^■^^-^sj/j^n- Fatty Acid Metabolism in Microorganisms E. R. SQUIBB LECTURES ON Presented at the Institute of Microbiology Rutgers, the State University of New Jersey F. M. Strong, Topics in Microbial Chemistry, 1956 F. H. Stodola, Chemical Transformations by Microorganisms, 1957 V. H. Cheldelin, Metabolic Pathways in Microorganisms, 1960 K. Hofmann, Fatty Acid Metabolism in Microorganisms, 1962 CHEMISTRY OF MICROBIAL PRODUCTS MARINE BIOLOGICAL LABORATORY LIBRARY WOODS HOLE, MASS. W. H. 0. \. Fatty Acid Metabolism in Microorganisms By KLAUS HOFMANN Professor of Biochemistry University of Pittsburgh School of Medicine NEW YORK • LONDON, JOHN WILEY & SONS, INC. Copyright © 1963 by John Wiley & Sons, Inc. All rights reserved. This book or any part thereof must not be reproduced in any form without the written permission of the publisher. Library of Congress Catalog Card Number: 63-17481 Printed in the United States of America In recognition of the importance of cooperation between chemist and microbiologist the E. R. Squibb Lectures on Chemistry of Microbial Products were established with the support of The Squibb Institute for Medical Research in 1955. The lectures are presented annually in the fall at the Institute of Microbiology, Rutgers, the State University of New Jersey, New Brunswick, New Jersey. PREFACE The invitation to deliver the 1962 Squibb Lectures on Chemistry of Microbial Products was an honor which pro- vided me with a welcome opportunity to summarize our studies dealing with some phases of fatty acid metabolism in microorganisms and to bring them into focus with recent developments. The experimental work has been carried out in the chemistry and biochemistry departments of the Uni- versity of Pittsburgh since 1947. The book is divided into three chapters, which deal, respectively, with the discovery and chemistry of cyclopropane fatty acids, the chemical na- ture of monounsaturated fatty acids in bacteria, the quan- titative estimation of fatty acids in bacterial lipids, the bio- synthesis of the cyclopropane ring, and finally the anaerobic biosynthesis of monounsaturated fatty acids in microorgan- isms. The presentation is critical and influenced by the author's own point of view. No attempt is made to provide a comprehensive summary of the literature, and apologies PREFACE are offered to investigators whose contributions have been omitted. The experimental studies could not have been carried out without the devoted help of a number of former students and colleagues, and I wish to express my very sincere ap- preciation to Drs. Henis, Jucker, Liu, Lucas, Marco, Miller, O'Leary, Panos, Sax, Tausig, Yoho, Young, and the late Dr. Orochena for their untiring efforts. I also wish to express my thanks to Professors Axelrod and Jeffrey of the Univer- sity of Pittsburgh for many helpful discussions. This little book will fulfill its mission if it stimulates further inquiry into the neglected but intriguing field of microbial lipid metabolism. K. HOFMANN Pittsburgh, Pennsylvania April, 1963 ACKNOWLEDGMENT The author wishes to express his gratitude to Biochemistry, to the Journal of the American Chemical Society, to the Journal of Biological Chemistry, and to Federation Proceed- ings for their permission to reproduce certain figures and tables. K. H. Bio:. LABORATORY LIBRARY WOODS HGLE, MASS. ! W. H. 0. !. CONTENTS CHAPTER 1 Lactobacillic Acid, a Novel Microbial Metabolite 7. Discovery of Lactobacillic Acid, 1 2. Structure of Lactobacillic Acid, 7 3. Laboratory Synthesis of Long-Chain Fatty Acids Containing the Cyclopro- pane Ring, 8 4. X-ray Studies of Cyclopropane Fatty Acids, 22 3. Occurrence of Lactobacillic Acid and Its Homologs in Bacteria, 27 6. Microbiological Activity of Cyclopro- pane Fatty Acids, 28 CONTENTS CHAPTER 2 Biosynthesis of Cyclopropane Fatty Acids 32 1. Estimation of the Fatty Acid Compo- sition of Bacterial Lipids, 32 2. Chemical Nature of Monoethenoid Fatty Acids in Microorganisms, 55 3. Fatty Acid Interconversions in Lacto- bacilli, 38 4. Position of the Cyclopropane Ring in Lactobacillic Acid and Separation of Carbon Atom 19 from the Mole- cule, 42 5. Cyclopropane Ring Biosynthesis, 48 CHAPTER 3 Biosynthesis of Monounsaturated Fatty Acids by Microorganisms 57 L Growth Studies, 57 2. Studies with Labeled Compounds, 62 3. Metabolism of Monounsaturated Fatty Acids in Bacteria, 66 Index 73 CHAPTER LACTOBACILLIC ACID, A NOVEL MICROBIAL METABOLITE 1. DISCOVERY OF LACTOBACILLIC ACID The observation (1-7) that unsaturated fatty acids exert a marked sparing action on the biotin requirements of certain lactic acid organisms prompted initiation of our systematic studies on the chemical nature of bacterial fatty acids. These studies, which led to the discovery of lactoba- cillic acid and to the recognition of cis-va.ccenic acid as an important constituent of bacteria, provided the structural foundation for investigations of fatty acid metabolism in these lower forms of life. The chemical nature of the fatty acids of Lactobacillus arahinosus (8, 9), Lactobacillus casei (10), Agrobacterium (Phytomonas) tumefaciens (1 1), and of a group C Streptococ- cus species (12) was determined in detail. The organisms were grown on essentially lipid-free media and fatty acids were isolated in the usual manner. Autoclaving with dilute acid must precede extraction, since some 80% of the fatty acids are present in the bacteria in a "bound" form not soluble in mixtures of acetone and ether. The lipids were 1 FATTY ACID METABOLISM IN MICROORGANISMS 190 180 — J^ 170- E 160 E en ^ 150 o o ^140 130 120 190 185 I 180 175 170 p 165 ^ 160 155 150 1 1 1 1 1 1 . - ^ . . .^ T /- Qc/ '- — r Ci8 - J Curve I J" Ci6 i 1 1 1 1 1 1 1 1 1 4 5 6 Grams distilled 10 1 1 1 i 1 1 1 1 1 - ^^^^^'^^'ciT""'"''^ / Curve II - 1 1 111! 1 1 1 5 6 7 Grams distilled 10 Fig. 1.1. Separation of methyl esters of fatty acids derived from L. arabinosus (curve I); L. casei (curve II); Streptococcus hemolytkus, group C (curve III); Agrobacterium tumefaciens (curve IV). LACTOBACILLIC ACID 120 110 200 190 1^ 180 Q. E 170 E 00 - 160 CO Curve III h J \ \ L J L 1234 56 789 10 11 Grams distilled 150 140 130 - ' ' ' ' ' i 1 1 1 >- - . . . J^'^ 4- f i Curve IV - 1 1 1 1 1 1 1 1 1 123456789 10 Grams distilled Fig. 1.1 continued. 4 FATTY ACID METABOLISM IN MICROORGANISMS saponified, the fatty acids converted into the methyl esters, and the ensuing ester mixture was separated into various components by fractional distillation. Inspection of typical distillation curves (Fig. 1.1), relating boiling point to amount distilled, shows the prominent presence of fatty acids containing 16 and 18 carbon atoms with lower fatty acids being present in small proportions. The presence of esters with boiling points above methyl stearate in the ester mixture derived from L. arahinosuSj I casei, and A. tume- faciens is of particular interest. SapoL fