
walls was studied and, as mentioned previously, hydroxyproline was
found (Cameron and Taylor 1976). Folystiatus and Ustilago walls have
been analyzed and the above listed monosaccharides were found; galactose
was also found in Ustilago walls (Crook and Johnston 1962).
Deuteromycete Walls
These are the imperfect fungi and some are known only by their
asexual states (Alexopoulos 1952) . Enzyme degradation studies of
Aspergillus oryzae and Fusarn-um solani indicated the presence of chitin
and Bl,3 glucans. Wall degradation did not occur unless both enzymes
were present or unless there was a glucanase pretreatment , leading to the
speculation that the wall consists of a chitin-containing core masked by
the glucan (Skujins et al. 1965). Chemical analyses of the carbohydrate
content of isolated walls from Aspergillus sp. and A. niger showed that
50 to 60% of the wall is carbohydrate of which 4.3% is mannose, 5 to 14%
is galactose, and the remainder is glucose (Ruiz-Herrera 1967; Cole et al.
15
1979). The amount of chltin in both organisms is 15%. Studies of
several species of Ven-La-illiymy F. oxysporum, and Botvytis ainevea
revealed the same proportion and types of monosaccharides as found in
Aspergillus, although mannose was not universally found (Crook and
Johnston 1962; Pengra et al. 1969).
Total protein measurements revealed between 7 and 8% of the dry
weight of the wall. Amino acid analyses were performed on both
Aspergillus organisms and the usual spectrum was found (Crook and
Johnston 1962; Ruiz-Herrera 1967; Cole et al. 1979). Hydroxyproline was
not found. The lipid content of readily extractable and bound lipids
was assayed for two Aspergillus species. Extractable lipids were found
to be present as 7.3% of the wall while bound lipids varied between 7
and 12% depending on the study (Ruiz-Herrera 1967; Cole et al. 1979).
Because some of the lipid could only be extracted after acid treatment
of the walls, some of the lipoidal material present in the wall is
probably complexed with structural polysaccharides and/or proteins
(Ruiz-Herrera 1967). Ash was not found in the A. niger wall (Cole et
al. 1979), but was found in that of Aspergillus sp. (4%) (Ruiz-Herrera
1967) . Phosphorus content in both types of walls was found to be very
low (0.1%) (Ruiz-Herrera 1967; Cole et al. 1979).
Ascomycete Walls
An analysis of the neutral sugars isolated from Chaetomium globosim
and Neurospora sitophila indicated high glucose and low mannose and
galactose amounts. Glucosamine was found in the walls of both organisms,
and galactosamine was found in Neurospora (Crook and Johnston 1962). The
presence of chitin was established by the usual means. Treatment of the
16
wall with exo- and endo-61,3-glucanases indicated the presence of a
61,3 glucan with some 31,6 linked glucose residues. From this datiim it
was proposed that the wall consists of layers of 31,3 glucans overlying
a chitinous core (Potgieter and Alexander 1965) . It was also noted in
this study that, although there was noticeable wall thinning after
exhaustive enzyme treatment, the characteristic hyphal morphology remained
unaltered (Potgieter and Alexander 1965) .
More complete studies were performed on Nenrospora arassa walls
where analyses of wild type and single gene morphological mutants
("colonial") walls were performed. Nenrospora avassa walls were separated
into four fractions based on their solubilities in a variety of solvents
(Mahadevan and latum 1965). Changes in fraction I, basically consisting
of glucose, galact OS amine, and glucuronic acids, were felt to be the
predominant factor in influencing colonial morphology (Mahadevan and
latum 1965). Higher amounts of uronic acids in wild type walls suggested
that these compounds have a role in regulating linear hyphal growth,
possibly due to the increased water content accompanying these compounds
which may increase wall plasticity (Cardemil and Pincheira 1979) . An
increase in mannose and galactose in the mutants suggested that colonial
morphology may result from higher levels of a branching mannan component
allowing increased bonding and therefore more rigidity (Cardemil and
Pincheira 1979).
Five peptide fractions extracted from N. avassa walls with weak
alkali indicated the presence of all normally occurring amino acids
(Wrathall and latum 1973). Quantitative differences were found, but
there were similarities in the ratio of acidic to basic components and
in the proportions of hydrophilic residues. 0-glycosyl-serine linkages
17
were discovered which indicated that this component was part of a glyco-
protein which did not appear to be covalently linked to any other major
wall constituent. It was felt that this was evidence for a separate
glycoprotein reticxilum as a wall component, thus supporting the earlier
work of Hunsley and Burnett (1970).
The yeast cell wall has been studied in great detail and the follow-
ing will only briefly touch on the subject. Three fractions of the yeast
wall were obtained by extractions with anhydrous ethylenediamine (Kom
and Northcote 1960). Fraction A, soluble in water and ethylenediamine,
contained the total spectrum of amino acids plus mannose and glucosamine
(36% of the total amino sugar found) . This fraction was felt to represent
a mannan-protein complex with the amino sugar serving as a link between
the polysaccharide and protein components (Kom and Northcote 1960).
Fraction B, insoluble in water but soluble in ethylenediamine, was
similar to A, except that glucose was present. Fraction C, insoluble
in both solvents, contained 58% of the glucosamine in addition to chitin.
Subsequent work has confirmed and extended these data. Both the glucan
and mannan components have been characterized more completely. The
major portion of yeast glucan is a 31,3 linked polymer with some 31,6
linkages and the minor portion is mainly 31,6 linked with a few 31,3
linked chains which may occur as interchain or interresidue linkers
(Manners et al. 1973a, b) . It was thought that these glucans provide a
structural function with, the 31,3 component forming an inner fibrillar
layer (Cabib 1975). Yeast mannan is a polymer of one protein and two
carbohydrate moieties, and may have both immunological and structural
functions (Cabib 1975).
18
Two models have been proposed for the yeast cell wall. In one
model (Lampen 1968) the wall is made up of phosphomannans which are
located in the outer layer of the wall. Wall-bound enzymes exist in this
portion of the wall and release of these enzymes or cleavage of this
fraction occurs by the action of an enzyme, the PR-factor, a "mannosidase. "
A smaller mannan is linked to the phosphomannan complex and also to
glucan fibrils located in the inner portion of the wall. Protein
molecules bound together by disulphide bridges make up part of the glucan
portion of the wall. Observations by Kidby and Davies (1970) of enzyme
release by sonication or thiol treatment in addition to previous studies
by Bacon et at. (1965) led to a slight alteration of this model. In the
altered model enzymes are inserted between the outer and middle layers
and are held by non-chemical means. The structural integrity of the
external wall layer is maintained by disulphide bridges. In this model
the middle layer is a mannan-glucan associated with disulphide-linked
proteins which are bound to a glucan chain which lies just outside the
plasma membrane.
Phycomycete Walls
Initial wall analysis of Allomyces maavogynus (a uniflagellate
Phycomycete) (Aronson and Machlis 1959) indicated the presence of chitin,
glucan, ash, and protein, the latter depending, however, on the method of
wall purification. Chemically cleaned walls contain 68% chitin, 8% glucan,
and 10% ash, while walls cleaned with buffer and water contain 58%
chitin, 16% glucan, 8% ash, and 10% protein. It is obvious that the
two methods of cleaning resulted in modifications of the wall constituents.
Amino acid analysis of a polypeptide fraction revealed a wide range of
19
these compounds (Youatt 1977). al,4 and al,6 linkages were found in the
hyphal walls and 61,3 in the walls of discharge plugs (Youatt 1977).
Rhiziomyaes sp. , another uniflagellate form, has both cellulose and
chitin in its walls as determined by x-ray diffraction studies (Fuller
and Barshad 1960) .
Zygomycete walls
Zygorhynohus vui-llem-ini-i walls contain galactose, mannose, fucose
(the most abundant monosaccharide found) , and glucosamine plus the usual
assortment of amino acids (Crook and Johnston 1962) . Two acidic poly-
saccharides, mucoran and mucoric acid, were isolated and analyzed from
Mucor rouxii (Bartnicki-Garcia and Reyes 1968) . Mucoran is made up of
2 fucose:3 mannose:5 glucuronic acid and mucoric acid is a homopolymer
of glucuronic acid. It was felt that these components made up a single
heteropolymer (Bartnicki-Garcia and Reyes 1968). Later studies of the
walls of M. mucedo revealed a glycuronan made up of 5 fucose :1 mannose:
1 galactose: 6 glucuronic acid non-covalently bound to glucosamine polymers
(Datemaet at. 1977a). The homopolymeric glucuronic acid part of the
isolated glycuronan is thought to be associated with the glucosamine
polymers (Datema et al. 1977a). Mucor walls also contain weakly
acetylated chitin, chitin, and chitosan (Bartnicki-Garcia and Nickerson
1962; Bartnicki-Garcia 1968; Datema er al. 1977b). On a percentage w/w
basis the composition of the hyphal wall of M. rrcucedo is 7% neutral
sugar, 12% uronic acid, 16% phosphate, 32% hexosamine, 13% protein, 10%
amino acids, and 13% ash (Datema et al. \311a., b).
Oomycete walls (Leptomitales)
Analyses of buffer-water washed sonicated walls of Sapromyaes
elongatus indicated a typical Oomycete wall, containing 91% glucan.
20
4% protein, and 0.1% ash with glucose as the only monosaccharide (Pao and
Aronson 1970). 31,3, SI, 4, and 31,6 linkages were found. Weakly
crystalline cellulose I was present but chitin was not detected (Pao and
Aronson 1970). The walls contained the full complement of amino acids
with aspartic acid, glutamic acid, serine, and threonine the most abundant
(46% of the wall protein). Hydroxyproline is 2.5% of the total amino
acid content. No lipids were found. Apodaahlya sp. and A. bvaahynema
walls differ from those of Sapromyaes in having glucosamine (Sietsma
et al. 1969; Lin et al. 1976). X-ray diffraction studies and stains
for chitin indicated the presence of both weakly crystalline cellulose I
and chitin in these walls (Lin and Aronson 1970) . The walls of A.
braahynema contain phospholipids, fatty acids, and triglycerides (Sietsma
et al. 1969). Linkage studies of A. bvaahynema revealed that 4% of the
dry weight of the wall is soluble in Schweitzer's reagent and consists
solely of 31,4 linkages, and 52% was found to be a branched 31,3 and
31,6 linked glucan, and 32% a linear 31,3 linked glucan (Sietsma et al.
1968). Apodachyla sp. walls contain 67% total glucose, 18% chitin, 9%
cellulose, 6.4% protein, 1.5% acid-soluble hexosamine, and 3.1% alkali-
soluble hexosamine (Lin and Aronson 1970; Lin et al. 1976). Analysis
of the hyphal wall chemistry of Leptomitus laateus indicated similarity
to the walls of Sapromyces and Apodaahlya, especially in regard to the
linkage pattern (Aronson and Lin 1978) .
Oomycete walls (Peronosporales)
Wall chemistry analyses of a number of species of Phytophthora
indicated the presence of 90% glucan, 4% protein (10% reported in one
species), 2% lipid, 0.4% phosphorus plus small amounts of mannose,
glucosamine, arabinose, xylose, galactose, rhamnose, ribose, and
21
galactosamine (Bartnicki-Garcia 1966; Bartnicki-Garcia and Lippman 1967;
Novaes-Ledieu et al. 1967; Tokunaga and Bartnicki-Garcia 1971). Weakly
crystalline cellulose I makes up about 25% of the wall (Novaes-Ledieu
et al. 1967) . About 5% of the total amino acid content is hydroxyproline
(Bartnicki-Garcia 1966) . Walls of various Fyth-ium species have also been
characterized and similar compositions have been reported, although the
total glucan and cellulose is lower (82% and 20% respectively) and the
lipid is higher (8%) (Cooper and Aronson 1967; Novaes-Ledieu et al. 1967;
Sietsma et al. 1969). All the common amino acids including hydroxyproline
have been reported (Novaes-Ledieu et al. 1967). Low levels of chitin have
also been found (Dietrich 1973) .
The same type of linkage pattern was found in Phytophthcra and
Pyth-iTMn walls as was described for the Leptomitales (Bartnicki-Garcia
1966; Bartnicki-Garcia and Lippman 1966, 1967; Aronson et al. 1967;
Cooper and Aronson 1967; Novaes-Ledieu et al. 1967; Eveleigh st al. 1968;
Novaes-Ledieu and Jimenez-Martinez 1969; Sietsma et al. 1969, 1975;
Zevenhuisen and Bartnicki-Garcia 1969; Tokunaga and Bartnicki-Garcia
1971; Yamada and Miyazaki 1976). The basic pattern which emerged from
a number of studies is that of a highly branched glucan of 6l,3 and
61,6 linkages covering and firmly bound to a Bl,4 linked linear glucan
(cellulose) . There are varying opinions as to the degree of branching
and as to which linkage groups serve as main chains and which as branches
(Eveleigh et al. 1968; Novaes-Ledieu and Jimenez-Martinez 1969; Sietsma
et al. 1969, 1975; Zevenhuisen and Bartnicki-Garcia 1969; Yamada and
Miyazaki 1976). Both components, the branched glucan and the cellulosic,
are reported to be slightly contaminated by linkages of the other (Novaes-
Ledieu and Jimenez-Martinez 1969; Zevenhuisen and Bartnicki-Garcia 1969;
22
Sietsma et at. 1975). A Bl,2 glucan was reported for the walls of one
species of 'Pythivm (Mitchell and Sabar 1966) and an al,3 glucan was
reported for a species of Phytophthora (Miyazaki et at. 1974).
Oomycete walls (Saprolegniales)
The basic pattern which has been described for the Leptomitales and
the Peronosporales is also seen in the Saprolegniales, the principle
differences lying in the relative quantities of the various components.
The walls of four species of Saprolegnia have been analyzed. The
predominant monosaccharide of S. fevcuz is glucose while considerably
smaller amounts of glucosamine, mannose, rhamnose, and ribose have been
found (Crook and Johnston 1962; Parker et at. 1963; Novaes-Ledieu et at.
1967). Quantitative studies revealed 93 or 85% total carbohydrate, 3 or
1.1% protein, 1.7 or 2.7% hexosamines, and 1 or 5% lipids depending on
the study (Novaes-Ledieu et at. 1967; Sietsma et at. 1969). All the
studies indicated that cellulose is present in these walls; however, there
are vast quantitative differences ranging from 42% (Novaes-Ledieu et at.
1967) to 18% (Sietsma et at. 1969) to 15% (Parker et at. 1963). In one
study an attempt was made to estimate the proportion of linkages and it
was found that 18% are 61,4, 44% are branched 01,3 with SI, 6 linkages,
and 20% are linear 61,3 (Sietsma et at. 1969). The usual amino acid
composition was found (Crook and Johnston 1962; Novaes-Ledieu et at.
1967). Other species of Saprotegnia, S. titovatis, S. monoiaa, and
S. diatina reveal essentially the same pattern, although uronic acids
were reported in S. titovalis (Parker et at. 1963) and S. diatina
(Cameron and Taylor 1976) . Some quantitative differences were found in
S. diatina possibly reflecting differences in wall preparation. These
23
walls consist of ll.S'A neutral sugars, 0.9% amino sugars, 3% uronic
acids, 8.5% protein, and 12% lipid (Cameron and Taylor 1976).
Wall chemistry of Aahyla flagellata^ A., raoemosa, A. amhisexuxzlis ,
Brevilegnia unisperma var. deliaa, B. bispora, Diatyuchus ster-ilis and
D-iatyuchus sp. is similar to Saprolegnia in all respects (Parker et at.
1963; Sietsma et at. 1969). The general linkage pattern common to all
these fungi was established for A. ambisexualis and D. stevilis (Aronson
et at. 1967; Sietsma et at. 1969). An attempt to determine the proportion
of the linkages was made for d. stevzlis and was found to be similar to
that of 5. fevax {Stetsma et at. 1969). Dietrich (1973), studying four
Oomycete genera, found hexosamine in all the walls (the three AcKlya
species studied had the highest content: 2.4, 3.1, and 3.8%) and upon
treatment of these walls with snail gut enzyme, N-acetylglucosamine at
1 and 2% levels was obtained. These results led to the speculation of
the presence of a chitin/chitosan component in these heretofore considered
chitinless walls (Dietrich 1973). An indirect indication of the presence
of chitin in Aahyla walls stems from the observation of Wang and LeJohn
(1974) of the absence of a UTP requirement for glutamic dehydrogenase;
UTP has been found to be necessary for activation of the enzyme in
organisms with chitinless walls. The walls of the marine fungus,
Atkinsiella dub-ia, have also been studied and in general these walls are
similar to those described above (Aronson et al. 1967; Aronson and
Fuller 1969). Notable differences are the protein content (13.7% in
these fungi) and a very high level of hydroxyproline (20.4% of the total
amino acid content and 2% of the dry weight of the wall) (Aronson and
Fuller 1969).
24
Fungal Walls, Physical Structure
The only polymers whose structures will be described here are those
which occur in Oomycete walls, i.e., those with 31,3; 61,4; and 31,6
linkages. Cellulose has been studied extensively. Glucose units in
cellulose are joined by 31,4 glycosidic bonds and it is because of this
type of linkage that the polymer can be described as a flat ribbon (Rees
1977; Preston 1979). The chain is stabilized by hydrogen bonds which
form between the third carbon of a glucose molecule and the ring oxygen
of the adjacent glucose unit (Preston 1979). Each ribbon-like chain has
numerous potentially hydrogen binding hydroxyl groups along each edge, so
when two chains come in contact many hydrogen bonds form creating a
stable structure (Preston 1979). In native cellulose, usually termed
cellulose I, it is thought that the chains lie parallel with each other
and parallel to the surface of the wall in staggered layers. In
regenerated cellulose, usually termed cellulose II, the chains are
antiparallel and lie in regularly stacked layers (Rees 1977; Preston
1979). The highly ordered arrangement of chains, known as a microfibril,
creates a crystalline structure amenable to x-ray diffraction and
polarizing light microscope studies. A microfibril is described as
containing a central crystalline core (5 to 7 nm wide in higher plants)
surrounded by a paracrystalline cortex. The cortex is made up of
molecular chains lying parallel to the microfibril length. The chains,
however, are not in a crystalline arrangement because of their mixed
cellulose and hemicellulose content (Preston 1974a) . With the cortex
added to the core, the width of the microfibril is about 10 nm (Preston
1974a). It is felt that the chains in the cortex become increasingly
25
more hemicellulosic with increasing distance from the core
(Preston 1974a) .
Polymeric glucan chains linked together by 61,3 bonds exist in a
hollow helical pattern (Rees 1977; Preston 1979). Three suggestions have
been made as to how the hollow area is filled: 1) by the formation of an
inclusion complex with appropriately sized molecules; 2) by the formation
of a double or triple helix with other 31,3 linked chains, and 3) by the
nesting of a number of 61,3 linked chains (Rees 1977). The structural
importance of these polymers is their ability to twist around each other
forming a network which is effective in entangling other polysaccharides
or in itself creating a strong, but flexible, assemblage of molecules
(Rees 1977; Preston 1979). The helical conformation will also exist, even
if the 61,3 bonds are interrupted by 61,4 bonds (Preston 1979).
Loosely jointed linkages and chains are formed when glucans are
held together by 61,6 bonds. There is a lot of freedom of rotation of
molecules involved in this type of bonding because of the separation of
the monomeric units by three bonds rather than two, placing the sugar
rings further apart (Rees 1977). Commonly individual glucans such as
these are not found in nature. Instead these glucans exist as branches
on other types of polysaccharides (Rees 1977). It is speculated that
the flexibility of these linkages may allow the molecules involved to
aid in various biological interactions such as the entry and exit of
enzymes (Rees 1977) .
Fungal Walls, Morphology
There is general agreement that hyphal walls consist of an inner
microfibrillar core of randomly oriented chitinous or cellulosic fibrils
26
covered by an amorphous matrix of varying chemical constituency (Aronson
and Preston 1960). The walls of sporangial, spore, and the sexual
apparatus may differ either by having an outer microfibrillar component
or an outer melanin layer (Tokunaga and Bartnicki-Garcia 1971; Hegnauer
and Hohl 1978; Cole et at. 1979; Hawes 1979; Mendoza et at. 1979).
Information on the morphology of hyphal walls, treated chemically
and enzymatically, comes from ultrastructural studies of surface
replicas and thin sectioned material. The classic study of wall morphology
is that of Hunsley and Burnett (1970) who compared walls after sequential
enzyme treatments of three different fungi, each representing one of
the major taxa. Live hyphae were used for two reasons: 1) any artifacts
brought about by wall isolation were eliminated; and 2) confidence that
enzymatic digestion occurred from the outside in. Models for each of
the three major groups were then developed from these data.
Walls of SchizophyllioTi aorrmune, the representative Basidiomycete,
have a four-layered structure which none of the three enzymes, laminarinase,
pronase, or chitinase, could hydrolyze. However, preliminary treatment
with KOH was effective in removing the outer protective (S-glucan) layer.
Subsequent treatment with laminarinase revealed a microfibrillar outline
which was clarified by the addition of pronase. Chitinase treatment
following that of laminarinase and pronase removed the microfibrillar
component. The model which was derived from this study depicts the wall
as having an outer S-glucan layer bounded internally by R-glucan, The
R-glucan is bordered on the inside by a thin, but discrete, proteinaceous
sheet which in turn is complexed and intermixed with the chitinous micro-
fibrils (Hunsley and Burnett 197Q). Van der Valk and Wessels (1977) using
27
isolated walls did a similar study and found that pronase had no effect
on the R-glucan-chitin portion of the wall. This led to the belief that
there is no protein layer and no protein-chitin complex. Carbon-
platinum replicas of the S-glucan revealed a surface composed of randomly
oriented parallel arrays of short rodlets and a filamentous surface of
the mucilage (Wessels et al. 1972) .
Neurospora avassa is the representative Ascomycete which Hunsley
and Burnett (1970) studied. In thin section the wall appeared three-
layered. Laminarinase treatment removed the outer amorphous layer
revealing a coarsely stranded network, more clearly resolved by the
addition of pronase, filled with a matrix material. Chitinase in
conjunction with the other two enzymes resulted in dissolution of the
wall. Neither chitinase nor pronase added alone or in sequence had any
effect at all. The model derived from these data envisions the wall as
having an outer layer of 61,3, SI, 6 glucan with an inner layer of protein
in which is embedded coarse strands of a glycoprotein (glucan-peptide-
galactosamine) reticulum. A discrete protein layer lies between the
reticulum and the chitinous microfibrils which are embedded in a protein
"matrix" and lie in the innermost part of the wall (Hunsley and Burnett
1970). A similar study by Mahadevan and Tatum (1967) indicated that the
wall consists of an outer coarse fibrillar layer (glucan-peptide-
galactosamine) and an inner layer of primarily 81,3 glucan with an
embedded core of fine chitin fibrils.
Phytophthora parasit-iaa walls were studied in order to derive a
model for a Phycomycete wall. The two- layered wall has a finely granular
amorphous surface which is unaffected by treatment with cellulase or
28
pronase or both. Laminarinase treatment resulted in the exposure of
microfibrils whose outlines were more pronounced if pronase treatment
followed. Laminarinase and cellulase treatment resulted in almost total
digestion. The model of the wall designed from these studies describes
an outer amorphous layer of 31,3; 31,6 linked glucan and an inner layer
of cellulose embedded in protein. Similar layering has been seen in the
hyphal walls of Phytophthora palmivova and Pythium aaanthioum (Tokunaga
and Bartnicki-Garcia 1971; Sietsma et at. 1975; Hegnauer and Hohl 1978).
Chemical removal of the outer amorphous layer of isolated walls of
Saprolegnia litoralis and Atkinsi-ella dubia and chemical and enzymatic
removal of this layer in Sccpromyces elongatus revealed a distinctly
microfibrillar layer (Parker et at. 1963; Aronson and Fuller 1969;
Pao and Aronson 1970).
Isolated walls of Choanephova GUourbiixiri.TMn , another Phycomycete,
were found to exist in two layers, an outer thick layer of randomly
oriented microfibrils made up of a mixture of chitosan, protein, and
lipids, and an inner thin layer of chitinous microfibrils oriented in a
parallel fashion (Letourneau et al. 1976). Microfibrillar orientation
of the hyphal walls of Lindevina pennispova is longitudinal except in
the most interior portion of the wall where it is random (Young 1970) .
Sporangiophore walls in this same organism are similar except for the
existence of spicules covering the outer surface of the wall (Young
1970).
Fungal Walls, Growth
Burnett (1968) presented a diagram of his views of apical and
subapical wall organization and how it is altered in response to growth.
29
The hyphal tip is thin-walled and non- extensible, but the area directly
behind the tip is thicker-walled and it is here that maximum intussusception
takes place. Distal to this zone lies a second thick-walled area known
as the region of maximum extensibility. In the most distal region
described, the wall reaches its maximum thickness and becomes rigid.
It has been suggested that the subapical wall in Phytophthora parasitiaa
has more protein in which the microfibrils are embedded and a greater
degree of microfibrillar aggregation than is found in the apical
(Hunsley and Burnett 1970) , which may account for the increasing rigidity
of this part of the wall. In this most distally described area of the
wall the arrangement of the microfibrils is longitudinal as compared
to the transverse arrangement nearer the tip. Growth is explained by a
change in the balance between synthetic and lytic enzymes (Bartnicki-
Garcia 1973) which allows for turgor driven apical expans?.on (Thomas
1970; Bartnicki-Garcia and Lippman 1972).
Autoradiographic studies indicated that the sites of growth are
at the tip (Van der Valk and Wessels 1977), although some wall thicken-
ing and modification is seen subapically (Bartnicki-Garcia 1973) . It
has been hypothesized that vesicles play a role in wall synthesis based
on the observation of their accumulation at growing tips (Heath et at.
1971; Van der Valk and Wessels 1976; Beakes and Gay 1978; Hawes 1979)
and at the sites of antheridial initials (Mullins and Ellis 1974) . The
suggestion has been made that these vesicles carry wall degrading
enzymes (Mullins and Ellis 1974; Fevre 1977) and materials for plasmalemma
and wall synthesis (Bartnicki-Garcia 1973).
MATERIALS AND METHODS
Organism and Culturlng Techniques
Strain E 87 male of Aahlya ambisexualis Raper (Barksdale 1960)
obtained from Dr. J. T. Mullins was the organism used in this study.
Mycelia were grown on defined media (Mullins and Barksdale 1965; Kane
1971) on agar plates or in liquid culture on a reciprocating shaker
(100 rpm) at 25°C. Two day old mycelium, grown on agar, was sporulated
in 0.5 mM CaCl„ on a reciprocating shaker (100 rpm) for 20 hr at 25°C
with one change of solution after the first 2 hr. An inoculum of 200 000
zoospores was added to 20 ml of defined liquid medium and grown for 48
hr. Mycelium was harvested by vacuum filtration and washed two times
with either 0.05 M potassium phosphate buffer pH 7.0 or 0.1 M tris-HCl
buffer pH 7.5 depending on the subsequent method of hyphal wall prepara-
tion. Harvested washed mycelium was quick frozen at -70°C in a Revco
Ultra Low freezer.
Hyphal Wall Isolation and Purification
In all cases hyphal walls were isolated by grinding in a chilled
mortar and pestle 10 gm fresh weight frozen mycelial lots until a fine
powder was obtained. The entire procedure was performed at 0-4 "C. The
appropriate buffer (final amount 20 ml) was added and grinding was con-
tinued. The resultant "slush" was centrifuged (Sorval RC-2B Automatic
Refrigerated Centrifuge) at 1085 x g and the pellet was saved for
further purification.
30
31
Two methods of cleaning hyphal walls were followed. In one, the
walls were cleaned chemically by a modification of Tokunaga and Bartnicki-
Garcia (1971) and, in the other, they were cleaned by repeated washings
with buffer and water (Lin et at. 1976). In the first method the
pellet was washed with phosphate buffer and then sonicated in 10 ml
2% sodium lauryl sulfate for one minute at 15 watts (Heat Systems-
Ultrasonics Sonifier Cell Disruptor, Model W 185, fitted with a standard
microtip) . After sonication the suspension was placed in a 90°C water
bath for 30 min. It was then centrifuged and the pellet was treated with
60 ml of a 2 95% ethanol: 1 2 N KOH solution three times for 10 min
each in a boiling water bath. The resulting pellet was washed with
distilled water three or four times or until the washings showed a
neutral pH. Wall purity was determined by phase and electron microscopy
and the decreasing level of protein found in the washings. The second
method followed closely that described by Lin et dl. (1976) with a 4 min
sonication at 30 watts. Cleaned walls were dried by lyophilization
(Virtis Research Equipment) and stored over desiccant until further use.
All subsequent analyses were begun with 100 mg samples of this material.
Total glucan was determined on each preparation of cleaned walls by
the anthrone method (Morris 1948; Dische 1962).
Chemical Fractionation of the Wall
The cleaned and freeze-dried walls were chemically fractionated by
three successive treatments. The first was acid (0.5 N HCl) (Aronson
et at. 1967; Sietsma et al. 1969) and the second was alkali (2 N KOH).
The third treatment used was either Schweitzer's reagent (personal
32
communication Dr. J. M. Aronson) or cadoxen (Jayme and Neuschaffer 1957;
Jayme and Lang 1963), both known cellulose solvents. The acid-soluble
fraction was obtained by five 30 min treatments at 70°C of 100 mg wall
material in 50 ml 0.5 N HCl. The supematants from each treatment were
pooled and brought to a final concentration of 85% ethanol and allowed to
stand overnight at 4°C. The ethanol precipitated polysaccharide was
collected the next day by centrifugation, freeze-dried, and stored over
desiccant until further use. The pellet which remained from the acid
extraction was washed with distilled water until neutral, and then treated
with 2 N KOH in the same manner as the acid treatment. This became the
alkali- soluble fraction. The remaining pellet was dissolved in either
one of the two cellulose solvents, with cadoxen being favored because
of its colorless and odorless nature (Ladisch et al. 1978). The basic
procedure for cellulose dissolution was the same with both solvents and
both appeared equally effective. The pellet remaining from the acid-
alkali extractions was treated overnight under N„ with 40 ml of freshly
prepared reagent at room temperature with stirring. Two additional 2 hr
extractions were performed and finally the supernatants were pooled and
treated with glacial acetic acid until neutral. The solution was
centrifuged at 48 300 x g for 20 min in a Beckman J2-21 refrigerated
centrifuge. The pellet was washed once with 1 N acetic acid, twice with
distilled water, twice with 22% NH.GH for the Schweitzer's reagent or
30% ethylenediamine for the cadoxen reagent (the first time for 30
min), once with 1 N acetic acid, and finally with distilled water. The
pellet was freeze-dried and stored over desiccant for further study.
This is the Schweitzer's or cadoxen reagent-soluble fraction, and is
termed cellulose II (regenerated cellulose) ( Preston 1974a) . The
33
pellet remaining was neutralized by washing with distilled water and
freeze-dried as above. This is the insoluble residuum.
Cellulose I or native cellulose and chitin were extracted from
frozen mycelia or isolated walls following the method of Aronson and Lin
(1978).
Chemical Analyses of Wall Constituents
Preparation of Material for Neutral Sugar Analyses
Enzyme hydrolysis
Lyophilized walls or their derived fractions were treated with
various enzymes and the products of hydrolysis were determined. The
enzymes used were laminarinase (E. C. 3.1.1.6, Sl,3-glucanase ex mollusca,
B grade, CalBiochem) , cellulase (E. C. 3.2.1.4, 61,4-glycanohydrolase
from Aspero-iZ-Z-us nigev. Type I, Sigma), chitinase (E. C. 3.2.1.14, chito-
dextrinase, poly(l,4-6- [2-acet-amido-2-deoxy]-D-glucoside) glycanohydrolase
from Streptomyaes gviseus, Sigma), protease (from 5. griseus , Type VI,
Sigma), and lipase (448 from hog pancreas, Nut. Biochem. Co.). The
buffer used for cellulase and laminarinase was 0.05 M sodium citrate
pH 5.0; for chitinase 0.05 M phosphate pH 6.0; for protease 0.05 M HEPES
pH 7.6 or 0.05 M sodium phosphate-citrate pH 7.6; and for lipase 0.05 M
sodium phosphate-citrate pH 6.2 Reactions were allowed to run 24 hr unless
otherwise specified. The chitinase and lipase reactions were carried out
at 25°C and the others at 37°C. The concentration of enzyme was 500 yg/
ml and that of the substrate 2 mg/ml. Bacterial contamination was
prevented by the addition of 100 Ug/ml streptomycin, 500 Ug/ml merthiolate,
or when gas chromatographic analyses were to follow, a toluene layer
34
covering the reaction mixture. Tests for enzyme purity were performed
by reacting the enzyme in question with a known substrate. They were:
laminar in, from Laminaria digitata, A grade, anhydroglucose 94%,
CalBiochem; cellulose, carboxymethylcellulose or acid swollen Whatman
Ashless Powder for chromatography (Reese and Mandels 1963b; Green 1963);
chitin, purified powder from crab, Sigma; Tween 20; and bovine albumin
powder, Fr. V, 96-99%, Sigma. The conditions of enzyme hydrolysis are
compiled in Table 1. Two of the enzymes were found to be active against
more than one substrate. Laminarinase was found to be active against
both laminarin and cellulose, and cellulase was active against both
protein and cellulose (Whitaker 1970). Thus, it was necessary to purify
these two enzymes before use (Sietsma et at. 1968).
Assays for enzyme activity
The activity of laminarinase was measured by determining the increase
in total reducing sugar or in glucose over that of the enzyme or sub-
strate alone, using the anthrone method (Morris 1948; Dische 1962) or
the glucostat test (Worthington Biochemical Corp.). Cellulase activity
was determined viscometrically (Thomas and Mullins 1969) . The production
of N-acetylglucosamine, as determined by the DMAB method (Reissig et dl,
1955) , was used to measure the chitinase activity. The activity of
protease was determined by the decrease in total protein, as measured
by the BioRad technique (BioRad Technical Bulletin #1051) . The release
of fatty acids, as determined by a change in pH, was used to measure the
activity of lipase (Bier 1955) .
This substrate contained 61,3 linkages only. Another laminarin (source
unknown) was also used which was found to contain 31,6 linkages in
addition to the 61,3.
35
•H U to
> u m
O IN
•H OJ -73 C
SO rH
W TJ tU O
O C -H -H
< 3 i4H -U
CO Q)
^ e
O M-t
m
OJ
M
3.
C
N
O
0)
C
O
o
w
in
c
o
U-(
>%
0)
CO
>
g
u
ao
3
•H
(U
W
60
3-0;
so
o :3.
o
so 00
I <r
o
o
in
I--^
g
CO
(U t~~^
oj 8
c
U3 "i^i
en
•H
CO CT)
to a
i-l
OJ
-H s:^
r-l t-^
to
B
3 >»
3 2i
c
>v
rH fX,
■H rs;
•H
N
iH 03
^ t>
6
C
0) ^
0) -^
to
u
u ^
u ^
hJ
CO
.
CO
4=
U-l u
;s
<-l
rS
S
•H O
^\>
>
Qi
U XI
CO
<N
CO
0)
3
•fj
•
•^
CU •"
i^
m
1^
!>^
C OJ
G^
C33
c
3 g
CO
cJ
CO
OJ
00 ^3
w
W
e C
n
w
>;>
00
^~ OJ
-v,
^
CO
a;
i=^
U 13
(U
o
X
n
o
•H d)
en
-M
CO
HOi
o
C -H
CO
-:>,
c
iJ,
in
3 y-i
OJ
w
•H
^«
•H
u
iin
4J
!h
n
O >j
o
-W
•H
■+i
(U
rg 3
M
^
^
<r,
a.
iH &
Oi
CJ
to r
a
36
Acid hydrolysis
Unfractionated walls and the wall fractions described previously
were hydrolyzed in sealed ampules for varying periods of time with 6 N
HCl under N^ at room temperature. The resulting hydrolysates were then
analyzed for mono- and disaccharides. Before analysis the hydrolysates
were diluted with distilled water and either dried over NaOH pellets
in vacuo until they were neutral or were deionized by passage through
a Dowex carbonate column and then dried in vacuo. The sample size was
2 mg per 0.5 ml 6 N HCl. Unfractionated walls were treated for 48 hr,
the acid- and alkali-soluble fractions and the insoluble residuum for
4 hr, and the cellulose II fraction for 96 hr. The anthrone reagent
was used to determine total glucan in these samples.
Neutral Sugar Analyses
All neutral sugar analyses were perfonned on chemically cleaned
walls or the fractions prepared from these walls.
Acid and enzyme hydrolysates of unfractionated walls and their
fractions were analyzed by both paper and gas- liquid chromatography, the
latter by the formation of trimethylsilyl derivatives (Sweeley et al.
1963; Zanetta 1972). Single dimension descending paper chromatography
was done on Whatman //I chromatography paper, 46 by 56 cm, in a solvent
saturated Chromatocab (Research Specialties Co.) at room temperature for
24 hr following known procedures (Kowkabany 1954; Block et al . 1958).
Material containing 100 yg or more carbohydrate was applied to the paper.
The solvent system used was 6 butanol: 4 pyridine: 3 water. Dried
chromatograms were developed in a 105 °C oven after having been sprayed
37
with aniline phthalate (Partridge 1944) . R values were calculated from
the resulting spots and compared with known standards.
Derivatization of samples and standards for GLC was done in the
following manner. Solutions containing known amounts of carbohydrate were
first lyophilized and then volatilized by a 10 min treatment with 0.2 ml
dried pyridine, 0.1 ml hexamethyldisilazane and 0.1 ml trimethylchloro-
silane when only monosaccharides were present, or for 3 hr when disac-
charides were (Ishizuka et al. 1966; Bhatti et at. 1970). The
reactants were mixed on a vortex and after gentle warming (Yamakawa and Ueta
1964a, b) the reactions were carried out at room temperature. At the
end of the reaction period, the mixture was dried with a stream of N„ in
a warm water bath, and the derivatives were extracted with 0.4 ml
methylene chloride (Mallinckrodt, nanograde) . Sample size varied from
2 to 6 yl depending on the amount of carbohydrate, and sample concentration
varied from 10 to 20 yg carbohydrate.
The gas chromatograph used was a Hewlett-Packard F & M 402 with
dual flame ionization detectors. The carrier gas was helium. The
columns were standard 1.8 m tubes with internal diameters of 3 mm. The
packing material was 3% (w/w) JXR on 100-120 mesh Gas Chrom Q. Two
different temperature programs were used. Monosaccharide separations
used a starting temperature of 170°C for 5 min, followed by a rise of
2°/min to 210°C where the temperature was held. Disaccharide separations
used a similar temperature regime, except that once 210° was reached the
program was changed to a 10°/min increase to 240° where the temperature
was held. The internal standard which was incorporated at the time of
acid or enzyme hydrolysis was myo- inositol in the monosaccharide program
38
and sucrose in the disaccharide. Peak areas were determined by the use
of a K & E Compensating Polar Planimeter (620 005) and the relative
quantities of the disaccharide components found were calculated on the
basis of the internal standards (Davison and Young 1969 ; Clamp et at. 1971) .
Linkage and branching analyses of the various polysaccharides
isolated from the wall were done by periodate oxidation. The following
procedure was modified from that of several previous ones (Dyer 1956;
Goldstein et al. 1965; Hay et at. 1965). A 36.7 mg sample was dissolved
in 25 ml of 0.04 M sodium metaperiodate (Sigma), which had been dissolved
in acidified water (pH 4.5), and placed in flasks which were covered
with black electric tape and aluminum foil. Aliquots were taken
immediately for T and analyzed. The materials to be oxidized were
placed on a wrist-arm shaker at h°C and aliquots were removed for
analysis every 24 hr for a total period of 120 hr. Periodate ion
consumption was determined by UV absorption at 222.5 nm of 0.1 ml
samples after a 250-fold dilution. Formic acid liberation was determined
by titration with 0.01 N NaOH on 1 ml samples, to which 0.1 ml acid free
ethylene glycol and, after 10 min at room temperature, 0.5 ml 0.02%
methyl red had been added. The amount of base necessary for neutrali-
zation was then correlated with the amount of formic acid in the sample.
Appropriate controls were also analyzed.
Live hyphae with developing branches plus samples of isolated
cleaned walls and their fractions were observed under a polarizing
light microscope. The pattern of birefringence was noted.
Samples of both Schweitzer's and cadoxen reagent-soluble material
were subjected to x-ray diffraction analyses by Dr. J. M. Aronson of the
39
Department of Botany and Microbiology, Arizona State University, Tempe,
Arizona.
Amino Sugar Analyses
Solubility of the hexosamine component of the wall
The procedure used was that of Lin et at. (1976). The three major
fractions were first lyophilized and then hydrolyzed with 16 ml of 4 N
HCl at 98°C under N„ in sealed ampules. After a 16 hr reaction period,
the ampules were opened and the acid was removed by rotoevaporation.
The remaining contents were washed three times with distilled water
and finally dissolved in 5 ml 0.01 N HCl for amino sugar analysis in
an automated Amino Acid Analyzer (Model JLC-6AH, Japan Electron Optics
Laboratory Co., Ltd., Tokyo, Japan).
Lugol's iodine detection of chitin
Lugol's iodine was prepared as a 1 iodine: 2 potassium iodide: 300
distilled water solution. The test material was placed in a depression
slide in a few drops of oxalate buffer ranging in pH from 1.6 to 4.0. A
few drops of the Lugol's iodine was added and the material was observed
under a light microscope to determine any color development (Prakasam and
Azariah 1975).
Uronic Acid Analysis
The procedure for isolating uronic acids follows that of Gancedo
et al. (1966). The presence and quantity of uronic acid was determined
by the carbazole test (Bitter and Muir 1962) .
40
Protein and Amino Acid Analyses
Total protein was determined on wall samples which had been washed
in buffer and water. They were then homogenized with a glass tissue
grinder (Kontes Glass Co.) in 1 N NaOH and the resulting homogenate was
placed at 50°C for 3 hr. Protein was determined by the BioRad method
(BioRad Technical Bulletin #1051 1977).
Amino acid profiles were determined on 200 and 20 mg samples of
chemically cleaned and of buffer-water washed walls, respectively, follow-
ing hydrolysis in 6 N HCl at 105°C. Similar profiles were also determined
on 20 mg samples of walls taken at various stages during chemical cleaning.
These stages were: (1) supernatant after initial pelleting subsequent
to grinding; (2) buffer washed once; (3) buffer washed once plus sonicated
and heated 30 min at 90°C in 2% SLS; and (4) walls from (3) treated once
10 min in boiling water with ethanolic KOH. After treatment for 48 hr
the acid was removed by rotoevaporation as described previously and the
amino acids were analyzed by an automated Amino Acid Analyzer.
Lipid Analysis
The procedure of extracting readily extractable lipids was that of
Kanfer and Kennedy (1963). The dried extract was spotted on Silica Gel G
plates activated with iodine and detection was by double bond formation
with iodine (Whitehouse et at. 1958). The solvent used for ascending
chromatography was 65 chloroform: 25 methanol: 8 glacial acetic acid
(Ames 1968). The pattern of spots suggested phospholipid. A Fiske-
Subbarow solution (Bartlett 1959) plus 0.5 ml 10 N H^SG, was sprayed
onto the dried plates to detect phosphorus.
41
Phosphorus Analysis
Total phosphorus was determined on wall samples which had been
combusted at 160°C in 10 N H^SO, and H2O2 for 48 hr. Phosphorus was
measured by the Fiske-Subbarow method (Barlett 1959).
Hydrolysis of Buffer-Water Washed Walls
by A. ambisexuatis Cellulase
A sample of the enzyme cellulase was extracted with acetone from
medium in which Aahtya had grown on the enriched formula (Kane 1971) for
48 hr. Enzyme precipitation was achieved by adding 2 volumes of acetone
to the medium, followed by centrifugation at 12 100 x g for 20 min. The
pellet was then resuspended in distilled water at a ratio of 1 ml per gm
fresh weight of mycelium, and centrifuged at 18 800 x g for 15 min. The
supernatant was dialyzed for 24 hr against a 0.018 M citrate-NaOH buffer
pH 5.0 with 0.05% merthiolate to remove glucose present in the original
medium. Viscometric assay of this enzyme solution revealed an activity
of 5 units/ml (Thomas and Mullins 1969) . Isolated buffer-water washed
walls were prepared as usual except that they were not lyophilized, and
a final washing with the above citrate-NaOH buffer was made. One ml of
the enzyme solution and 2.5 ml of the wall suspension were added to a
15 ml conical centrifuge tube and placed at 30°C for 168 hr. Aliquots
of 0.2 ml were taken at 24 hr intervals and the total reducing sugar was
measured in the supernatant by the anthrone method (Morris 1948; Dische
196 2).
42
Ultrastructural Studies
Surface structure of live hyphae, isolated walls, and wall fractions
was studied under a number of varying regimes of chemical and enzymatic
treatments.
Surface Structure of Chemically Treated Walls
Isolated walls were first treated with 0.5 N HCl and placed in a
70°C water bath for 30 min. This treatment was repeated five times.
Surface replicas were made of samples of the wall which remained. The
rest of the remaining wall was treated similarly but with 2 N KOH.
Samples of the wall left from this treatment were taken for surface
replication. The residual wall material was treated with cadoxen reagent
and surface replicas of the insoluble material were made. The cadoxen-
soluble material was treated with acid to regenerate cellulose II and
surface replicas were again made of this component.
Surface Structure of Wall Fractions
Surface replicas of each of the wall fractions described in the
section on Chemical Fractionation of the Wall were made.
Surface Structure of Chemically Treated Live Hyphae
Live hyphae were treated in the same manner as the isolated walls
and replicas were made of the wall surfaces of the hyphal samples after
each treatment. Replicas were not made, however, of material which was
solubilized in cadoxen.
43
Surface Structure of Enzymatically Treated Live Hyphae
Very small amounts of 48 hr old mycelium were placed in the wells
of a plastic Tissue Culture Cluster Chamber (Costar) and 0.2 ml of the
various enzyme solutions containing merthiolate were added. Sterile
cotton, soaked in sterile water, was placed in nearby wells to prevent
desiccation. The reaction mixtures were placed at 37°C for 48 hr. At
the end of the incubation time hyphae were removed from the well, washed
with sterile water, and placed on freshly cleaved mica for drying and
eventual surface replication. Hyphae, which were to be treated with a
second enzyme, were washed and returned to the well and the second enzyme
was added. If a third enzyme was to be added, the same procedure was
repeated. Assay conditions were the same as those described in the
section on enzyme hydrolysis. The activities of the enzymes used were:
laminarinase (purified) , 620 ug reducing sugar (as glucose) released
from 2 mg cell wall/ml enzyme; Aspergillus nigev cellulase (purified),
12 units /ml; Aahlya ambisexualis cellulase, 5 units /ml;
and protease, 500 yg/ml. Controls for each sample contained boiled
enzyme.
Diameter of Microfibrils
The width of microfibrils was determined from negatives of carbon-
platinum surface replicas taken at 33 K and 50 K magnification under
varying preparative conditions. These conditions were treatment with
buffer (untreated), laminarinase, laminarinase-protease, and 0.5 N HCl
followed by 2 N KOH. The diameter of cellulose I microfibrils was also
determined.
44
Preparation of Replicas
Single-stage carbon-platinum replicas (Pease 1964; Bradley 1965)
were made of the wall surface by the following procedure. Samples of
the treated wall were air-dried on mica and were shadowed with platinum
at an angle of 45° in a Balzer's High Vacuum Coating Unit Micro-BA 3 or
a Balzer's BA 360 Freeze Etch Device. After shadowing, the specimens
were coated with carbon. Biological material and the mica were removed
from the replicas by floating on 40% chromic acid solution. The replicas
were washed twice with distilled water and allowed to sit overnight in
50% chlorox. The chlorox was washed off by two 15 min washes with
distilled water. The replicas were placed on 100 mesh copper formvar
coated grids and were examined by a Hitachi HU-llE or a Jeolco JEM-100
Cx electron microscope.
RESULTS
Criteria for Wall Purity
Observations of both chemically cleaned and buffer-water washed
walls with phase and electron microscopy revealed that they were
relatively free of cytoplasmic contaminants (Fig. 1 a and b). In
addition, there was a decrease in the protein content of the wall
washings during the successive stages in the chemical cleaning process
as shown in Fig. 2.
The two methods of wall cleaning gave quite different amounts of
dried cleaned walls per original gm fresh weight of mycelium. About
twice as much wall material was obtained after buffer-water washing as
after chemical cleaning, 6.82 mg and 3.20 mg dried walls /gm fresh weight
of mycelium. Total glucan content of both preparations, as measured by
the anthrone method, was quite similar: 52. 80 mg and 56.00 mg/100 mg dried
walls for buffer-water washed and chemically cleaned, respectively.
Chemical Fractionation of the Wall
Table 2 gives the results of the chemical fractionation of the
carbohydrate component of the wall. That portion of the wall soluble in
weak acid was 37.58%, while only 7.07% of the remaining wall was soluble
in alkali. Some 20.83% was soluble in cellulose solvents, leaving a
5.58% residuum. These four fractions account for 71.06% of the wall
andwere shown to consist of the following: (1) acid-soluble = Bl,3
45
Figure 1. Phase contrast photographs of cleaned walls.
(a) Chemical. X 800. (b) Buffer-water. X 800.
47
48
4-,
WASHES
1.
2.
3.
4.
5.
6.
7.
Buffer suspension after grinding
Detergent treated, sonicated, and heated
2 ethancl:
2 ethanol :
2 ethanol :
2 ethanol:
Water, IX
Water, 2X
Water, 3X
KOH
KOH,
KOH,
KOH,
10 min boiling,
10 min boiling,
10 min boiling,
IX
2X
3X
Figure 2. Decrease in total protein as a measure of wall purity.
determined by the Lowry method
49
Table 2. Carbohydrate fractions of A. ambisexuatis wall.
Fraction mg
Acid-soluble 37.58
Alkali-soluble 7.07
Cellulose II 20.83
Insoluble residuum 5.58
Cellulose I 19.06
Chitin 0.63
Total glucan^ 52,8*^, 56.0^
average of three determinations based on 100 mg samples
determined (as glucose) with anthrone on unhydrolyzed
walls; calculated as anhydroglucose
c
buffer-water washed walls
chemically cleaned walls
50
and 61,6 glucan; (2) alkali-soluble = 61,3; 61,4 and 61,6 glucan; (3)
cellulose II = 61,4 glucan; and (4) insoluble residuum = 61,3; 61,4
and 61,6 glucan.
The amount of cellulose I was 19.06% of the wall and this is 2%
less than the value for cellulose II. A very small portion of chitin was
found (0.63%) and some additional tests were made to support this
identification. A two week incubation of this component with chitinase
released about the same level of N-acetylglucosamine as did a known
chitin substrate. A 700 yg sample of wall material released 58 yg
of N-acetylglucosamine and a 2000 yg sample of crab chitin released
152 yg. A spot identified as N-acetylglucosamine was obtained with paper
chromatography from both enzyme hydrolysates (Table 3) . Cytological
staining with Lugol's iodine compared favorably with known samples of
chitosan and deacetylated chitin.
Oiemical Analyses of Wall Constituents
Neutral Sugars
The above fractions (Table 2) were then analyzed for their mono-
saccharide composition, types of glycosidic linkage, and pattern of
branching. The acid-soluble fraction was hydrolyzed and the products
separated by paper chromatography. They consisted of the monosaccharide
glucose and the disaccharides laminaribiose and gentiobiose (Table 4) .
Positive identification of a probable trisaccharide spot was not possible
because of conflicting R values between laminaritriose and cellotriose.
The two disaccharides found indicated the presence of two different
linkages, 61,3 and 61,6, respectively. The alkali-soluble fraction
51
X
o.
OJ
CB
u
^1
CO
00
M
o
>^^
J=
o
<D
>-i
e
<3J
>.
D.
N
nl
c
a
QJ
>^
Uh
XI
O
CO
c
•r^
o
i~~i
•H
f^
03
^^
)-i
V^^
nj
CO
O-
•r-i
OJ
.Ai
H -^i:
M e
s +
I +
I +
+ +
+ I
+ +
CO
CTI
c
a;
c
H
tfl
•H
!-l
-H ca
Ul
CT!
r-l rH
tn
C
C3 3
c
H
3 rH
•H
ti
iH
i=
cfl
,-1 QJ
CO
3^ •
4-1 J=
O ft,
to
to u
52
4- +
o
i~j,
•H
«
U
nj
H
M
^\^
ca
CO
a.
•i-i
<U rii
CO
^
<u
j:
H
•^
53
revealed glucose, laminaribiose, gentiobiose, and cellobiose. Both
the cellulose I and II fractions gave glucose and cellobiose with the
same analysis. The residutmi, remaining after removal of the three
above fractions, produced small amounts of glucose, laminaribiose,
gentiobiose, and cellobiose upon hydrolysis. The unf ractionated wall
produced all of the above sugars except laminaribiose, even after a
short (2 hr) hydrolysis period. Both the unfractionated wall and
cellulose II contained a compound which was unidentifiable by R value.
Before presenting the results of the enzyme hydrolysis studies,
some comments on the properties of the purified enzymes will be made.
The purified laminarinase (see Materials and Methods) when reacted with
a laminarin containing only 31,3 linkages, produced glucose, laminaribiose,
and laminaritriose. When, however, it was presented with a laminarin
containing mixed linkages of 31,3 and 31,6, gentiobiose appeared with
the above products. This enzyme preparation thus behaves like both an
exo-31,3-glucanase and an endo-31,3-glucanase. The purified cellulase
preparation obtained from Aspergillus n-igev exhibited activity against
carboxymethylcellulose in the viscometric assay and is thus classified
as an endo- or random splitting enzyme (Reese and Mandels 1963a) . When
this enzyme was reacted with wall fractions or the intact wall no
chromatographable compounds were formed in the reaction time used.
However, treatment of the wall or its fraction with unpurified enzyme
yielded glucose. The unpurified enzyme preparation also is able to
hydrolyze sucrose, as this disaccharide was never obtained in GLC
analysis, even though it was added as an internal standard at the time
of hydrolysis (Fig. 3e) .
Figure 3. GLC of the TMS derivatives of the monosaccharides released
by hydrolysis of the wall fractions or the total wall by
unpurified A. nigev cellulase. (a) Acid-soluble. Glucose
is the only product. (b) Boiled enzyme control. Small
peaks of mannose and glucose are contaminants of the enzyme
and substrate preparations respectively. (c) Cellulose II.
Glucose is the only product. (d) Boiled enzyme control,
(e) Total wall. Glucose is the only sugar produced. The
fructose and some of the glucose result from the action of
3-glucosidase, present as a contaminant in the enzyme pre-
paration, on sucrose added as the internal disaccharide
standard. (f) Boiled enzyme control.
55
f.
TIME (min)
TIME (min)
56
Table 3 gives the results of enzyme hydrolysis of the various
wall fractions. Neither lipase nor protease showed activity against
any of the wall fractions. Purfied laminarinase hydrolyzed the acid-
soluble portion of the wall releasing glucose, laminaribiose, and
gentiobiose. Glucose was released from all the fractions, except the
chitinous one. Laminaribiose was found in the unfractionated wall after
treatment with laminarinase. This disaccharide was also found in
laminarinase hydrolysat.es of the alkali-soluble fraction and the
insoluble residuum. Based on the R value, cellobiose was also found
g
with laminarinase treatment of this residuum.
In a number of cases an unidentified spot appeared with a variable
R value. It was always higher than laminaribiose but lower than
glucose. The R value of this spot was a little higher than that found
in some of the acid hydrolysates. All the control enzyme and substrate
solutions were chromatographed, and no spots were found. From the GLC
studies, a compound which cochromatographed with mannose, was found
associated with both laminarinase and cellulase; however, the R value
g
of authentic mannose is higher than glucose for this solvent system.
The data obtained from the GLC studies is similar to that found
with paper chromatography. Acid hydrolysates (Fig. 4a-c) of the acid-
soluble and cellulose II fractions plus the unfractionated wall yielded
a mono- and disaccharide pattern similar to that found in the paper
chromatographs. Some differences, however, were found. The hydrolysate
of cellulose II consisted of not only glucose and cellobiose, but also
small amounts of laminaribiose and gentiobiose (Fig. 4b). The gentio-
biose component of this fraction was small, but it was difficult
Figure 4. GLC of the TMS derivatives of the mono- and disaccharides
released by acid hydrolysis of wall fractions and total
wall. (a) Acid-soluble. Products are glucose, laminaribiose
and gentiobiose. (b) Cellulose II. Products are glucose,
cellobiose, laminaribiose, and gentiobiose. (c) Total
wall. Products are the same as in (b) .
58
b.
Mi
Sue
TIME (min)
59
to determine the amount of the laminaribiose portion because there was
an overlap between the two laminaribiose peaks and the second of the
cellobiose. Acid treatment of the unf ractionated wall revealed a
pattern similar to that of the acid-soluble fraction, except for the
presence of a small cellobiose peak. (Fig. 4c). Although laminaribiose
was not found in the paper chromatographic analysis of this hydrolysate,
it was found in the GLC analysis.
Laminarinase treatment of fractionated and unfractionated walls
is presented in Fig, 5a- f . Products from the enzymatic hydrolysis of
the total wall and the acid-soluble fraction were glucose, laminaribiose,
and gentiobiose (Fig. 5a and e) . No products were obtained from
laminarinase hydrolysis of cellulose II. Control samples containing
boiled enzyme were also analyzed, and the results indicated that this
treatment produced inactivation (Fig. 5b, d and f ) . The only substrate
which had any residual sugar was the acid-soluble fraction which gave
a very small glucose peak upon analysis. Treatment of the various
fractions with cellulase gave results which were similar to the paper
chromatographic studies (Fig. 3a-f ) . Glucose was the primary product
found in these hydrolyses, and for this reason only the monosaccharide
portions of these chromatographs are included.
Both laminarinase and cellulase were reacted with the unfractionated
wall and only glucose was obtained (Fig. 6a and b) . For some reason the
mannose component was absent.
Since glucose was the only monosaccharide found, this datum was not
quantified. The disaccharide components were quantitated based on their
peak areas and that of a known standard (sucrose) . The molar relative
response factors for each of the components found were not calculated
Figure 5. GLC of the TMS derivatives of the mono- and disaccharides
released by hydrolysis of the wall fractions or the total
wall by laminarinase. (a) Acid-soluble. Products are
glucose, laminaribiose, and gentiobiose. (b) Boiled enzyme
control. Mannose is a contaminant of the enzyme preparation,
(c) Cellulose II. (d) Boiled enzyme control. (e) Total
wall. Products are glucose, laminaribiose, and gentiobiose.
(f) Boiled enzyme control.
61
0 5
TIME (min)
Figure 5. Continued.
63
e.
f.
Mann
Mi
Sue
T — i — I — I r//^ .
Mi Sue
Mann
Glc
Glc
I r
pL
I" r
Mann
Mi
Sue
1 r
'^
0 5 10 15
v" I I 1 1 r
20 25 35 40 45 50 55
TIME (min)
64
5 10 15 20
TIME (min)
b.
5 10 15 20 25
TIME (min)
Figure 6. GLC of the TMS derivatives of the monosaccharides
released by hydrolysis of the total wall with
laminarinase and unpurified A. nigev cellulase.
(a) Total wall. (b) Boiled enzyme control.
65
because of variabilities in the system observed from day to day. It
was felt that greater accuracy was obtained by comparing the areas of
the peaks in question to known standards run simultaneously. The ratio
of laminaribiose to gentiobiose in the acid-soluble fraction following
acid hydrolysis was 1:0.72 and following enzyme hydrolysis was 1:0.57.
In the unfractionated wall acid hydrolysis gave 1:1.02 and enzyme
hydrolysis 1:0.9.
Periodate oxidation studies were done in order to gain some knowledge
of the linkage and branching patterns in the various wall fractions. In
addition to the studies of the wall fractions, three standard poly-
saccharides of known linkage patterns were also analyzed. These were
cellulose powder (Whatman Ashless Powder, Chromatographic Grade) and
the two different laminarins described previously. Table 5 gives the
values of periodate consumption and formate liberation for the various
wall fractions, and Table 6 for the polysaccharide standards.
Observations of unfractionated walls, wall fractions, and live
hyphae under polarizing light revealed strong birefringence in the
cellulose II fraction but none in the acid-soluble. Both live hyphae
and isolated walls showed birefringence.
The results of x-ray diffraction analysis of cellulose II isolated
by dissolution with Schweitzer's reagent or with cadoxen are presented
in Table 7 and Fig. 7a and b. The lattice spacings of both preparations
were the same as those found for the avicel cellulose II standard.
Amino Sugars
Because of the relatively large amount of glucosamine found in
the samples analyzed for amino acids, a more detailed study of this
66
Table 5. Periodate consumption and formate liberation
of A. ambisexualis wall fractions.
Fraction
Acid-soluble
Alkali-soluble
Cellulose II
Insoluble residuum
Total Wall
Cellulose I
moles per mole glucose
values after 96 hr of treatment
values after 48 hr of treatment
Periodate^'^
Formate
0.917
0.427
0.609
0.091
0.329
0.031
0.610'^
0.031^^
0.963
0.213
0.366
0.061
67
Table 6. Periodate consumption and formate liberation of
known polysaccharides.
Polysaccharide
Periodate
b
a,b
Formate '
Whatman cellulose powder
0.370
0.031
Laminar in (31,3 linked)
0.159
0.152
Laminar in (31,6; 31,3
linked)
0.329
0.152
moles per mole glucose
values after 96 hr of treatment
68
Table 7. X-ray diffraction analysis of Schweitzer's
and cadoxen reagent-soluble fractions of
A. cmbisexualis wall.
Samples
Lattice spacings in A
Avicel standard
7.37 4.46 4.08
Schweitzer's
7.37 4.46 4.08
Cadoxen
7.37 4.46 4.08
Figure 7. X-ray diffraction patterns of cellulose II isolated
from A. amhisexualis walls, (a) Cellulose dissolved
with Schweitzer's reagent. (b) Cellulose dissolved
with cadoxen.
70
r5
71
monosaccharide was done. The solubility characteristics of the glucosa-
mine component of the wall were studied and the results are given in
Table 8. A large portion, 98.5%, of the glucosamine was insoluble in
both dilute acid and base. The preliminary characterization of a small
chitinous component isolated from the wall has already been described.
Uronic Acids
Preliminary studies of the uronic acid content of the wall gave 0.03
mg/100 mg dried wall prepared by buffer-water washing. No attempt was
made to identify which uronic acids were present.
Protein and Amino Acids
Total protein of the untreated wall was 6 mg/100 mg dried wall
after preparation by buffer-water washing. If, however, these walls
were washed with 1 N NaOH and placed in a 50°C water bath for 3 hr, the
total protein value increased to IQ mg/100 mg wall. Total protein was
also measured in the various wall fractions. The acid-soluble fraction
was the only one which showed the presence of measurable protein, 1.5 mg/
100 mg dried wall. Traces of protein were found in the cellulose II
fraction and in the insoluble residuum. Much of the protein was
probably destroyed or washed away during the fractionation processes.
Amino acid analyses performed on both types of wall preparations
showed distinct differences (Table 9) . The chemically cleaned walls
contained very low levels of amino acids and in some cases certain
expected ones were missing, even when as much as a 200 mg wall sample
was used. Walls which were buffer-water washed contained the whole
spectrum of amino acids, including hydroxyproline (3%) (Table 9). A
72
Table 8. Analysis of solubility of glucosamine from
unfractionated walls of A. ambisexualis .
Treatment mg Glucosamine
1 N NH^OH, 25 "C
1 N acetic acid, 98°C
Insoluble
Total glucosamine 2.626
mg/100 mg dried wall, buffer-water washed
0
039
0
000
2
587
73
Table 9. Amino acid profile of the total wall of A.
ambisexuatis after chemical or buffer-water
cleaning.
Amino Acid or
Amino Sugar^
Chemical
Buffer-water
Glucosamine (average)
0.513
1.224
Lysine
0.009
0.298
Histidine
0.016
0.085
NH3
0.629
0.205
Arginine
0.015
0.207
Hydroxyproline
0.103
Aspartate
0.009
0.187
Threonine
0.002
0.308
Serine
0.002
0.222
Glutamate
0.007
0.419
Proline
T
0.162
Glycine
0.007
0.166
Alanine
0.058
0.226
Cysteine
0.043
Valine
0.006
0.206
Methionine
0.004
0.045
Isoleucine
0.004
0.172
Leucine
0.009
0.296
Tryosine
0.104
Phenylalanine
0.008
0.168
Total amino acid
0.156
3.417
mg/lOO mg dried wall
200 mg sample used for hydrolysis
'20 mg sample used for hydrolysis
74
comparison of the total amounts of amino acids/100 mg dried wall revealed
that the buffer-water washed walls contained nearly 22 times more amino
acids than the chemically cleaned walls. The level of total protein
in the buffer-water washed walls was much higher than the total amino
acid content (10 mg and 3.42 mg respectively).
Examination of Table 10 and Fig. 8 indicates that the amino acid
profile of the wall changed drastically during the chemical cleaning
process. Detergent treated walls which were washed once with ethanolic:
KOH followed by a 10 min incubation in a boiling water bath had a greatly
reduced amino acid content. In a 20 mg sample only measurable amounts
of lysine and methionine were present, and only trace amounts of
aspartate, threonine, serine, glutamate, glycine, alanine, isoleucine,
and leucine. Even detergent treated walls, which theoretically contained
all covalently bound amino acids but not others, had a generally low
level. These walls were also missing hydroxyproline, an amino acid
generally found associated with cellulosic walls. The buffer washed
walls (one washing) did not appear clean and most likely contained a lot
of contaminating membrane proteins. The level of hydroxyproline was
very low in these walls. Observations of Fig. 8 indicate that the
profile in the buffer-water washed walls (washed until clean micro-
scopically) in general follows that of the single buffer washing. The
amino acid content of the supernatant after the initial pelleting
represented only those amino acids which were soluble, as this fraction
was not treated with any solubilizing agent. Significantly, hydroxy-
proline was missing from this sample. Strangely, histidine was also
missing, and proline and cyteine were present in only trace amounts.
In addition, glucosamine was not found.
75
U)
OJ
<u
00
rH
to
P4
4-1
£2
cn
CO
en
en
3
U-l
0
O
•H
^
en
CO
OJ
>
to ^<i
o 5
C H
1-1 ta
0 CO
o ^5;
.
cn
u
•H U-l
c
M C
•H
CO
C
CirH
CO
R ^
<U
O CO
iH
u 3
O
<U
CO
XI
A
OJ
to
0)
u
CO
a
H
rs
•H
H
4J
V-i
c
(U
OJ
(U
1+-I
c
at
U-l
o
M
0) 0) T-l
4-1 j: H
iM tn
3 CO 0)
PQ 3 C
O
4J
CO CO
c
•H W
CO
■u C
4J
•H -H
CO
C JJ
C
M 0)
1-1
tH
Q)
I-J rH
A
0) 0)
3
•u pu
c/1
y-i
<;
o
00
rsi
tJ^
CN
O
o
o
in
o
o
00
CM
00
o
o
00
o
00
H O
o
CM
00
00
H O
o
O
U1
CM
1— 1
u-l
H O
o
O
O
o
o
O
o
o
o
o
O
O
o
O
o
O
iH
O ^ t^ ^ -^
CM tT\ >£i m c-g
i-i r^ CM CM vo H
CJ^ o m
m vo o
vO u-l CM
<3-CTivOOr~»C<rcOtncO
tH o o o o
iHOOrHOOOOOOOOOO
0^ CM
00 in
O iH
CM
in
CM
in
o
in
H
O
CM
m
O
in
CM
CM
O
CM
00
O
tJN
m
iH
o o
o
O
o
o
o
o
O
o
O
O
o
O
<U
C>0
(0
v^
<u
>
CO
c
a)
rH
c
o
•H
CU
%j <U
0)
dJ
R
c
0)
a -u
C
4-1
CO
•H
c
>i CO
•H
to
OJ
(U
en
(U
-n
•H
X 4-)
fi
eu
R
C
3
o
c
•H
c
O ^1
o
C
CO
•H
•H
tj
•H
4-1
•H
U CO
CU
•H
4-J
1-4
O
3
en
en
ro 00 -O &.
Vj
M
3
O
>.
t-l
>,
•H
33
>-l
>. en
^
0)
iH
u
iH
O
1-1
ffi
Z
<
33 <
H
U2
O
PU
U
C'H 0) O 3 C-Wr-I
•H -H (U c -H OJ T-l en ■
C 4-1 -H J3 tH O O
caenrH4-io3>,aj
iHr-i>.taaicn<DMj3
■ <u>s;i-ihJHPH
0)
to
c
•H
o
c
3
crt
•H
1— 1
e
to
CO
>>
1— (
c
CO
P-
0)
ti
•H
to
)-l
en
Ta
Of)
00
S
e
s s
to jQ
H
3
>J
n)
rO
4-1 d
S
C 4-1
M 1
OJ 14-
iH
c
60 =
to
•H •
U X
U
c 1
(U
O
tfl
4-1
4-1
0) .
<u
^
-o
0)
U 1
4=
A
u
rH ••>
0)
cd ta
e
<+-!
a ao -H 1
O
•H c
4J
CO
oj w
tU CO
OJ
JZ 0)
c •v.
rH
O iH
O 0;
O.
iH
4-
id
u^ 0)
T3 c:
n)
o a.
Qi QJ
to
J2 ^-1
CO rH
CO 4J
M-4
0) CO
CO
O
ao'H
? M
cO -u
C
to
4J -H
U «
CU CO C 0) I
y-i 3 >-i 3 iH
O O 0) ^ o
D. M
<4-l .
CO •
CO
CO
J=U3
T) >
•
■w C
•H
4-1
(U CO
a ao
c •
, 1J
CO a
CO
T—l
•H
4-1 •
1 '^
O (-1
CO
C 3
fl •'
1 iH
•H T3
^1 CO
•H
g
0) oj
4-1
CO CO
ft e
1 c
•<>'
3 -H
1 3
14-1 T<1
CO 4J
Q «
1 "^
!^
1 '^
(U
C H
c
J=
O Q)
• O
• • to
CO CO
•
CO CO
•H -t^
1 -
"tJ >
^4 r£i
T1
OJ
CO g
'. OJ
4-1 tJ
ft 8
,• -^
CO 0)
a
CO
01 4-1
o .
CO
>-( CO
U -q;
^ 3
4J S
77
ONIl^V UO aiOV ONIl^V^oi
78
Lipid
Only trace amounts of extractable lipids were found by the method
used. The pattern of spots seen on thin layer chromatography plates
suggested mainly phospholipids, and this was confirmed by a colormetric
spray test specific for these compounds.
Phosphorus
The level of phosphorus per 100 mg dried wall was 0.15 mg.
Total Wall Composition
The composition of the total wall is given in Table 11. When all
the components were added the total represented 86.44% of the wall. The
remaining 13.56% represented unidentified constituents or experimental
losses.
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase
The results of the hydrolysis of isolated walls by A. canbisexualis
cellulase can be seen in Figs. 9 and 10. In the first 24 hr of hydrolysis,
there was a significant increase in total reducing sugars in the treated
wall samples as compared with the controls. This increase continued for
another 72 to 96 hr, but at a reduced rate, and ceased between 96 and
120 hr.
Ultrastructural Studies
Surface Structure of Chemically Treated Walls
The replica of an untreated wall reveals a smooth surface (Fig.
11a). Walls treated with 0.5 N HCl appear somewhat less smooth than the
79
Table 11. Chemical constituents of the buffex-water
washed walls of A. amb-isexualis .
Constituent % Dry Weight
1. Glucan^ 52.80
2. Glucan 50.23
3. Cellulose II 20.83
4. Alkali-soluble hexosamine 0.04
5. Insoluble hexosamine 2.59
6. Protein 10.00
7. Total amino acids 3.42
8. Uronic acids 0.03
9. Phosphorus 0.15
10. Readily extrac table lipids T
86.44%
c/C
determined (as glucose) with anthrone on unhydrolyzed
walls; calculated as anhydroglucose
sum of acid- and alkali-soluble portions and the
insoluble residuum of the wall
'^sum of 1, 3, 4, 5, 6, 8, and 9
o o •>
01
01
N
u
en
a
cd
iH
a
6C
3
p
1— 1
A
05
M
CJ
C
•H
CO
a
•r^
3
r<»
tJ
«
0)
u
H
«<
•^^^
•
c
to
y*^
•H
•r-i
• «
,
ri.1
(U
OJ
(U
u
iJ
03
^^
m
nJ
00
^j
J-i
<U
u
-u
U
"^
05
05
U
ja
ja
c
>,
3
3
M
Ji
05
05
81
f
\
\ <
\
\
\
\
<3 \.
\
\
en
\
\
\
N.
\
I r
CVJ —
-r
o
CD
T"
1^
*. o
o
Ld
CO
to
o
h-CM
LU
lO ^
yvons 9NI0nG38 yO\X\^/^f^
o
!-i
T)
T}
c
>-,
CO
J2
dJ
U
f=
0)
s
JJ
N
14-1
c
CO
0)
i-l
M
iH
o
crt
4-1
S
T3
•T3
<U
cu
t:
W
CO
a
u
CO
3
CO
iH
o
M
3
n
3
.H
u
cn
tH
^
0)
CJ
M
u
CO
C
XI
•H
03
O
•'SI
rH
3
«1
o
t3
«
)4
Q)
■u
U
u
C
w
o
C
03
o
83
yvons 9Niona3d ,01 x\^/^^
Figure 11. Surface replicas of isolated walls treated chemically,
(a) Untreated. The surface is relatively smooth with a
few microfibrils. X 26 000. (b) 0.5 N HCl treatment.
Microfibrils are indistinct in both the control (inset)
and the treated, X 30 000. (c) Treatment with 0.5 N
HCl followed by 2 N KOH. Microfibrils, not seen in the
control (inset), are evident after treatment. X 30 000.
(d) Sequential treatment of 0.5 N HCl, 2 N KOH, and
cadoxen. Control material (inset) appears intact and
smooth, while in the treated only scattered pieces of
amorphous material remain. X 27 000 (control) , x 30 000
(treated). (e) Cadoxen-soluble wall material reconstituted.
No recognizable microfibrillar pattern is seen, x 30 000.
85
86
untreated, but no microfibrillar structure is revealed (Fig. lib). Walls
treated with 0.5 N HCl followed by 2 N KOH show a pattern of microfibrils
(Fig. lie). Sequential treatment of walls with acid, alkali, and cadoxen
results in a general disintegration with only pieces of amorphous material
remaining (Fig. lid). When the cadoxen-soluble material was reconstituted
as cellulose II, a definite linear pattern resulted (Fig. lie). This
pattern does not resemble the microfibrillar one seen after acid and
alkali treatment.
Surface Structure of Wall Fractions
A surface replica of the acid-soluble fraction has an amorphous
appearance (Fig. 12a) , while that of the alkali-soluble fraction appears
weakly microfibrillar (Fig. 12b). The surface of the cellulose II
fraction does not appear microfibrillar in Fig. 12c, but does in
Fig. 12d. Cellulose I has a microfibrillar pattern similar to that
seen after acid and alkali treatment (Fig. 12e) . The insoluble residuum
reveals faint microfibrils (Fig. 12f ) . The chitinous portion of the
wall is microfibrillar, and it appears that the individual microfibrils
are bound together in bundles (Fig. 12g) . These bundles are arranged
longitudinally, and there are a few microfibrillar groups which seem
to run perpendicularly to the longitudinal ones.
Surface Structure of Chemically Treated Live Hyphae
The smooth surface of an untreated hyphae is seen in Fig. 13a.
Mild acid (0.5 N HCl) treatment suggests an underlying pattern (Fig. 13b).
Treatment with 0.5 N HCl followed by 2 N KOH produces a dramatic change
with microfibrils becoming very evident (Fig. 13c) . The surfaces of
Figure 12. Surface replicas of wall fractions. (a) Acid-soluble
fraction. The surface is amorphous. X 30 000. (b)
Alkali-soluble fraction. Some microfibrils are visible.
X 30 000. (c) Cellulose II. No microfibrillar pattern
is evident. X 32 000. (d) Cellulose II. Microfibrils
are seen in apparent aggregations. X 32 000. (e)
Cellulose I. Microfibrils are evident. X 32 000 (f)
Insoluble residuum. A faint microfibrillar pattern is
seen. X 32 000. (g) Chitin. Distinct arrangements
of microfibrils are seen. X 32 000.
Figure 13. Surface replicas of live hyphae after chemical treatment,
(a) Untreated. The surface is smooth. X 27 000. (b)
0.5 N HCl treatment. The surfaces of both the control
(inset) and the treated samples appear amorphous. Faint
microfibrils are seen in both samples. X 32 000. (c)
Treatment with 0.5 N HCl followed by 2 N KOH. The control
(inset) surface is amorphous, while that of the sample
shows distinct microfibrils. X 30 000. (d) Sequential
treatment of 0.5 N HCl, 2 N KOH, and cadoxen. The surface
of the control (inset) is smooth, while that of the
sample is striated with a suggestion of fibrillar material.
X 30 000.
90
91
hyphae after acid, alkali and cadoxen treatment appear textured with a
striated pattern and faint microfibrils can be seen (Fig. 13d) .
Surface Structure of Enzymatically Treated Live Hyphae
Walls treated wtih laminarinase show distinct microfibrils while
those treated with boiled enzjme resemble untreated walls (Fig. 14a) . The
cellulase prepared from Aspergillus niger or Aohlya ambisexualis gives a
smooth surface in both cases (Fig. 14b and c) . The protease treated
walls resemble their controls (Fig. 14d) .
Sequential treatment with laminarinase and protease reveals
microfibrils somewhat more sharply than treatment with laminarinase
alone (Fig. 15a). Treatment with laminarinase followed by A-. niger
cellulase produces naked microfibrils (Fig. 15b) . Laminarinase followed
by A. ambisexualis cellulase results in a fragmented appearance with no
long sections resembling hyphae remaining. The resulting pieces show
scattered microfibrils (Fig. 15c) .
Results from the sequential treatment with laminarinase, protease,
and then cellulase from either A. niger or A. ambisexualis are given
in Fig. 16a and b. The treatment containing cellulase from A. niger
showed that drastic digestion had occurred and it was difficult to find
structures resembling hyphae. When found, such pieces give the appearance
of scattered microfibrils (Fig. 16a). Walls treated with the mixture
containing A. ambisexualis cellulase reveal a pattern similar to that
seen in the laminarinase plus A. ambisexualis cellulase treatment (Figs.
16b and 15c). Nothing resembling a hyphal structure was found.
Microfibrillar Diameter
The diameter of the few microfibrils detected in buffer treated
samples was 11.23 nm while those of the laminarinase or
Figure 14. Surface replicas of live hyphae after single enzyme
treatment. (a) Laminarinase. Microfibrils, not seen in
the control (inset) , are very evident in the treated
sample. X 26 000. (b) A. nigev cellulase. Both the
control (inset) and the treated surfaces are amorphous.
X 30 000. (c) A. amb-tsexualis cellulase. Both the
control (inset) and the treated surfaces are amorphous.
X 26 000 (control), X 30 000 (treated). (d) Protease.
Both the control (inset) and the treated surfaces are
amorphous. X 30 000.
93
•kf'irti
Figure 15. Surface replicas of live hyphae after sequential enzyme
treatment. (a) Laminarinase and protease. The control
(inset) appears amorphous while the treated is distinctly
microfibrillar. X 30 000. (b) Laminarinase and A. nigev
cellulase. The control (inset) is smooth, but microfibrils
are evident in the treated hyphae. X 26 400. (c)
Laminarinase and A. ambisexualis cellulase. The control
(inset) appears normal, but the treated hyphae are almost
totally destroyed. One of the few remaining pieces shows
some microfibrils. X 26 000 (control), X 30 000 (treated).
95
Figure 16. Surface replicas of live hyphae after sequential enzyme
treatment. (a) Laminarinase, protease, and A. niger
cellulase. The control (inset) appears amorphous. The
treated hyphae are disrupted with only scattered patches
of microfibrils remaining. X 30 000. (b) Laminarinase,
protease, and A. ambisexualis cellulase. The control
(inset) is smooth. The remaining pieces of the treated
hyphae reveal few microfibrils. X 30 000 (control). X
24 900 (treated).
97
98
laminarinase-protease treated were 15.96 nm and 15.43 iim respectively
(Table 12) . The diameter of microfibrils revealed in hyphae treated
with 0.5 N HCl and 2 N KOH was 20.29 nm (Table 12). Measurements of
cellulose I microfibrils indicated that the width was 21.76 nm (Table
12).
99
Table 12. Microfibrillar diameter of various prepar-
ations from A. ambisexnatis.
Treatment of
Live Hyphae Microfibrillar diameter'
Buffer 11.23
Laminarinase 15.96
Laminarinase-protease 15.42
0.5 N HCl- 2 N KOH 20.29
Wall fraction-Cellulose I 21.76
nm; final number is average of between 20 and 60
measurements
DISCUSSION
The Preparation of Wall Samples
A comparison of the two methods of wall preparation reveals
certain major differences in the chemical composition. A large dis-
crepancy in total amino acids and amino sugars is evident (Table 9).
The values for buffer-water washed walls were 3.42 mg amino acids and
1.22 mg amino sugars/100 mg dried walls, and for chemically cleaned walls
0.16 mg and 0.51 mg, respectively. The quality of amino acids was also
different as judged by the lack of hydroxyproline, cysteine, and tyrosine
in the chemically cleaned walls (Table 9). Proline was present in these
preparations in only trace amounts. The combination of detergent
treatment and extraction with ethanolic KOH in boiling water removed
certain amino acids and perhaps proteins as well. This could be
significant since protein may be a structural component of the wall
(Hunsley and Burnett 1970; Wrathall and Tatum 1973). These results
and others (Cameron and Taylor 1976) indicate that a given method of
wall preparation clearly influences the values obtained.
Chemical Fractionation of the Wall
The fractionation procedure used in this study has been employed in
analyses of the walls of Atkinsiella dub-La (Aronson and Fuller 1969),
Pythium dehamjamm (Yamada and Miyazaki 1976), and two species of
Phytophthora (Bartnicki-Garcia 1966). The value of 37.58% for the
100
101
acid-soluble portion of the wall in A. ambisexualis (Table 2) compares
with 16.3% for A. dubia (2.90% had already been solubilized in boiling
water) (Aronson and Fuller 1969) and 6 3% for both Phytophthora sp.
(Bartnicki-Garcia 1966) and P. debccryanvm (Yamada and Miyazaki 1976) .
The value of 7.07% for the alkali-soluble portion compares with 20% for
A. dubia (Aronson and Fuller 1969) and P. debaryanum (Yamada and Miyazaki
1976), and 3.1% for Phytophthora sp. (Bartnicki-Garcia 1966).
In this study the first fraction which was removed from the wall
was the acid-soluble one and this was the major wall component. Analyses
of enzyme and acid hydrolysates of this fraction by paper (Tables 3 and
4) and gas chromatography (Figs. 3, 4, 5, and 6) gave laminaribiose and
gentiobiose, thereby demonstrating the presence of both 61,3 and 61,6
linkages (Aronson et at. 1967). No cellobiose was found. The data on
periodate consumption and formate liberation (Table 5) plus the solubility
in water or dilute acid are consistent with a highly branched glucan
structure (Sietsma et al. 1969) . To account for the ratio of laminari-
biose: gentiobiose found in the GLC analysis, a structure with a 61,3
backbone with single glucose units linked through the sixth carbon atom
occurring every fifth glucose residue is suggested. Glucans with single
unit branches have been postulated for the walls of S. fevax and D.
sterilis (Sietsma et al. 1969) as well as Pythiwn sp. (Eveleigh et al.
1968) and Pythium aaanthicum (Sietsma et al. 1975). In S. fevax and
D. stevi-lis this fraction makes up 43% of the wall (Sietsma et al.
1969). It is possible that the 61,6 linked side chains are longer than
one unit (Zevenhuisen and Bartnicki-Garcia 1969) or that parallel homo-
pol3rmeric chains of 61,3 and 61,6 linkages are present. A purified
102
3l,6-glucanase and a known SI, 6 glucan would be useful in testing this
possibility. Several variations of this type of glucan have been
suggested. In Phytophthora sp. and Pythium sp. the side branches are
31,3 linked and a few 81,4 linkages are also found in the acid-soluble
complex (Novaes-Ledieu and Jimenez-Martinez 1969) . In Pythium debaryanum
the acid-soluble fraction is described as a 61,3 branched glucan (Yamada
and Miyazaki 1976) . In Phytophthora cinnamomi the portion of the wall
insoluble in Schweitzer's reagent {i.e., the 31,3 and 61,6 glucans) is
said to consist of a core of 61,3 or 61,6 or mixed linked main chains
with short (four or five units long) 61,3 linked side branches (Zevenhuisen
and Bartnicki-Garcia 1969). A few 61,4 linked chains are also thought
to exist in this fraction (Zevenhuisen and Bartnicki-Garcia 1969).
The second fraction isolated from the wall was soluble in 2 N KOH.
This fraction released glucose, laminar ib lose, gentiobiose, and cellobiose
upon acid hydrolysis (Table 4) . Treatment with laminarinase released
glucose and laminaribiose (Table 3) . These results indicate three
different linkages, 61,3, 61,4, and 61,6. The high amount of periodate
consumption relative to formate liberation plus the insolubility in
water and dilute acid suggest that this component is rich in 61,4 linked
glucans (Table 5) . The observation of faint spots representing gentio-
biose also suggest 61,6 linked units. A similar preparation from
P. aoanthiaim also contained all three of these linkages (Sietsma et al.
1975), while the one from P. debaryanum had only 61,3 and 61,6 forming
highly branched glucans (Yamada and Miyazaki 1976) . The proportion of
61,4 glucans in this fraction suggests that it may represent material
from the cortex of the microfibrils, thought to consist of cellulosic
103
and hemicellulosic components thus differing from the cellulosic core
(Preston 1974a).
The third fraction isolated wais soluble in Schweitzer's or cadoxen
reagent and is thus cellulose II, a 31,4 glucan. Both x-ray diffraction
analysis (Table 7 and Fig. 7) , which is considered definitive evidence
for cellulose (Weijman 1979) , and polarizing light microscopy are
indicative of cellulose II. The presence of glucose and cellobiose in
acid hydrolysates of cellulose I, v.e. , not solubilized and regenerated
(Table 4), provide additional evidence for cellulose. X-ray diffraction
studies would be useful in confirming the identity of this component.
The amounts of isolated cellulose I and solubilized cellulose II are
similar (Table 2). When these two substances are compared to a cellulose
standard by periodate oxidation they are similar (Table 5). Cellulose
I liberated about twice as much formate as cellulose II or the known
cellulose standard, indicating a shorter chain length. The presence of
laminaribiose and gentiobiose (Fig. 4) indicated that the cellulose II
fraction was not pure. This result is not surprising in light of Preston's
(1974a) concept of microfibrillar structure.
The insoluble residuum is similar to the alkali-soluble component.
A difference is seen in the ratio of periodate consumption to formate
liberation (Table 5) suggesting that the polymeric chains of this
fraction are longer than those of the alkali-soluble. Based on the
fact that this fraction remains after acid, alkali, and cadoxen treat-
ments, it is postulated that this residuum lies adjacent and is tightly
bound to the plasmalemma. Its extreme insolubility may be due to
extensive cross-linking of the polymeric chains and its function may
104
be protective. Or, it is possible that this fraction is part of the
others and is the result of incomplete solubilization.
Analyses of enzyme and acid hydrolysates of the unfractionated wall
(Tables 3 and 4, and Figs. 3, 4, 5, and 6) indicated the presence of a
majority of 31,3 and 31,6 linkages and a minority of 31,4. High
periodate consumption and a moderate amount of formate liberation were
seen (Table 5). It is hard to interpret this datum, especially when the
ratio of laminaribiose:gentiobiose, as determined from the GLC data, is
close to one, a deviation from that of the acid-soluble fraction. A
possible explanation for the large periodate consumption as compared with
formate liberation is the existence of 31,4 linkages.
The linking and branching data have been used to prepare a model
of the carbohydrate portion of the wall (Fig. 17) . Speculation on the
location of the protein component is also included in this model. A
cellulosic backbone, hydrogen bonded both within and between polymers,
is bound, again by hydrogen bonds, and possibly hydroxyproline-rich
protein (Novaes-Ledieu and Jimenez-Martinez 1969), to the 31,4 glucan
portion of the alkali-soluble fraction of the wall. The 31,3 glucan
segment of the alkali-soluble fraction "nests" with like components of
adjacent polymers forming a strong pliant structure. The outer 31,3
linked glucans of the nest are part of the acid-soluble fraction of
the wall and are characterized by the presence of 31,6 linked branch
points occurring about every fifth glucose residue. The function of
uronic acids, the glucosamine units from the small alkali-soluble
component (Table 8) (Novaes-Ledieu and Jimenez-Martinez 1969) , and the
31,6 linked branch points may be to serve as bridges between the polymeric
components.
en
>^ fl
to
X -H
0)
O
iH
^
M
^1 T3
1-1
CO
CO
T3 lU
to
tu
>^'a
»
(U
c
4= 3
J=
■H
iH
r-i
H
rH
!-i a
ta
o a
,c
u
14-1 -H
a.
•
a
>%
M
<u
CO 4-1
•
j=
to
u
eu O
c
M
CO
■u C
•H
(U
3
•r-l
•H
CO
j::
a
TD
m to
4-1
4J
01
CO
•H
!-i
1— 1
fH
0)
U-l
o
C4-I
to B
a
o
E
O
•H 3
c
Vj
4J 3
3
c
0)
60
c -a
o
4-1
c
(U -H
en
•H
c
•H
4-1 to
•H
4J
■H
U
O OJ
M
Cfl
a M
■H
o
-o
<u
rH
0-
c
C
OJ <u
CO
CO
43 -H
3
0)
o;
43
4-J
1
j=
0 3
OJ
to
CO
w
U tH
-C
u
u
O
4-1
•V
u
P>>T3 CO
>^
c
43
0) C
3
J=
•H
^ T-l
•H
O
TJ
0)
43
4=
eu
-a 0)
c
Ul
U
j-i
•H 43
o
to
O
CO
m H
•H
o
XI
0)
c
4-J
^
o
•H
OJ
A
CJ
o •
eo
x:
to
CO
o
4-1
T3
(U
OJ c
a.
C
M
1-4 -H
U-l
O
CO
CO 0)
CO
O
42
■u
4-1
—
T3 O
•H
iH
c
S
C M
0)
(U
o
to a
eo
•a
60 -H
CO
O
O
rH
to 4=
S
M
o
C O
A
■a
X
CO -H
iH
M
>.
^
a ^j
0)
CO
^
3 1
T3
iH
-a
rH OJ
Q
3
a>
60 C
g
U
iH
•H
0)
(U
C^ r-l
CO
iH
43
« o
•H
Q
to
iH V4
x:
s
■H
CO. a
4J
106
UJ
ixl
2
CO
< <
o
2 cr
_J
C/) 03
z>
1- ^ a^.
m, !■_ '
«4 <i ■^—
_1 UJ
_1
Q- 2
UJ
o
Q
.- UJ
m _j <
t-T "-T _]
QQ OD CD
UJ <
rO to v; CJ,
<-• ^ £ _i
CD CD _l O
or
o
ir
UJ
V
X
UJ
107
Chemical Analyses of Wall Constituents
Neutral Sugars
Comparison on a percentage basis of the chemical components of the
A. amb-isexualis wall with other members of the Saprolegniales indicates
a fundamental similarity. Glucose was the only neutral sugar found in
the A. amb-isexualis wall. In general this is the pattern for other
members of this order, although trace amounts of several other monomers
have been reported (Crook and Johnston 1962; Parker et at. 1963; Novaes-
Ledieu et al. 1967). While the total glucan of unhydrolyzed walls is
52.8% and of hydrolyzed walls, including the insoluble residuum,
50.23%, this does not account for the entire glucan component of the
wall (Table 11) . The cellulosic component of the wall is excluded from
the above figures, as it has been found that the quantitation of glucan
of this fraction cannot be determined by the anthrone method. This
results from its extreme insolubility, even in the sulfuric acid medium
of the anthrone reagent. If the assayable glucan and the cellulose
portions are added together the wall consists of 73.63% glucan. This
figure is in agreement with that found for Sapvolegnia dialina (72.6%)
(Cameron and Taylor 1976) and Atkinsiella dubia (78.4%) (Aronson and
Fuller 1969), but is lower than that for some other species of
Sapvolegnia (Novaes-Ledieu et al. 1967; Sietsma et al. 1969). The
cellulose content of A. ambisexualis walls is 20.83% which agrees quite
closely with the 18% found in Saprolegnia ferax and Diatyuohus stevilis
(Sietsma et al. 1969) and the 15% found in various species of Saprolegnia,
Achlya, Bvevilegnia, and Diatyuohus (Parker et al. 1963). Other workers
108
have found much higher amounts (42%) in the walls of S. ferax (Novaes-
Ledieu et al. 1967).
Amino Sugars
The hexosamine content of the wall in A. ambisexualis is 2.6%
(Table 8) and is similar to that found by Dietrich (1973) for three
other species of Aahyla and that found in S. ferax and D. steriZis
(Sietsma et al. 1969). Studies of other members of this order revealed
lower concentrations of amino sugars (Novaes-Ledieu et al. 1967;
Cameron and Taylor 1976) . Hexosamines are very susceptible to acid
destruction as can be seen in the different amounts found in 4 N HCl
(2.60% after 16 hours) and 6 N HCl (1.65% after 12 hours). Thus the
actual amount of this sugar is probably higher than reported values.
The hexosamine component in A. ambisexualis walls is glucosamine as
identified by standards run with the samples in the amino sugar analyses.
Solubility in 1 N acids is one of the methods used to distinguish
chitosan from chitin, the former being soluble while the latter is
not (Muzzarelli 1977; Mendoza et al. 1979). Chitin is insoluble in both
acid and base (Tracey 1955). The isolated chitinous component from A.
ambisexualis walls is not chitosan based on solubility characteristics
plus hydrolysis by chitinase. Lin et al. (1976) reported that in
Apodaohyla insoluble glucosamine is indicative of the presence of
chitin in the wall. This was based on former studies identifying the
presence of chitin by x-ray diffraction (Lin and Aronson 1970) . Perhaps
deacetylation occurred under the conditions of hydrolysis (Muzzarelli
1977) used prior to analysis of the insoluble glucosamine component and
the glucosamine content recorded is really N-acetylglucosamine. Most
109
likely the chitin isolated from the wall (0.63 mg/lOO mg dried walls)
is part of the larger insoluble glucosamine component (2.59 mg/100 mg
dried walls). The procedure for isolating chitin from the wall is quite
extensive, and it is possible that a considerable amount was lost. Since
the component from A. amb-isexuatis showed a positive reaction with
Lugol's iodine, a test for chitosan, it appears to be a weakly acetylated
chitin. X-ray diffraction analysis is necessary for the correct
identification of this wall constituent. N-acetylglucosamine has been
reported in the walls of S. fevax (Sietsma et at. 1969).
Uronic Acids
Uronic acids are labile in acids and are thus overlooked in some
studies or reported at levels lower than the actual value (Rosenberger
1976). For this reason it is possible that the value (0.03%) found for
A. ambisexuatis walls is lower than the true value. Walls of S. d-ialina
have been reported to contain uronic acids at the level of 1.3%
(Cameron and Taylor 1976).
Protein and Amino Acids
The 10% protein found in A. canb-isexualis walls is similar to the
8.5% found in S. di-alina walls by Cameron and Taylor (1976). These
figures are much higher than those found in earlier studies, i.e., 3% in
Sapvolegnia fevax (Sietsma et al. 1969), except in the case of A. dubia
where total protein was 13.7% (Aronson and Fuller 1969). The hydroxy-
proline content of the A. ambisexrj.alis wall is only 0,1% (Table 9),
although this is higher than the 0.02% found in S. diclina walls
(Cameron and Taylor 1976) . The value found for A. ambisexuatis walls
110
may be lower than the true value because ninhydrin was used as the
detector and this compound is known to underestimate the amounts of both
hydroxyproline and proline (Tristram and Smith 1963) . Separate analysis
for this imino acid should be done in order to get a more reliable value.
The presence of cysteine in A. ambisexualis at 0.04% (Table 9) is similar
to that reported for A. dubia (Aronson and Fuller 1969) . It was not
found in S. diclina (Cameron and Taylor 1976) . A comparison of the
total content of amino acids (Table 9) and the total protein indicated
a loss of 70% of the amino acids during hydrolysis and/or analysis. It
was noted during the hydrolysis that humin and NH„ were produced indicat-
ing destruction (Lippman et at. 1974; Taylor and Cameron 1973). It is
also known that the predominance of polysaccharides in the reaction
mixture will negatively influence the recovery of amino acids, except
for serine and threonine whose stability is increased (Tristram and
Smith 1963; Taylor and Cameron 1973).
Lipids
Only trace amounts of readily extractable lipids were found in
A. ambisexiAalis walls. This differs from the 8.2% reported in S. diclina
walls (Cameron and Taylor 1976) . Other analyses have found between 1
and 5% in the walls of related organisms (Noveas-Ledieu et at. 1967;
Sietsma et at. 1969). The trace of lipids found in this study was
separated by thin layer chromatography and upon staining gave a positive
test for phosphorus. Thus they may be phospholipids and the result of
membrane contamination. Bound lipids were not extracted in this study
but they were from the walls of S. dialina and found at the level of
3.75% (Cameron and Taylor 1976). Why there is such a wide discrepancy
Ill
in the amounts of this component in the walls of closely related organisms
is not known, unless it is an artifact of preparation.
Total Wall Composition
The remaining problem in these analyses of the wall components of
A. ambisexualis is the unidentified 13.5% (Table 11). Part of this
unidentified fraction probably represents loss of material during
hydrolysis. Another portion may be bound lipid and some may be ash.
The ash component was not identified beyond phosphorus, which was
analyzed and accounted for 0.15% of the wall. Ash content of closely
related species include S. fsrax at 3.2%, D. sterilis at 2.1% (Sietsma
et at. 1969), S. dialina at 2.48% (Cameron and Taylor 1976), and
Atkinsiella dubia at 1.5% (Aronson and Fuller 1969).
Hydrolysis of Buffer-Water Washed Walls by
A. ambisexualis Cellulase
A study of the digestion of isolated walls by A. ambisexualis
cellulase indicated that over a limited period of time there was an
increase in reducing sugars produced (Figs. 9 and 10). This enzyme is
active against carboxymethylcellulose and therefore is classed as an
endo-enzyme (Reese and Mandels 1963a) , It is evident from the description
of the cellulase from Aspergillus niger that there are several 6-glucosi-
dases in the preparation (Hirayama et a Z. 1976), but the ^. ambisexualis
cellulase is reported to lack exocellulase and B-glucosidase activity
(Thomas and Mullins 1969) . The leveling off of enzyme activity seen
here, even though visible pieces of wall remained in the reaction
solution (Fig. 9), corroborates the supposition that the A. ambisexualis
112
cellulase is an endo-enzyme. The reduced level of substrate is thought
to be responsible for the reduction of enzyme activity (Reese and Mandels
1963a) . For the total enzymatic hydrolysis of cellulose the combination
of endo- and exo-glucanases and 6-glucosidases is required; the endo-
enzyme is necessary for the generation of short pieces of cellulose on
which the other enzymes can act (Okazaki and Moo-Young 1978; Ghose and
Bisaria 1979) . In the consideration of the activity of any cellulase
knowledge of the structure of the cellulosic substrate is important.
Such characteristics as degree of polymerization, substitution, porosity,
and the distribution of crystalline and amorphous regions are important
in determining the level of activity of a reacting cellulase (Cowling
1963; Berg 1978; Sasaki et at. 1979; Fan et al. 1980).
Ultrastructural Studies
Surface Structure of Enzymatically Treated Live Hyphae
Replicas of A., amb-tsexualis cellulase-treated live hyphae indicate
no change in 48 hr of incubation (Fig. 14c) , but treatment of live hyphae
with laminarinase followed by A. ambisexualis enzyme show that drastic
hydrolysis has taken place (Fig. 15c). No long hyphae were seen in a
thorough examination of the replicas; only pieces remained. It seems
as if once the matrix is removed the enzyme is able to hydrolyze the
wall quite effectively, much better in fact than the A. niger cellulase
(Fig. 15b). The fact that the A. ambisexualis enzyme is more effective
than the one from A. niger, even though the latter appears more active
based on viscometric data, may be because the former is adapted to
hydrolyze the wall from within prior to lateral branch induction (Thomas
and Mullins 1969).
113
The effect of protease is interesting. It does appear that there
is a slightly better resolution of the microfibrils in live hyphae
treated with laminarinase followed by protease (Figs. 15a and 14a),
thereby confirming the observations made by Hunsley and Burnett (1970) .
Hyphae treated with the sequence of laminarinase-protease-/!. nigev
cellulase appear to be almost totally destroyed (Fig. 16a) . It would
seem from these data that the cellulosic-protein linkages proposed by
Hunsley and Burnett (1970) may in fact occur, as microfibrillar aggre-
gations were not touched by the sequential application of laminarinase-
A. nigev cellulase but were, once protease was added. The "ghosts" or
proteinaceous shells described by Hunsley and Burnett (1970) in
Phytovhthora after laminarinase-cellulase treatment were not observed
here after similar enzyme application ( laminar inase-4. nigev cellulase)
(Fig. 15b). Wall destruction is seen as the result of hydrolysis of the
protecting protein by the protease followed by cellulase hydrolysis of
the cellulosic component. Whether or not the proteinaceous shell of
Hunsley and Burnett (1970) is the same as the protective proteinaceous
component described here is unknown. In the case of laminarinase-/l.
ambisexualis cellulase treatment, total hydrolysis occurs without added
protease. It must be remembered, however, that this enzyme may contain
protease activity itself, as this type of contamination is commonly found
in crude cellulase preparations (Whitaker 1970). The contaminating
protease activity had been removed from the A. nigev cellulase.
It would appear from these studies that the bonds of interest,
those responsible for wall integrity, lie in both the matrix and the
microfibrillar components, as laminar inase-/4. ambisexualis cellulase
or laminarinase-protease-.4. nigev cellulase are required for hyphal
114
demise. However, though single enzyme treatments do not seem to affect
hyphal morphology, one cannot be sure that the cellulosic component was
reached by the externally applied cellulase because of the protective
matrix cover. Therefore, it cannot be ruled out that the bonds of
interest may lie solely in the cellulosic component of the wall (Mullins
1979). In addition, it must be remembered that cellulase, produced by
the organism itself, hydrolyzes from the inside. If the above morphological
data are true, the enzyme acts directly on the cellulosic portion of the
wall during the process of wall softening prior to branching. Therefore,
it would seem that wall integrity, at least in branching, lies in the
microfibrils or between the microfibrils and another component (Mullins
1979), perhaps protein. It is felt in general, though, that both the
matrix and microfibrillar components are necessary for the maintenance
of integrity (Bartnicki-Garcia and Lippman 1967; Hunsley and Burnett
1970); however, this conclusion is based on studies of wall hydrolysis
of externally applied enzymes.
Surface Structure of Chemically Treated Walls and Live Hyphae
Treatment of isolated walls and live hyphae with mild acid demonstrates
in both cases that the material (the acid-soluble fraction) which was
removed does not itself directly cover the microfibrillar component of
the wall (Figs, lib and 13b). However, subsequent treatment with 2 N
KOH removes the material (the alkali-soluble fraction) which does directly
cover the microfibrils, as they are revealed with clarity comparable to
the laminarinase-protease treatment (Figs, lie, 13c, and 15a). Walls
which were treated with acid, alkali, and cadoxen appear almost totally
disintegrated (Fig. lid), while live hyphae treated similarly retain
115
their morphology and have a striated appearance (Fig. 13d) . The surface
seen here may be that of the insoluble residuum described previously.
The cadoxen-soluble material from the treatment of isolated walls was
regenerated audit does not show the normal microfibrillar pattern seen
after acid-alkali treatment (Fig. lie). This material is comparable
to the cellulose II fraction, in which some material has reformed into
recognizable linear microfibrils (Fig. 12d) .
Surface Structure of Wall Fractions
Surface replicas of the acid-soluble fraction appear amorphous, and
it may be this fraction which is seen on the surface of untreated walls
(Fig. 12a) . The alkali-soluble fraction shows some microfibrillar
structure which may be caused by the long stretches of 61,4 linked
glucose residues thought to exist in this component (Fig. 12b). This
fraction may be comparable to the "short fibrils" seen after extensive
exo-31,3-glucanase treatment by Sietsma et al. (1975). The insoluble
residuum material appears weakly microfibrillar (Fig. 12f ) . Its
insolubility in cadoxen may be because the 81,4 linkages are inter-
spersed with other linkage groups.
Microfibrillar Diameter
The measurements (Table 12) made in this study are similar to those
of ethanolic-KOH cleaned walls (10-15 nm) (Tokunaga and Bartnicki-Garcia
1971) and untreated cyst walls (12-17 nm) (Desjardins et al. 1973) in
Phytophthora. A discrepancy is seen in the measurements from the isolated
microfibrillar portions of Phytopltbhora walls (Hunsley and Burnett 1968).
The value reported is 13 nm for the microfibrils from distal walls (lower
values were obtained for apical walls). It is thought that the apparent
116
increase in microfibrillar diameter seen in cellulose I could be due to
the increasing dissolution of the matrix material thus revealing more
and more of the microfibril (Fig. 18) or to acid swelling during the
preparation.
There are criticisms of measuring microfibrillar diameter from
shadowed material because of the variable amount of deposit added during
the shadowing procedure (Preston 1974b). Mcrof ibrillar diameters of
material which have been negatively stained are much lower, as seen in
the 3 nm widths of the microfibrils of Aphanomyces astaai. (Nyhlen and
Unestam 1978). A criticism of this method is that the stain penetrates
the cortex area of the microfibril and therefore the only portion that is
measured is the core (Preston 1974b) . It has also been suggested that
nascent microfibrils are wider than those after dehydration (Leppard
et al. 1975).
Based on ultrastructural studies, particularly those using live
hyphae where it is easier to determine which side of the wall is being
acted upon, the amorphous layer hydrolyzed by laminarinase or dilute
acid exists as the outer boundary of the wall. Moving inward the
next layer is also hydrolyzed by laminarinase or alkali and appears to
have an outer amorphous region and an inner one with a weakly micro-
fibrillar pattern. The innermost layer of the wall is made up largely
of cellulosic microfibrils which may be cross-linked with protein. The
insoluble residuum remaining after wall fractionation is weakly micro-
fibrillar and may be found between the plasma membrane and the micro-
fibrillar portion of the wall. The idea of Phycomycete walls consisting
of an amorphous outer layer and an inner microfibrillar one is not new
117
MATRfX
MATERIAL
UNTREATED
1 l.23nm
LAMINARINASE
15.96 nm
LAMINARINASE-
PROTEASE
1 5.43nm
0.5NHCI-2NK0H
Z0.29nm
CELLULOSE I
2i,76nm
Figure 18. Scheme for explaining the apparent increase in
microfibrillar width as a result of enzymatic or
chemical treatment.
118
(Hunsley and Burnett 1970; Tokunaga and Bartnicki-Garcia 1971; Sietsma
et al. 1975), Ultrastructural data suggest two layers for Phytophthora
walls (Hunsley and Burnett 1970) , but a more accurate description for
A. ambisexualis walls might be two layers with a gradual change from
one to the other as seen in the alkali-soluble fraction. A gradual
change in wall layers in Phycomycetes has been suggested by Bartnicki-
Garcia (1973). A schematic drawing of the layers of the hyphal wall,
demonstrating the gradual change from one to the next, is seen in Fig.
19.
119
Figure 19. Diagrammatical representation of the hyphal wall based on
ultrastructural evidence. (a) Acid-soluble and laminarinase
hydrolyzed. (b) Alkali-soluble and laminarinase hydrolyzed.
It should be noted that the upper side, corresponding to the
outer portion, is fairly similar to (a) , but that there is a
gradual change to a more fibrous condition, similar to (c) ,
on the lower side. (c) Cadoxen-soluble, and cellulase and
protease hydrolyzed. Distinct microfibrils can be seen,
(d) Insoluble residuum. The exact location of this fraction
is uncertain, but it is thought that it may lie next to the
plasma membrane.
CONCLUSION
In conclusion this analysis of the hyphal wall of A. ambisexualis
has confirmed the results of previous studies on other members of the
Phycomycetes. Some of the chemical differences noted may be the results
of different procedures rather than actual variations between the walls
themselves. Chemical fractionation of the carbohydrate wall constituents
yielded four fractions based on solubility characteristics. The major
component, soluble in weak acid, was found to consist of a Bl,3 glucan
with numerous 81,6 branch points. A smaller component, soluble in alkali,
was determined to be a linear glucan with mixed Bl,3 and 61,4 linkages
with occasional 31,6 branches. The microfibrillar component, soluble
in Schweitzer's or cadoxen reagents, was examined and found to be
cellulose. After these treatments, a small portion of mixed 61,3 and
61,4 linkages with a few 61,6 remained. It is speculated that this
material lies adjacent to the plasma membrane. Protein was also found
and amino acid analyses revealed the usual spectrum of amino acids,
including hydroxyproline, commonly found in cellulosic cell walls. The
only known report of the probable existence of a chitinous wall component
(Dietrich 1973) was confirmed.
Morphologically, the wall consists of an outer matrix of 61,3 and
61,6 glucans covering an inner cellulosic-proteinaceous core. A dia-
grammatic model (Fig. 19) synthesized from the chemical and morphological
data is proposed. In addition, a molecular model (Fig. 17) of the hyphal
wall, utilizing the data from the extensive study of the linkage and
branching pattern of the wall fractions, is presented.
120
121
Based on the enzymological studies performed here, it would seem
that the bonds which are necessary for wall integrity lie in both the
matrix and the microfibrillar regions. However, the enzymes were
applied from the outside, so it is possible that the microfibrillar
component was never reached by the cellulase. Thus the bonds of
interest may be in this constituent, or between it and another,
possibly protein.
APPENDICES,
APPENDIX A
TECHNIQUES
Buffer-Water Washing of Isolated Walls (Lin et at, 1976)
Isolated walls were washed with 0.1 N tris-HCl buffer six times
with low speed (1085 x g) centrifugations in between. The pellet,
resuspended in 20 ml of the same buffer, was sonicated for 4 min at
30 watts. After sonication the pellet was washed six more times with
the buffer, homogenized with a glass tissue grinder, and washed twelve
times with distilled water or until the walls appeared clean micro-
scopically and the supernatant from the washings was clear. Walls were
then lyophilized and stored over desiccant.
Chitin Isolation (Aronson and Lin 1978)
Either lyophilized chemically cleaned walls (100 mg) or frozen
mycelia (20 gm fresh weight) were used for this extraction. If the
latter was used, it was homogenized with a mortar and pestle in 5% KOH
in 80% methanol and placed at 98°C for 15 min. The resulting solution
was centrifuged at low speed (1085 x g) and the pellet was treated as
above two more times. The residue was washed twice with distilled
water, once with 0.5 N acetic acid, and twice more with distilled
water. It was then treated five times with 1 N acetic acid at 98°C
for 15 min each time; the remaining residue was washed with water until
it was acid free. Chitin was extracted from chemically cleaned walls
or from mycelia prepared as above by the following procedure: treatment
123
124
with 50 ml 2% (w/v) KMnO^ at 25°C for 18 hr with intermittant stirring
followed by centrifugation and washing twice with distilled water and
three times with 2% (w/v) oxalic acid containing a few drops of 1 N
H^SO^. Five additional washings were done. The residue was then stirred
continuously under N™ for three 1 hr treatments at room temperature in
Schweitzer's reagent; the remaining residue ("chitin") was washed four
times with 1 N acetic acid and then with water until acid free. The
material was freeze dried and stored over desiccant until further
analysis.
Cellulose I Isolation (Aronson and Lin 1978)
Cellulose I was extracted from chemically cleaned walls or from
mycelia prepared as above (see Chitin Isolation) by treatment with 1 N
KOH at 25°C for 1 hr, followed by three water washings and treatment with
1 N acetic acid at 98 "C for 15 min followed by three more water washings.
The alkali extraction and water washings were repeated and the residual
material was suspended in H^O -acetic acid (equal volumes of 30% H„0
and glacial acetic acid) and placed at 98°C for 30 min and then washed
with distilled water three times. The insoluble material was treated
with 500 )Jg/ml chitinase in 0.05 M potassium phosphate buffer at pH 6.0
for 72 hr at 25°C with shaking. The remaining pellet was washed three
times with water and stirred for 90 min in 5.25% NaOCl with 5% KOH.
The pellet was then washed once with the chlorox solution, once with
distilled water, once with 1 N acetic acid, and three times with distilled
water. Then it was lyophilized and stored over desiccant.
125
Preparation of Acid Swollen Cellulose
(Green 1963; Reese and Mand els 1963b)
A 500 mg sample of Whatman No. 1 cellulose powder was treated with
15 ml 85% H^PO, and stirred occasionally with a glass rod over a period
of 65 hr. After this period of time all the cellulose appeared dissolved;
five volumes of distilled water were added. This solution was filtered
leaving a gelatinous mass on the filter paper which was washed twice
with 95% ethanol and twice with xylene and dried overnight in a vacuum
desiccator. This process is called WAN drying (water/alcohol/non-polar
hydrocarbon) .
Enzyme Purification (Sietsma et at. 1968)
Two enzymes, laminarinase and cellulase, were found to be impure
and were subjected to the following purification process. Degassed,
washed DEAE-cellulose (medium mesh, capacity 0.94 meq/gm, Sigma) was
poured into a 23 cm column (I.D. 1.5 cm) and the enzyme solution to be
purified (1 mg/ml in 0.005 M potassium phosphate buffer pH 7.5) was
layered on top. The enzymes were eluted with a linear gradient of increas-
ing NaCl concentration (0.0 to 0.8 M) in phosphate buffer. The salt
concentration was measured by conductivity. Preliminary studies
indicated that in the purification of cellulase most of the enzyme
activity was found in the fractions which were eluted with a lower salt
concentration; the enzyme eluted with 0.4 M salt was free of proteolytic
activity. It was decided to use a stepwise elution process when it was
found that the contaminating proteolytic enzymes were eluted at 0.2 M
NaCl concentration. Thus two eluants were used, 0.2 M and 0.4 M NaCl
in equal proportions. The 0.2 M fraction and the first 10% of the 0.4 M
126
were discarded. The remaining 0.4 M was saved and dialyzed for 48 hr
against a 0.05 M sodium citrate buffer pH 5.0 (the buffer was changed
six times during the dialysis period) to remove salt and to place the
enzyme in an appropriate buffer for reaction. The enzyme was concentrated
with an immersible molecular separator (Millipore Corporation) with a
pellicon membrane of 10 000 nominal molecular weight limit or over
Ficoll or carbowax. In general purified cellulase had an activity of
about 12 units /ml.
The purification of laminarinase was carried out in the same manner.
It was found that the bulk of laminarinase activity came out in the
fractions eluted with salt concentrations lying between 0.25 M and
0.35 M, However, the fractions with salt concentrations in the vicinity
of 0.35Malso contained cellulase activity, so it was decided, since more
laminarinase activity was found in the 0.25 M fraction, to use only the
enzyme from this fraction. From the above information it was decided
that purfified laminarinase could be obtained by eluting the column with
0.25 M NaCl. Dialysis and concentration of the 0.25 M fraction was the
same as for the purified cellulase.
Enzymatic Hydrolysis of Laminarin
Two different sources of laminarin were used as substrates for the
generation of Bl,3 linked di- and trisaccharides to be used as reference
compounds for gas and paper chromatographic studies. Briefly, the
procedure involved treating each of the substrates with purified lami-
narinase and incubating the reaction mixture at S^C for 6 hr in 0.05 M
citrate buffer pH 5.0. Undigested material was pelleted and aliquots
of the supernatant were used as reference compounds. It was found that
127
the Calbiochem laminarin contained 61,3 linkages while the other poly-
saccharide (source unknown) contained both 61,3 and 61,6.
Hydrolysis of the Unf ractionated Wall with H2SO4
A 20 mg sample of dried walls was treated with 0.4 ml of 12 N
H2SO, under N- for 12 hr at room temperature. The acid solution was
diluted to 1 N and placed at 105°C for 4 hr. Then it was diluted 10-fold
and neutralized with BaOH, and the BaSO, precipitate was removed by
centrifugation. The resulting supernatant was lyophilized and the
residue was redissolved in 2.5 ml distilled water and after derivatization
was analyzed by gas chromatography (personal communication Dr. R.
Michael Roberts) .
Description of Analyses Used for the Detection of
Neutral Sugars
Paper Chromatography
The technique of paper chromatography involves the separation of
substances based on their relative solubilities in water and an organic
solvent (Kowkabany 1954; Bloch et at. 1958). The filter paper is a
support for the water-rich stationary phase over which runs the organic
solvent, the mobile phase. The substances to be analyzed were spotted
6.35 cm apart on a line near the top of the paper in concentrations of
about 100 yg. The concentration of the standards was 50 yg. The spotted
paper was hung from a trough in a presaturated chromatocab; 40 ml of the
solvent was poured into the trough and the separation was run for 24 hr.
At the end of this time, the chromatograms were removed from the chamber,
dried in a hood, sprayed with an aniline phthalate spray for the detection
128
of reducing sugars, and developed in a 105 °C oven. Unknown spots were
identified by their similarity to R, or R values of known standards.
R- is defined as the ratio of the movement of the spot to the movement
of the solvent front; R is the ratio of the movement of the spot to
that of known glucose and is more reliable if the solvent has run off
the paper. For sugars the best resolution of separation occurs with
solvents which give R^ values between 0.2 and 0.3 The butanol:
pyridine: water solvent chosen gave the best results of those tried.
Other solvents were 5 ethyl acetate: 5 pyridine :1 acetic acid: 3 water
v/v, 2 ethyl acetate:l pyridine:2 water and 8 ethyl acetate:2 pyridine:
1 water.
Gas-Liquid Chromatography
The principle of gas-liquid chromatography is basically the same
as that of paper. Substances are separated into their component parts
between the mobile gas phase and the stationary liquid phase based on
their partition coefficients (Bishop 1964; White et al. 1964; McNair
and Bonelli 1968), The solubility of substances is dependent on many
factors such as molecular weight, degree of substitution of side groups,
polarity, and stearic factors. The stationary liquid phase is spread
thinly over an inert solid, the support phase, generally a silanized
diatomaceous substance, and then the gas phase moves through the station-
ary bed (McNair and Bonelli 1968; Pierce 1968). The components to be
separated are carried by an inert gas, the carrier gas, and the sample
is partitioned as described above. The separated samples leave the
column in the carrier gas and in this case are sensed by a flame ionization
detector (FID). The FID consists of a mixture of hydrogen and air which
129
produces a flame over which is placed an electrode which measures the
conductivity of the flame. Pure hydrogen has low conductivity, but as
the organic compounds in the carrier gas pass over the flame and are
combusted there is an increase in conductivity which is amplified and
recorded (McNair and Bonelli 1968) . Partition coefficients are directly
proportional to retention volumes, therefore the time at which a
particular component comes out of the column is a good indication of its
identity as compared with known standards. Temperature programming is a
method by which an investigator can maximize separations and yet shorten
the time substances remain on the column thereby obtaining peaks which
are reasonably sharp. Generally, a temperature program must be arrived
at empirically for there is much variation in the systems used and the
components to be analyzed.
Derivatization of Samples
There are several ways of volatilizing carbohydrates for this type
of analysis. The formation of trimethylsilyl groups was used in this
study because of its ease and the relatively low number of components
found in the substances to be separated. The reaction involved in the
formation of these compounds is the replacement of the active hydrogen
of the free hydroxyl group in polysaccharides by a silyl group; the use
of both TMCS and HMDS insures that complete silylation will take place.
Pyridine is used as a solvent (Pierce 1968) . Hydroxyl hydrogens are
replaced in order to prevent hydrogen bonding between free hydrogen
groups and also to decrease the polarity of the compounds to be studied
(Pierce 1968; Clamp et al. 1971).
130
Periodate Oxidation
Periodate oxidation studies were done in order to gain some knowledge
as to the pattern of linkages and branching in the isolated fractions of
the wall. The principle involved in these studies is that in long chain
polymers there is under controlled conditions a specific pattern of
periodate consumption and formate liberation which upon analysis gives
information as to linkage and branching arrangements in the polymer
(Hay et at. 1965). If the manner in which a chain is linked leaves two
adjacent free hydroxyl groups per monomeric residue then it will take
one molecular proportion of periodate to cleave the carbon chain and
no formate will be released in the reaction. This is the case with
glucan chains which are linked 01,4. However, if the situation exists
where there are three adjacent free hydroxyl groups, then it will take
two molecular proportions of periodate to cleave the chain and one
molecular proportion of formate will be released. This situation occurs
at non-reducing terminal points and with 61,6 linked non-terminal units.
Periodate does not affect residues which joined such that there are no
adjacent hydroxyl groups as is the case with 01,3 linked glucans. It
is not well understood what happens at the reducing end, but it is
thought that two molecular proportions of formate are given off unless
a formate ester is formed, in which case one proportion is given off
(Bobbitt 1956; Smith and Montgomery 1956). Data from these studies can
be seen in Tables 5 and 6, and Figs. 20, 21, and 22.
Polarized Light Microscopy
Polarized light microscopy is used to detect the presence of highly
ordered systems, in this case cellulose. Polarized light, light vibrating
131
UJ
o
<
GD
q:
o
en
CD
<
1.7-
i.3-
03-1
0-H
a.
NalO^
NalO-
24
48 72
HOURS
96
20
b.
O
2 C.33
1 0.33-
0
NaI04
NalO.
24
4 9 72
HOURS
96
120
Figure 20. Periodate and iodate oxidation. (a) Periodate consumption,
(b) Formate liberation.
132
.7-.
24
48 72
HOURS
96
120
Figure 21.
Periodate consumption of the wall fractions and the total
wall. (a) Acid-soluble. (b) Alkali-soluble. (c)
Celluloase II. (d) Insoluble residuum. (e) Total
wall. (f) Cellulose I.
0.67-
0.33-
o
2 0.33-
"I 0.33
0.67-
0.33-
0.67-
0.3>
b.
c.
d.
e.
f.
24
48
I
72
— r-
96
HOURS
133
"120
Figure 22. Formate liberation of the wall fractions and the total
wall. (a) Ac id- soluble. (b) Alkali-soluble. (c)
Cellulose II. (d) Insoluble residuum. (e) Total
wall. (f) Cellulose I.
134
in one plane, is "bent" to shine in a new plane by encountering solids
of well-ordered molecular arrangements (Wolfe 1972) . The phenomenon is
known as birefringence.
X-ray Diffraction Analysis
X-ray diffraction analysis, by studying the angles and intensities
of the scattering of x-rays at a given wavelength by the electrons which
surround each atom, is used to measure the lattice spacings of crystal-
line structures (Lehninger 1975). Atoms with higher electron densities
produce more diffractions than those with lower densities and with this
knowledge various patterns of atomic arrangement can be discerned. Photo-
graphs of x-ray diffraction studies can be made by placing the crystal
in question in a known orientation in the path of monochromatic x-rays.
The x-rays scattered by the crystal hit a photographic plate behind the
crystal. The three dimensional structure of crystals can be obtained by
doing a series of electron density photographs in different planes
(White et at. 1964). Cellulose, having a well ordered molecular arrange-
ment, is well suited to such studies.
Solubility Analysis of the Hexosamine Component of the
Wall (Aronson and Lin 1978)
A 40 mg sample of buffer-water washed walls was treated with 4 ml
1 N NH^OH for 8 hr at room temperature and then centrifuged; the pellet
was treated again with 2 ml 1 N NH.OH followed by a 2 ml water wash.
All the supematants were combined and dried in vacuo; these constituted
the alkali-soluble portion of the hexosamine component. The remaining
pellet was treated twice for 15 min with 4 ml 1 N acetic acid at 98°C,
and once with 2 ml 1 N acetic acid at room temperature. Again all the
135
supematants were combined and dried in vacuo ; this was the acid-soluble
hexosamine fraction. The pellet which remained was washed twice with
95% ethanol and dried under a stream of N«. All these fractions were
hydrolyzed as described and analyzed for hexosamine content in an auto-
mated Amino Acid Analyzer,
Uronic Acid Analysis (Gancedo et al. 1966)
A 100 mg sample of buffer-water washed walls was hydrolyzed with
1% HCl for 2 hr in a boiling water bath. The hydrolysate was centrifuged
and the supernatant was passed through an Amberlite (Mallinckrodt) IR-45
column to remove the mineral acid. About 65 ml was collected and this
was run through a Dowex-I formate column which bound the uronic acid.
The uronic acid was eluted with 0.5 N formic acid; 16 6 ml fractions
were collected and analyzed for uronic acid content by the Carbazole
test (Bitter and Muir 1962).
Lipid Extraction and Analysis (Whitehouse et at.
1958; Bartlett 1959; Ames 1968; Kanfer and Kennedy 1963)
A 40 mg sample of buffer-water washed walls was treated with 8 ml
methanol and heated to 50''C for 30 min. The mixture was cooled to room
temperature and 16 ml chloroform were added and the mixture was shaken
for 30 sec. Then 24 ml 2N KCl were added, the mixture was shaken
vigorously, and the phases were allowed to separate for 30 min. The
top layer (methanol-salt) was aspirated off, distilled water added, and
the solution was mixed. The phases were allowed to separate and again
the top layer was aspirated off. The tubes were spun and the water layer
(top) was removed; the remainder was dried under vacuum. The dried
residue ("lipid") was spotted on Silica gel G plates as described pre-
viously.
136
Phosphorus Analysis (Bartlett 1959)
A 10 mg sample of buffer-water washed walls homogenized with a
tissue grinder plus 0.5 ml 10 N H2SO, was combusted in a 160''C oven for
3 hr, after which time 2 drops 30% H„0- were added and the solution was
returned to the oven. Periodically (every 3 hr or overnight) the solution
was removed from the oven, treated with the H„0„ and returned to the
oven. This was continued for 48 hr until the solution was clear and
colorless, then a 5% urea solution was added and the tubes were combusted
for 1 hr longer to insure that all the H„0„ was consumed (personal communi-
cation Dr. Thomas E. Humphreys). The Fiske-SubbaRow assay for phosphorus
was performed on the resulting solution.
Ultrastructural Studies - Thin Section
Thin sections of untreated walls of live hyphae were made and
measurements of the width of the wall were taken. Material was fixed in
5% glutaraldehyde for 1 hr and post-fixed with 1% osmium tetraoxide for
another hour. The specimens were dehydrated and embedded in Mollenhauer
Mixture No. 2 (Mollenhauer 1964). Specimens were sectioned with a
Huxley LKB microtome and placed on 100 mesh copper grids. They were
then post-stained with 0.5% uranyl acetate and then treated with lead
citrate. Microscopic observations were done with a Hitachi HU-llE
electron microscope.
APPENDIX B
RECIPES
Growth Media for A. ambisexual-is
Agar
(ENR)
Ingredient
Liquid gm/1
(Kane 1971) gm/1
Monosodium glutamate
0.4
0.5
Glucose
2.0
14.0
Tris (hydroxymethyl) aminoethane
1.2
1.2
Combined liquid stock^
17.5
ml/1
17.5 ml/1
Distilled water to
1.0
1
1.0
1
Agar
0.0
25.0
Combined liquid stock
ml/1
ml/1
1-Methionine (15 mg/ml in
10% HCl)
1.0
1.0
KCl (2 M)
1.0
1.0
MgS04-7H20 (0.5 M)
1.0
1.0
CaCl2 (0.5 M)
1.0
1.0
HEDTA (10 mg/ml)
2.0
2.0
KH2PO4 (1 M)
1.5
1.5
Metal mix #4 (2 mg/ml) ^
10.0
10.0
Metal mix #4; grind togeth
ler
Fe(NH4)2-(S04)-6H20
28.9
gm
28.9
gm
Zn(S04)'7H20
8.8
gm
8.8
gm
Mn(S04)-H20
3.1
gm
3.1
gm
As used this metal mix provides Zn at 1.0 mg/1, Fe at 2.0 mg/1, and
Mn at 0.5 mg/1.
Cadoxen Reagent (Jayme and Neuschaffer 1957; Jayme and Lang 1963)
100 ml 30% ethylenediamine
4 gm cadmium oxide (saturated solution)
The ethylenediamine-cadmium oxide solution was stirred under the
hood at room temperature for 30 min. Excess cadmium oxide was removed
by centrifugation (3020 x g, 10 min) . The supernatant was used for
cellulose dissolution; it was best when used freshly made.
137
138
Schweitzer's Reagent (Cuoxam Solution) (Jayme and Lang 1963)
54 gm C.P. copper (II)
10% NH4OH
22% NH4OH
Distilled water
The copper was heated to boiling in 300 ml distilled water. About
55 ml of the 10% NH.GH was stirred in until a bluish color appeared. The
precipitate was allowed to settle and the supernatant was decanted. The
precipitate was washed with 3 or 4 100 ml portions of distilled water and
the washings were decanted. The precipitate was rinsed with 22% NH4OH
into a dark bottle and diluted to one liter with 22% NH,OH. The solution
4
was stored at room temperature in the dark. The final concentration of
copper was 13.5 gm/1.
Anthrone Reagent (Morris 1948; Dische 1962)
0.2 gm anthrone (Sigma)
100 ml concentrated H„SO,
2 4
To be prepared daily and chilled for 2 hr before use.
Material in the standard, blank, and sample tubes was brought to
a final volume of 1.25 ml and chilled. Aliquots of 2.5 ml of the anthrone
reagent were layered on and the tubes were covered with parafilm. The
tubes were mixed with a vortex and chilled. The parafilm was then
removed and a marble was placed on top of the tubes which were then put
in a boiling water bath for 16 min. After removal from the boiling water
bath tubes were set in an ice bath for 2 to 3 min and then allowed to
stand at room temperature for 5 to 10 min. They were then read at 620
nm against a reagent blank in a Gilford 240 spectrophotometer; glucose
was used as the standard.
139
Glucostat Test (Worthlngton Biochemical Corporation, Freehold. NJ OlllQ)
Principle: 3-I^glucose + O2 + H2O glucose oxidase ^^^^ ^
D-glucono-6-lactone
^2^2 "*" ^^'^"^^^ chromagen ^ oxidized chromogen
+ H2O
Chromagen
Glucostat reagent
4 N HCl
Dissolved chromagen and the glucostat reagent were mixed according
to the package instructions and the volume was brought to 80 ml (macro
method) . Aliquots of 8 ml of this solution were added to 2 ml volumes
in the standard, blank, and sample tubes at 45 sec intervals. The tubes
were mixed and allowed to stand 10 min. At 45 sec intervals 1 drop 4 N
HCl was added to each tube. The tubes were mixed and were allowed to
stand at least 5 min. The absorbance was read at 420 nm in a Gilford
240 spectrophotometer; standards were glucose.
A procedure for deproteinization was necessary before the assay-
could be done successfully.
0.14 N NaOH
2% ZnS0,-7H„0
4 2
The standard, blank, and samples were treated with 1 ml of the
NaOH solution and 1 ml of the Zn solution diluted 1:4 with distilled
water, and after mixing, were centrifuged at 755 x y- for 5 min. A sample
of 2 ml of the supernatant was used for the glucostat test.
Cellulase Viscometric Assay (Thomas and Mullins 1969)
Carboxymethylcellulose substrate:
1.2% CMC
0.018 M citrate-NaOH buffer pH 5.0
0.05% merthiolate
distilled water to one liter
140
Portions of 5 ml of the substrate were added to size 300 Ostwald-
Fenske Viscosity tubes and placed in a preheated 30°C water bath. A 1 ml
sample of the enzyme solution or the appropriate blank was added and the
resulting solution was mixed by gentle suction. The solution was then
drawn by suction into a glass bulb near the top of the tube and the rate
at which it fell back into the bottom tube was measured. Similar measure-
ments were taken at time intervals. A cellulase unit is equal to the
percent decrease in flow time per hr divided by 10%.
DMAS Assay (Reissig et al. 1955)
0.8 M KBO4
p-dimethylaminobenzaldehyde (Sigma) diluted with 9 volumes of
glacial acetic acid
A 0.1 ml aliquot of the KBO, solution was added to 0.5 ml of the
sample, blank, and standard tubes and these were heated in a vigorously
boiling water bath for 3 min. After cooling in tap water, 3 ml of the
DMAB solution were added and the samples were mixed with a vortex and
placed in a 36 to 38°C water bath for 20 min. After cooling in tap water
the absorbance was read immediately at 544 nm on a Gilford 240 spectro-
photometer. N-acetylglucosamine solutions were used as standards.
The preparation of KBO, solution is described by the following
procedure:
0.9 M H3BO3
2 N KOH
A total of 35 ml of 0.9 M H_BO„ was stirred on a hot plate and
2 N KOH was added with stirring until the pH was raised to 9.2. The
volume was brought to 50 ml which decreased the molarity to about 0.8 M.
141
Folln Test (Lowry et at. 1951)
0.5% CUSO4
2% sodiijm tartrate
2% Na2C03
Folin reagent
0.1 N NaOH
Blanks, standard, and sample volumes were all brought to 1 ml with
0.1 N NaOH (Reiskind 1970), A solution of 0.5 ml CuSO,, 0.5 ml sodium
tartrate, and 50 ml ^a.^ZQ^ \ia.s made and 1 ml of this was added at 45 sec
intervals to the solutions to be tested which were mixed immediately
with a vortex. After exactly 10 min 0.1 ml of the Folin reagent was
added at 45 sec intervals and mixed immediately. The absorbance of the
tubes was read 30 min later at 45 sec intervals at 750 nm on a Gilford
240 spectrophotometer against a 0.1 N blank. The protein standards were
made with bovine serum albumen (Sigma).
BioRad Protein Assay (BioRad Technical Bulletin No, 1051)
To 0.1 ml blank, standard, and sample solutions was added 5.0 ml of
diluted BioRad dye reagent (1 dye reagent: 4 distilled water using
filtration to remove the precipitate) and the solutions were mixed on a
vortex. They were allowed to stand for a minimum of 5 min and a maximum
of 60 min, and their absorbance was read against the appropriate blank
at 595 nm in a Gilford 240 spectrophotometer. Gamma globulin was used
as a protein standard.
Lipase Assay (Bier 1955)
Tween 20
0,2 M sodium phosphate-citrate pH 6,2 buffer
0,02% phenol red
octyl alcohol
0.02 N NaOH
142
To 5 ml substrate solution (1 ml tween 20 + 2.0 ml 0.2 M buffer
+ 1.8 ml H„0 + 0.2 ml phenol red) is added 1 ml enzyme solution. The
tubes were stoppered and placed in a 20°C water bath for 9 min. One drop
octyl alcohol was added to prevent foaming. The solution was titrated
with 0.02 N NaOH under a N_ stream with stirring such that the end point
was reached at 10 min after the initial addition of the enzyme to the
substrate. Enzyme activity was determined by calculating the difference
between the alkali consumed in the test solutions and the sum of the two
blanks. A ml of titrant equals 100 lipase units. In the tests of
lipase activity against various substrates, the reaction mixtures after
24 hr of incubation were titrated directly.
Carbazole Test (Bitter and Muir 1962)
0.025 M sodium tetraborate' IOH2O
concentrated H^SO,
Carbazole (Eastman Kodak)
Methanol, analytical grade
In screw-capped culture test tubes 5 ml of a solution of sodium
tetraborate in concentrated sulfuric acid was cooled to 4°C, after which
1 ml sample, blank, or standard solution was layered onto the acid.
The caps of the tubes were loosely screwed and the tubes were placed
in an ice bath and shaken gently at first and then vigorously; the
temperature was never allowed to exceed room temperature. The tubes
were then placed for 10 min in a vigorously boiling water bath and then
were cooled to room temperature. An aliquot of 0.2 ml carbazole (0.125%
carbazole in methanol) reagent was added to the tubes which were shaken
and heated in a boiling water bath for 15 more minutes. The tubes were
then cooled to room temperature and their absorbance was read at 530 nm
in a Gilford 240 spectrophotometer. The standard used was glucuronolactone.
143
Carbazole reagent is stable for 12 weeks if stored at 4°C in the
dark.
Fiske-Subbarow Assay (Bartlett 1959)
0.22% (M4)6Mo7024-4H20
Fiske-Subbarow reagent (Sigma)
To the combusted material (initial volume not to exceed 2 ml) was
added 4.6 ml of the molybdate solution and 0.2 ml of the Fiske-Subbarow
reagent. The solution was mixed thoroughly and heated for 7 min in a
boiling water bath with marbles covering the tubes. The absorbance of
the cooled tubes was read at 830 nm on a Gilford 240 spectrophotometer.
The standard was made from dilutions of a 1 mM solution of Na^HPO, such
Z 4
that the final concentration of phosphorus in the assay mixture ranged
from about 0.2 to 0.02 yM.
APPENDIX C
PERIPHERAL STUDIES
Dry Weight Determination of Washed Mycelium
Dry weights of freshly harvested 48 hr washed mycelium were
determined on material which had been dried in tared weighing pans in
vacuo over desiccant for three days. Percent dry weight of fresh weight
was determined. Percent dried wall of the dried weight was also calcu-
lated. Mycelial dry weight was estimated to be about 1.33% of the fresh
weight of washed mycelium. Of this 51.3% or 0.682 gm is wall estimated
from buffer-water washed material.
GLC Analyses of H2SO4 Hydrolysates of Unfractionated Walls
In general the results of the sulfuric acid hydrolysis of the wall
were disappointing, mainly because none of the expected dissacharides
appeared. The one interesting fact about these chromatograms (Fig. 23)
was that a small amount of measurable glucosamine was found while only
trace amounts of this sugar appeared in some of the HCl hydrolysates.
Congo Red Stain of the Wall and its Fractions
Congo red, a specific stain for cellulose, was used to determine
the presence or absence of cellulose in the various wall fractions and
in the wall itself. The acid-soluble fraction did not take up the stain
at all while the wall itself was stained moderatel^r. Cellulose I
and II were stained heavily and both the alkali-soluble fraction and the
insoluble residuum were stained moderately.
144
145
10 15
TIME (min)
r
20
1
25
Figure 23. GLC of the TMS derivatives of the monosaccharides
released by H2SO4 hydrolysis of the unfractionated
wall. In addition to glucose small peaks of
glucosamine are also seen.
146
Wall Width as Measured from Thin Section Micrographs
The width of the wall was measured from sections near the tip and
those more distal (Fig. 24a and b) . Sections of wall on each micrograph
were divided into fifths and measurements were taken at each fifth.
Examination of the micrographs (Figs. 24a and b) reveals that the wall is
thicker in some areas than in others; it is not known if the added thickness
is an artifact produced during the preparation of the sections or not.
However, the measurements made of the wall width include the added thick-
ness if that was the situation where the measurements were made. Widths
were taken from three different micrographs of apical and subapical
walls. The average width of the wall near the tip was 198.0 nm + 32.15
and the more distal 299.2 nm + 37.58. This is in partial agreement with
Hill (Thesis 1978) who found the width to be 178 nm + 100,
Observations of Enzymatically Treated Material by-
Phase Microscopy
The material from which surface replicas were made was observed
under phase microscopy. Because of the relatively low magnification
used in these studies compared with that of the electron microscope,
it was difficult to ascertain very accurately the effect of the enzymes.
In general it can be said that the walls treated with single enzymes
appeared normal. Those treated with the two enzyme sequence appeared
a little thinner than those treated with one. The walls treated with
the three enzyme sequence definitely appeared thinner and in some cases
parts of the wall were missing and the characteristic tubular hyphal
shape was destroyed.
Figure 24. Apical and subapical sections of an A. ambisexualis
hypha. (a) Apical, X 25 500. (b) Subapical, X 13 300.
148
. y-at^'m.
149
Observations of Hyphal Branching by Polarizing
Light Microscopy
As mentioned in the main body of this study observations of hyphae
with polarized light demonstrated birefringence. However, birefringence
was not seen in antheridial branches nor in the areas where branching
occurred. Very strong birefringence was noted though in the corners
between the parent hyphae and the branch points, as if the "softened"
wall in the parent hyphae was pushed aside, possibly by turgor pressure,
to allow the incipient branch to develop (Thomas 1970). Therefore,
it seems in branching as if the cellulosic component of the wall is
changed to accommodate the developing branch, but what happens to the
matrix of the wall is not known. Is it softened, stretched or dissolved
to make way for the new branch? Perhaps its function is to hold the wall
together in the isolated areas of branch induction after cellulosic
"softening. "
REFERENCES
Albersheim, P. 1974. The walls of growing plant cells. Sci. Amer.
80: 81-95.
Albersheim, P., W. D. Bauer, K. Keegstra, and K. W. Talmadge, 1973.
The structure of the walls of suspension-cultured Sycamore cells.
In Biogenesis of plant cell wall polysaccharides. Edited by
F. Loewus. Academic Press, N.Y. pp. 117-147.
Alexopoulos, C. 1952. Introductory mycology. John Wiley and Sons,
Inc. , N.Y. 482 pp.
Ames, B. N. 1968. Lipids of Salmonella typhimurium and Escheriahia
aoli: structure and metabolism. J. Bacterid. 93: 833-843.
Aronson, J. M. 1965. The cell wall. In The fungi. Vol. I. Edited by
G. C. Ainsworth and A. S. Sussman. Academic Press, N.Y. pp. 49-76.
Aronson, J. M. , B. A. Cooper, and M. S. Fuller. 1967. Glucans of
Oomycete cell walls. Science, 155: 332-335.
Aronson, J. M, , and M. S. Fuller. 1969. Cell wall structure of the
marine fungus, Atkinsiella dubia. Arch. Mikrobiol. 68: 395-305.
Aronson, J. M. , and C. C. Lin. 1978. Hyphal wall chemistry of
Leptomitus laoteus. Mycologia, 70: 363-369.
Aronson, J. M. , and L. Machlis. 1959. The chemical composition of the
hyphal walls of the fungus Allomyoes. Amer. J. Bot. 46: 292-300.
Aronson, J. M. , and R. D. Preston. 1960. An electronnicroscopic and
x-ray analysis of the walls of selected lower Phycomycetes.
Proc. Roy. Soc. B, 152: 346-352.
Arsenault, G. P., K. Biemann, A. W. Barksdale, and T. C. McMorris. 1968.
The structure of antheridiol, a sex hormone in Achlya bisexualis.
J. Amer. Chem. Soc. 90: 5635-5636.
Aspinall, G. 0. 1973. Carbohydrate polymers of plant cell walls. In
Biogenesis of plant cell wall polysaccharides. Edited by F. Loewus.
Academic Press, N.Y. pp. 95-115.
Bacon, J. S. D. , B. D. Milne, I. F. Taylor, and D. M. Webley. 1965.
Features of the cell wall structure of yeast revealed by the action
of enzymes from a non-fruiting myxobacterium (Cytophaaa johnsonii) .
Biochem. J. 95: 28c-30c.
150
151
Barksdale, A. W. 1960. Interthallic sexual relations in Aahlya, a
genus of aquatic fungi. Amer. J. Bot. 47: 14-23.
Bartlett, G, R. 1959. Phosphorus assay in column chromatography. J.
Biol. Chem. 234: 466-468.
Bartnicki-Garcia, S. 1966. Chemistry of hyphal walls of Phytophthora.
J. Gen. Microbiol. 42: 57-69.
Bartnicki-Garcia, S. 1968. Cell wall chemistry, morphogenesis, and
taxonomy of fungi. Ann. Rev. Microbiol. 22: 87-108.
Bartnicki-Garcia, S. 1973. Fundamental aspects of hyphal morphogenesis.
Symp. Soc. Gen. Microbiol. 23: 245-267.
Bartnicki-Garcia, S. , and E. Lippman. 1966. Liberation of protoplasts
from the mycelium of Phytophthora. J. Gen. Microbiol. 42: 411-416.
Bartnicki-Garcia, S. , and E. Lippman. 1967. Enzymic digestion and
glucan structure of hyphal walls of Phytophthora ainnamomi.
Biochim. Biophys. Acta, 136: 533-543.
Bartnicki-Garcia, S., and E. Lippman. 1972. The bursting tendency of
hyphal tips of fungi: presumptive evidence for a delicate balance
between wall synthesis and wall lysis in apical growth. J. Gen.
Microbiol. 73: 487-500.
Bartnicki-Garcia, S., and W. J, Nickerson. 1962. Isolation, composition,
and structure of the cell walls of filamentous and yeast-like forms
of Muoor ronxii. Biochim. Biophys. Acta, 58: 102-119.
Bartnicki-Garcia, S. , and E. Reyes. 1964. Chemistry of spore wall
differentiation in Muoor rouxii. Arch. Biochem. Biophys. 108:
125-133.
Bartnicki-Garcia, S., and E. Reyes. 1968. Polyuronides in the cell
walls of Muoor rouxii. Biochim. Biophys. Acta, 170: 54-62.
Bauer, W. D. , K. W. Talmadge, K. Keegstra, and P. Albersheim. 1973.
The structure of plant cell walls. II. The hemicellulose of the
walls of suspension-cultured Sycamore cells. PI. Physiol. 51:
174-187.
Beakes, G. W. , J. L. Gay. 1978. Light and electron microscopy of
oospore maturation in Saprolegnia furoata. Part 2. Wall develop-
ment. Trans. Brit. Mycol. Soc. 71: 25-36.
Berg, B. 1978. Cellulose degradation and cellulase formation by
Phialophora malorum. Arch. Microbiol. 118: 61-65.
Bhatti, T., R. E. Chambers, and J. R. Clamp. 1970. The gas chromatographic
properties of biologically important N-acetylglucosamine derivatives,
monosaccharides, disaccharides , trisaccharides, tetrasaccharides, and
pentasaccharides. Biochem. Biophys, Acta, 222: 339-347.
152
Bier, M. 1955. Lipases. In Methods in enzymology. Vol. I. Edited by
S. P. Colowick and N. 0. Kaplan. Academic Press, N.Y. pp. 627-642.
BioRad Technical Bulletin #1051. 1977. BioRad Laboratories. Richmond,
California.
Bishop, C. T. 1964. Gas-liquid chromatography of carbohydrate deriva-
tives. Adv. Carbohy. Chera. 19: 95-147.
Bitter, T. , and H. M. Muir. 1962. A modified uronic acid carbazole
reaction. Anal. Biochem. 4: 330-334.
Block, R. J., E. L. Durrum, and G. Zweig. 1958. A manual of paper
chromatography and paper electrophoresis. Academic Press, N.Y.
710 pp.
Bobbitt, J. M. 1956. Periodate oxidation of carbohydrates. In Advances
of carbohydrate chemistry, Vol. II. Edited by H. I. Wolfrom.
Academic Press, N.Y. pp. 1-41.
Bradley, D. E, 1965. Replica and shadowing techniques. In Techniques
for electron microscopy, 2nd Edition. Edited by D. Kay. F. A.
Davis Co., Philadelphia, pp. 96-152.
Burnett, J. H. 1968. Fundamentals of mycology. Edward Arnold, Ltd.,
London. 548 pp.
Cabib, E. 1975. Molecular aspects of yeast morphogenesis. Ann. Rev.
Microbiol. 29: 191-214.
Cameron, D. W. , and I. E. P. Taylor. 1976. Quantitative microanalysis
of cell walls of Saprolegnia dialina Humphrey and TpemeZ-Za
mesenteviaa Fries. Biochim. Biophys, Acta, 444: 212-222.
Cardemil, L. , and G. Pincheira. 1979. Characterization of the carbohydrate
component of fraction I in the Neuvospora crassa cell wall. J.
Bacterioi. 137: 1067-1072.
Clamp, J. R. , T. Bhatti, and R. E. Chambers. 1971. The determination
of carbohydrate in biological materials by gas-liquid chromatography.
In Methods of biochemical analysis. Vol. 19. Edited by D. Click.
John Wiley and Sons, N.Y. pp. 229-344.
Cole, G. T. , Sekiya, R. Kasai, T. Kokoyama, and Y. Nozawa. 1979.
Surface ultrastructure and chemical composition of the cell walls
on conidial fungi. Exp. Mycol. 3: 132-156.
Cooper, B. A., and J. M. Aronson. 1967. Cell wall structure of
Pythivm debaryanum. Mycologia, 59: 658-670.
153
Cowling, E. B. 1963. Structural features of cellulose that influence
its susceptibility to enzymatic hydrolysis. In Advances in enzymic
hydrolysis of cellulose and related materials. Edited by E. T.
Reese. Macmillan Company, N.Y. pp. 1-32.
Crook, E. M. , and I. R. Johnston- 1962. The qualitative analysis of the
cell walls of selected species of fungi. Biochem. J. 82: 325-331.
Datema, R. , H. Van den Ende, and J. G. H. Wessels. 1977a. The hyphal
wall of Muaor mucedo. I. Polyanionic polymers. Eur. J. Biochem.
80: 611-619.
Datema, R. , J. G. H. Wessels, and H. Van den Ende. 1977b. The hyphal
wall of Muaor muaedo. 2. Hexosamine-containing poljnners. Eur. J.
Biochem. 80: 621-626.
Davison, P. K. , and R. Young. 1969. Gas chromatography of carbohydrates.
The quantitative determination of the free sugars of plants as their
trimethylsilyl ethers. J. Chromatog. 41: 12-21.
Desjardins, P. R. , M. C. Wang, and S. Bartnicki-Garcia. 1973. Electron
microscopy of zoosprores and cysts of Phytophthora palmivora:
morphology and surface texture. Arch. Mikrobiol. 88: 61-70.
deVries, 0. M. H. , and J. G. H. Wessels. 1972. Release of protoplasts
from Sahizophyllum commune by a lytic enzyme preparation from
Triahoderma vivide. J. Gen. Microbiol. 73: 13-22.
deVries, 0. M. H. , and J. G. H. Wessels. 1973a. Effectiveness of a
lytic enzyme preparation from Tr-iohoderma vivide in releasing
spheroplasts from fungi, particularly basidiomycetes. Antonie
van Leeuwenhoek, 39: 397-400.
deVries, 0. M. H. , and J. G. H. Wessels. 1973b. Release of protoplasts
from Sahizophyllum commune by combined action of purified 61,3-
glucanase and chitinase derived from Triahcderma vivide. J. Gen.
Microbiol. 76: 319-330.
Dietrich, S. M. C. 1973. Carbohydrates from the hyphal walls of some
Oomycetes. Biochim. Biophys. Acta, 313: 95-98.
Dische, Z. 1962. General color reactions. In Methods in carbohydrate
chemistry. Vol. I. Edited hy R. L. Whistler and M. L. Wolfrom.
Academic Press, N.Y. pp. 477-512.
Dow, J. M. , and P. H. Rubery. 1977. Chemical fractionation of the cell
walls of mycelial and yeast-like forms of Muaor vouxii: a compar-
ative study of the polysaccharide and glycoprotein components.
J. Gen. Microbiol. 99: 29-41.
Dyer, J. R. 1956. Use of periodate oxidations in biochemical analysis.
In Methods of biochemical analysis, Vol. III. Edited by D. Click.
Interscience Publishers, Inc., N.Y. pp. 111-152.
154
Edwards, J. A., J. S. Mills, J. Sundeen, and J, H. Fried. 1969. The
synthesis of the fungal sex hormone antheridiol. J. Amer. Chem.
Soc. 91: 1248-1249.
Eveleigh, D. E. , J. H. Sietsma, and R. H. Haskins. 1968. The involvement
of cellulase and laminarinase in the formation of Pythi-nm proto-
plasts. J. Gen. Microbiol. 52: 89-97.
Fan, L. T. , Y. H. Lee, and D. H. Beardmore. 1980. Mechanism of the
enzymatic hydrolysis of cellulose: effects of major structural
features of cellulose on enzymatic hydrolysis. Biotech. Bioeng.
22: 177-199.
Farkas, V. 1979. Biosynthesis of cell walls of fungi. Microbiol. Rev.
43: 117-144.
Fevre, M. 1977, Subcellular localization of glucanase and cellulase in
Saprolegnia monoiaa Pringsheim. J. Gen. Microbiol. 103: 287-295.
Fuller, M. S. , and I. Barshad. 1960. Chitin and cellulose in the cell
walls of Rhizidiomyaes sp. Amer. J. Bot. 47: 105-109.
Gancedo, J. M. , C. Gancedo, and C. Asensio. 1966. Uronic acids in
fungal cell walls. Biochem. Zeitsch. 346: 328-332.
Gander, J. E. 1974. Fungal cell wall glycoproteins and peptido-
polysaccharides. Ann. Rev. Microbiol. 28: 103-119.
Ghose, T. , and V. S. Bisaria. 1979. Studies in the mechanism of enzymatic
hydrolysis of cellulosic substances. Biotech. Bioeng. 21: 131-146.
Goldstein, I. J., G. W. Hay, B. A. Lewis, and F. Smith. 1965. Controlled
degradation of polysaccharides by periodate oxidation, reduction,
and hydrolysis. In Methods in carbohydrate chemistry. Vol. V.
Edited by R. L. Whistler, B. C. Miller, and J. N. Wolfrom.
Academic Press, N.Y. pp. 361-370.
Green, J. W. 1963. Drying and reactivity of cellulose. In Methods of
carbohydrate chemistry. Vol. III. Edited by R. L. Whistler and
M. L. Wolfrom. Academic Press, N.Y. pp. 100-101.
Hawes, C. R. 1979. Conidium ultrastructure and wall architecture in
the chalara state of Ceratocystis adiposa. Trans. Brit. Mycol.
Soc. 72: 177-187.
Hay, G. W. , B. A. Lewis, and F. Smith. 1965. Periodate oxidation of
polysaccharides: general procedures. In Methods of carbohydrate
chemistry, Vol. V, Edited by R. L. Whistler, B. C. Miller, and
J. N. Wolfrom. Academic Press, N.Y. pp. 357-360.
Heath, I. B. , J. L. Gay, and A. D. Greenwood. 1971. Cell wall formation
in the Saprolegniales : cytoplasmic vesicles underlying developing
walls. J. Gen. Microbiol. 65: 225-232.
155
Hegnauer, H. , and H. R. Hohl. 1978. Cell wall architecture of sporangia,
chlamydospores, oogonia, and oospores in Phytophthora. Exp.
Mycol. 2: 216-233.
Hill, T. W. 1978. Isolation and characterization of vesicles involved
in hyphal tip growth of Aahlya. Dissertation. University of
Florida.
Hirayama, T. , H. Nagayama, and K. Matsuda. 1976. Studies on cellulases
of a phytopathogenic fungus, Pyviaularia ovyzae Cavara. III.
Multiplicity of 6-glucosidase, and purification and properties
of a second component. J. Biochem. 85: 591-599.
Horowitz, D. K. , and P. J, Russell. 1974. Hormone- induced differentiation
of antheridial branches in Aahyta ambiseayualis : dependence on
ribonucleic acid synthesis. Can. J. Microbiol. 20: 977-980.
Hunsley, D. , and J. H. Burnett. 1968. Dimensions of microfibrillar
elements in fungal walls. Nature, 218: 462-463.
Hunsley, D. , and J. H. Burnett. 1970. The ultrastructural architecture
of the walls of some hyphal fungi. J. Gen. Microbiol. 62: 203-218.
Ishizuka, I., N. Ueta, and T. Yamakawa. 1966. Gas chromatographic
studies of microbial components. II. Carbohydrate and fatty acid
constitution of the family Micrococcaceae. Jap. J. Exp. Med.
36: 73-83.
Jayme, G. , and F. Lang. 1963. Cellulose solvents. In Methods in
carbohydrate chemistry, Vol. III. Edited by R. L. Whistler and
M. L. Wolfrom. Academic Press, N.Y. pp. 75-83.
Jayme, G. , and K. Neuschaffer. 1957. Tri-(en)-cadmium-hydroxyd als
neues farbloses, waBriges Losungsmittel fiir cellulose. Die
Naturwissenschaften, 44: 62-63.
Kane, B. E. 1971. Regulatory mechanisms in cellulase synthesis during
hyphal morphogenesis in the water mold Aahlya. Dissertation.
University of Florida.
Kane, B. E. , J. B. Reiskind, and J. T. Mullins. 1973. Hormonal control
of sexual morphogenesis in Aahlya: dependence on protein and ribo-
nucleic acid syntheses. Science, 180: 1192-1193.
Kanfer, J., and E. P. Kennedy. 1963. Metabolism and function of
bacterial lipids. I. Metabolism of phospholipids in Esaheviahia
aoli B*. J. Biol. Chem. 238: 2919-2922.
Keegstra, K. , K. W. Talmadge, W. D. Bauer, and P. Albersheim. 1973.
The structure of plant cell walls. III. A model of the walls of
suspension-cultured Sycamore cells based on the interconnections
of the macromolecular components. PI. Physiol. 51: 188-196.
156
Kidby, D. K. , and R. Davies. 1970. Invertase and disulphide bridges
in the yeast wall. J. Gen, Microbiol. 61: 327-333.
Korn, E. D. , and D. H. Northcote. 1960. Physical and chemical
properties of polysaccharides and glycoproteins of the yeast-cell
wall. Biochem. J. 75: 12-17.
Kowkabany, G. N. 1954. Paper chromatography of carbohydrates and
related compounds. In Advances in carbohydrate chemistry, Vol. 9.
Edited by M. L. Wolfrom. Academic Press, N.Y. pp. 303-353.
Ladisch, M. R. , C. M. Ladisch, and G. T. Tsao. 1978. Cellulose to
sugars: new path gives quantitative yield. Science, 201: 743-745.
Lampen, J. 0. 1968. External enzymes of yeast: their nature and
formation. Antonie van Leeuwenhoek, 34: 1-18.
Lamport, D. T. A. 1965. The protein component of primary cell walls.
In Advances in botanical research. Edited by R. D. Preston.
Academic Press, N.Y. pp. 151-218.
Lamport, D. T. A. 1967. Hydroxyproline-o-glycosidic linkage of the
plant cell wall glycoprotein extensin. Nature, 216: 1322-1324.
Lamport, D. T. A. 1969. The isolation and partial characterization of
hydroxyproline-rich glycopeptides obtained by enzymic degradation
of primary cell walls. Biochem, 8: 1155-1163.
Lamport, D. T. A. 1970. Cell wall metabolism. Ann. Rev. PI. Physiol
21: 255-270.
Lamport, D. T. A. 1973. The glycopeptide linkages of extensin: 0-D-
galactosyl serine and 0-L-arabinosyl hydroxyproline. In Biogenesis
of plant cell wall polysaccharides. Edited by F, Loewus. Academic
Press, N.Y. pp. 147-164.
Lamport, D. T. A., and D. H. Northcote. 1960. Hydroxyproline in
primary cell walls of higher plants. Nature, 188: 665-666.
Lehninger, A. L. 1975. Biochemistry, 2nd edition. Worth Publishers
Inc., N.Y. 1104 pp.
Leppard, G. G. , L. C. Sowden, and J. R. Colvin. 1975. Nascent stage
of cellulose biosynthesis. Science, 189: 1094-1095.
Letoumeau, D. R. , J. M. Deven, and M, S. Manocha. 1976. Structure
and composition of the cell wall of Chocmephova auaurbitopum.
Can. J. Microbiol. 22: 486-494.
Lin, C. C, and J. M. Aronson. 1970. Chitin and cellulose in the cell
walls of the Oomycete, Apodaohlya sp. Arch, Mikrobiol. 72: 111-
114.
157
Lin, C. C. , C. Sicher, Jr., and J. M. Aronson, 1976. Hyphal wall
chemistry in Apodaahlya. Arch. Microbiol. 108: 85-91.
Lippman, E. , D. C. Erwin, and S. Bartnicki-Garcia. 1974. Isolation and
chemical composition of oospore-oogonium walls of Phytophthora
megasperma var. sojae. J. Gen. Microbiol. 80: 131-141.
Lowry, 0. H. , N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193: 265-275.
Mahadevan, P. R. , and E. L. latum. 1965. Relationship of the major
constituents of the Neurospora arassa cell wall to wild-type and
colonial morphology. J. Bacteriol. 90: 1073-1081.
Mahadevan, P. R. , and E. L. Tatum. 1967. Localization of structural
polymers in the cell wall of Neurospora arassa. J. Cell. Biol.
35: 295-302.
Manners, D. J., A. J. Masson, and J. C. Patterson. 1973a. The
structure of a 6(1^3)-D-glucan from yeast cell walls. Biochem. J.
135: 19-30.
Manners, D. J., A. J. Masson, J. C. Patterson, H. Bjorndal, and B. Lindberj
1973b. The structure of a 6-(l^)-D-glucan from yeast cell walls.
Biochem. J. 135: 31-36.
McMorris, T. C. , and A. W. Barksdale. 1967. Isolation of a sex hormone
from the water mold Aahlya hisexualis. Nature, 215: 320-321.
McMorris, T. C. , R. Seshadri, G. R. Weihe, G. P. Arsenault, and A. W.
Barksdale. 1975. Structures of oogoniol-1, -2, and -3, steroidal
sex hormones of the water mold, Aahlua. J. Amer. Chem. Soc. 97:
2544-2545.
McNair, H. M. , and E. J. Bonelli. 1968. Basic gas chromatography.
Varian Aerography, Walnut Creek, Cal. 306 pp.
Mendoza, C. G. , J. A. Leal, and M. Novaes-Ledieu. 1979. Studies of
the spore walls of Agariaus hisporus and Agariaus aampestris.
Can. J. Microbiol. 25: 32-39.
Mitchell, R. , and N. Sabar. 1966. Hyphal cell wall structure of two
species of Pythiim. Can. J. Microbiol. 12: 471-475.
Miyazaki, T. , M. Yamada, and T. Ohno. 1974. Isolation and structure
of al,3 linked glucan from hyphal wall of Phytophthora infestans.
Chem. Pharm. Bull. 22: 1666-1669.
Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron
microscopy. Stain Technology, 39: 111-114.
158
Monro, J, A., R. W. Bailey, and D. Penny. 1974. Cell wall hydroxy-
proline-polysaccharide associations in Lupinus hypocotyls.
Phytochemistry, 13: 375-382.
Monro, J. A., D. Penny, and R. W. Bailey. 1976. The organization and
growth of primary cell walls of lupin hypocotyl. Phytochemistry.
15: 1193-1198.
Morris, D. L. 1948. Quantitative determination of carbohydrates with
Dreywood's anthrone reagent. Science, 197: 254-255.
Mullins, J. T. 1979. A freeze-fracture study of hormone- induced branch-
ing in the fungus Aahtya. Tissue and Cell, 11: 585-595.
Mullins, J. T. , and A. W. Barksdale. 1965. Parasitism of the chytrid
Diatyomorpha dioiaa. Mycologia, 57: 352-359.
Mullins, J. T. , and E. A. Ellis. 1974. Sexual morphogenesis in Aahtya:
ultrastructural basis for the hormonal induction of antheridial
hyphae. Proc. Nat. Acad. Sci. U.S.A. 71: 1347-1350.
Muzzarelli, R. A. A. 1977. Chitin. Pergamon Press, N.Y. 309 pp.
Northcote, D. H. 1963. The nature of plant cell surfaces. In The
structure and function of the membranes and surfaces of cells.
Biochem. Soc. Symp. 22. Edited by D. J. Bell and J. K. Grant.
Cambridge University Press, Cambridge, pp. 105-125.
Novaes-Ledieu, M. , and A. Jimenez-Martinez. 1969. The structure of
cell walls of Phycomycetes. J. Gen. Microbiol. 54: 407-415.
Novaes-Ledieu, M. , A. Jimenez-Martinez, and J. R. Villaneuva. 1967.
Chemical composition of hyphal wall of Phycomycetes. J. Gen.
Microbiol. 47: 237-245.
Nyhlen, L. , and T. Unestam. 1978. Cyst and germ tube wall structure
in Aphxznomyces astaci, Oomvcetes. Can. J. Microbiol. 24: 1293-
1299.
Okazaki, M. , and M. Moo-Young. 1978. Kinetics of enzymatic hydrolysis
of cellulose: analytical description of a mechanistic model.
Biotech. Bioeng. 20: 637-663.
Pao, V. M. , and J. M. Aronson. 1970. Cell wall structure of Sapromyaes
elongatus. Mycologia, 62: 531-541.
Parker, B. C. , R. D. Preston, and G. E. Fogg. 1963. Studies of the
structure and chemical composition of the cell walls of Vaucheriaceae
and Saprolegniaceae. Proc. Roy. Soc. B, London, 158: 435-445.
Partridge, S. M. 1944. Aniline hydrogen phthalate as a spraying
reagent for chromatography of sugars. Nature, 164: 443.
159
Pease, 0. C. 1964, Histological techniques for electron microscopy.
2nd Edition. Academic Press, N.Y. 381 pp.
Pengra, R. M. , M. A. Cole, and M. Alexander. 1969. Cell walls and lysis
of Mortievella parvispora hyphae. J. Bacteriol. 97: 1056-1061.
Pierce, A. E. 1968. Silylation of organic compounds. Pierce Chemical
Company, Rockford, Illinois. 487 pp.
Potgieter, J. H. , and M. Alexander. 1965. Polysaccharide components of
NevLTOspora avassa hyphal walls. Can. J. Microbiol. 11: 122-125.
Prakasam, V. R. , and J. Azariah. 1975. An optimum pH for the demonstra-
tion of chitin in Peviplanata ameriaana using Lugol's iodine.
Acta histochem. 53: 238-240.
Preston, R. D. 1974a. The physical biology of plant cell walls.
Chapman and Hall, London. 491 pp.
Preston, R. D. 1974b. Plant cell walls. In Dynamic aspects of plant
ultrastructure. Edited by A. W. Robards. McGraw-Hill Book
Company, N.Y. pp. 256-309.
Preston, R. D. 1979. Polysaccharide conformation and cell wall function.
Ann. Rev. Physiol. 30: 55-78.
Raper, J. R. 1939a. Role of hormones in the sexual reaction of hetero-
thallic Achlyas. Science, NS. 89: 321-322.
Raper, J. R. 1939b. Sexual hormones in Aahlya. I. Indicative evidence
for a hormonal coordinating mechanism. Amer. J. Hot. 26: 639-650.
Raper, J. R. 1940. Sexual hormones in Achlya. II. Distance reactions,
conclusive evidence for a hormonal coordinating mechanism. Amer.
J. Hot. 27: 162-173.
Rees, D. A. 1977. Polysaccharide shapes. John Wiley and Sons, N.Y. 80
pp.
Reese, E. T. , and M. Mandels. 1963a. Enzjnnic hydrolysis of 6-glucans.
In Advances of enzymic hydrolysis of cellulose and related materials.
Edited by E. T. Reese. Macmillan Company, N.Y. pp. 197-234.
Reese, E. T. , and M. Mandels. 1963b. Enzymic hydrolysis of cellulose and
its derivatives. In Methods of carbohydrate chemistry. Vol. III.
Edited by R. L. Whistler and M. L. Wolfrom. Academic Press, N.Y.
pp. 139-143.
Reiskind, J. B. 1970. The role of translation in the hormonal induction
of sexual morphogenesis in Aahlya. Thesis. University of Florida.
Reissig, J. L. , J. L. Strominger, and L. F. Leloir. 1955. A modified
colorimetric method for the estimation of N-acetylamino sugars.
J. Biol. Chem. 217: 959-966.
160
Rosenberger, R. F. 1976, The cell wall. In The filamentous fungi.
Vol. 2. Edited by J. E. Smith and D. R. Berry. John Wiley and
Sons, N.Y. pp. 328-344.
Ruiz-Herrera, J. 1967. Chemical components of the cell wall of
Aspergillus species. Arch. Biochem. Biophys. 122: 118-125.
Sasaki, T. , and T. Tanaka, N. Naki, Y. Sato, and K. Kainuma. 1979.
Correlation between x-ray diffraction measurements of cellulose
crystalline structure and the susceptibility to microbial cellulase.
Biotech. Bioeng. 21: 1031-1043.
Sietsma, J. H. , D. E. Eveleigh, and R. H. Raskins. 1968. The purification
of cellulase and exo- laminar inase and their role in the formation of
Pythiian sp. protoplasts. Antonie van Leeuvenhoek, 34: 331-340.
Sietsma, J. H. , D. E. Eveleigh, and R. H. Haskins. 1969. Cell wall
composition and protoplast formation of some Oomycete species.
Biochim. Biophys. Acta, 184: 306-317.
Sietsma, J. H. , J. J. Child, L. R. Nesbitt, and R. H. Haskins. 1975.
Chemistry and ultrastructure of the hyphal walls of Pythium
acanthiaim. J. Gen. Microbiol. 86: 29-38.
Sietsma, J. H. , and J. G. H. Wessels. 1977. Chemical analysis of the
hyphal wall of Schizophyllum aommicne. Biochim. Biophys. Acta,
496: 225-239.
Sietsma, J. H. , and J. G. H. Wessels. 1979. Evidence for covalent
linkages between chitin and g-glucan in a fungal wall. J. Gen.
Microbiol. 114: 99-108.
Skujins, J. J., H. J. Potgieter, and M. Alexander. 1965. Dissolution
of fungal cell walls by a Streptomycete chitinase and 6- (1-^3)
glucanse. Arch. Biochem. Biophys. Ill: 358-364.
Smith, F., and R. Montgomery. 1956. End group analysis of polysaccharides.
In Methods of biochemical analysis, Vol. III. Edited by D. Click.
Interscience Publishers, Inc., N.Y. pp. 153-212.
Sturgeon, R. J. 1974. Chemical and biochemical aspects of fungal cell
walls. In Plant carbohydrate biochemistry. Edited by J. B.
Pridham. Academic Press, N.Y. pp. 219-233.
Sweeley, C. C. , R. Bentley, M. Makita, and W. W. Wells. 1963. Gas-
liquid chromatography of trimethylsilyl derivatives of sugars
and related substances. J. Amer. Chem. Soc, 85: 2497-2507.
Talmadge, K. W. , K. Keegstra, W. D. Bauer, and P. Albersheim. 1973. The
structure of plant cell walls. I. The macromolecular components of
the walls of suspension-cultured Sycamore cells with a detailed
analysis of the pectic polysaccharides. PI. Physiol. 51: 158-173.
161
Taylor, I. E. P., and D. S. Cameron. 1973. Preparation and quantitative
analysis of fungal cell walls: strategy and tactics. Ann. Rev.
Mcrobiol. 27: 243-257.
Thomas, D. des S. 1970. Effects of water stress on induction and
secretion of cellulase and on branching in the water mold Aahlya
ambisexualis. Can. J. Bot. 48: 977-979.
Thomas, D. des S. , and J. T. Mullins. 1969. Cellulase induction and
wall extension in the water mold Aohlya ambisexual-is . Physiol.
Plant. 22: 347-353.
Timberlake, W. E. 1976. Alterations in RNA and protein synthesis
associated with steroid hormone- induced sexual morphogenesis in
the water mold Aohlya, Devel. Biol. 51: 202-214.
Tokunaga, J., and S. Bartnicki-Garcia. 1971. Structure and differentia-
tion of the cell wall of Phytophthora palnrivora: cysts, hyphae and
sporangia. Arch. Mikrobiol. 79: 293-310.
Tracey, M. V. 1955. Chitin. In Modern methods of plant analysis, Vol.
II. Edited by K. Paech and M. V. Tracey. Springer-Verlag,
Berlin, pp. 264-274.
Tristram, G. R. , and R. H. Smith. 1963. The amino acid composition of
some purified proteins. In Advances in protein chemistry. Vol. 18.
Edited by C. B. Anfinsen, Jr. , M. L. Anson, and J. T. Edsall.
Academic Press, N.Y. pp. 227-318.
Van der Valk, P. , and J. G. H. Wessels. 1977. Light and electron
microscopic autoradiography of cell-wall regeneration by
Sahizophyltum aorrmune protoplasts. Acta Bot. Neerl. 26: 43-52.
Wang, H. S. , and H. B. LeJohn. 1974. Analogy and homology of the
dehydrogenases of Oomycetes. I. Regulation of glutamic dehydrogenases
and isozyme patterns. Can. J. Microbiol. 20: 567-574,
Weijman, A. C. M. 1979. Cell-wall composition and taxonomy of
Cephaloasaus fragrans and some Ophiostomataceae. Antonie van
Leeuwenhoek, 42: 315-324.
Wessels, J. G. H. , D. R. Kreger, R. Marchant, B. A. Regensburg, and
0. M. H. de Vries. 1972. Chemical and morphological characteri-
zation of the hyphal wall surface of the Basidiomycete Sohizophyllim
aorrmune. Biochim. Biophys. Acta, 273: 346-358.
Whitaker, D. R. 1970. Cellulases. In The enzymes. Vol. II, 3rd
edition. Edited by P. D. Boyer, Academic Press, N.Y. pp. 273-290.
White, A., P. Handler, and E. L. Smith. 1964. Principles of bio-
chemistry. McGraw-Hill Book Company, N.Y. 1106 pp.
162
Whitehouse, M. W. , A. E. Bresler, and E. Staple. 1958. The use of
iodine for the detection of lipids. J. Chr.omatog. 1: 385-386.
Wolfe, S. L. 1972. Biology of the cell. V7adsworth Publishing Company,
Inc., Belmont, California. 545 pp.
Worthington Glucostat Reagent Set, Worthington Biochemical Corporation,
Freehold, New Jersey.
Wrathall, C. R. , and E. L. latum. 1973. The peptides of the hyphal
wall of NevLTos-pora arassa. J. Gen. Iticrobiol. 78: 139-153.
Yamada, M. , and T. Miyazaki. 1976. Ultrastructure and chemical analysis
of the cell wall of Pythiion debary anion. Japan J, Microbiol. 20:
83-91.
Yamakawa, T. , and N. Ueta. 1964a. Gas-liquid chromatography of
carbohydrates. Japan J. Exp. Med. 34: 37-51.
Yamakawa, T. , and N. Ueta. 1964b. Gaschromatographic studies of microbial
components. I. Carbohydrate and fatty acid constitution of
Eeissevla. Jap. J. Exp. Med. 34: 361-374.
Youatt, J. 1977. Chemical nature of Allomyaes walls. Trans. Brit.
Mycol. Soc. 69: 187-190.
Young, T. W. K. 1970. Arrangement of microfibrils in walls of aerial
hyphae of Lirdevina. Trans. Brit. Mycol. Soc. 55: 29-35.
Zanetta, J. P., W. C. Brechenridge, and G. Vincedon. 1972. Analysis
of monosaccharides by gas-liquid chromatography of the 0-methyl
glycosides as trif luoroacetate derivatives. Application to
glycoproteins and glycolipids. J. Chromatog. 69: 291-304.
Zevenhuisen, L. P. T. M. , and S. Bartnicki-Garcia. 1969. Chemical
structure of the insoluble hyphal wall glucan of Phytophtiiora
ainncanomi . Biochem. 8: 1496-1502.
BIOGRAPHICAL SKETCH
Julia Barth Reiskind was born in Hackensack, New Jersey, on
June 26, 1941. She attended the public schools in Ridgewood, New
Jersey, and graduated from high school in 1959. She received a Bachelor
of Arts degree from Goucher College, Towson, Maryland, in biological
sciences in 1963. She then worked as a Research Assistant in the
laboratory of John R. Raper, Harvard University, Cambridge, Massachusetts,
until 1967. She enrolled in graduate school at the University of Florida
in 1968 and received a Master of Science in botany in 1970. She began
her pursuit of the Ph.D. in botany in 1973 as a part-time student and
has been working toward that end since that time. She is a member of
Phi Kappa Phi Honorary Society, Sigma Xi, the Mycological Society of
America, and the American Society of Plant Physiologists.
She is married to Dr. Jonathan Reiskind and has two children,
Julia Alexandra and Michael Hay, ages nine and six, respectively.
163
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
J(;y'rhomas Mullins , Ch/irman
Professor of Botany
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Mildred M. Griffith - '
Professor Emeritus of Botany
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Chesley B. Hall
Professor of Vegetable Crops
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Thomas E. Humphreys
Professor of Botany
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Paul H. Smith l^JJ
Professor of Microbiology and Cell
Science
This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
June 1980
■cuh
\t.^.
DeanyyCollege of AgricuJ,«rre
Dean, Graduate School
i#liii
Live Music Archive
Librivox Free Audio
Metropolitan Museum
Cleveland Museum of Art
Internet Arcade
Console Living Room
Books to Borrow
Open Library
TV News
Understanding 9/11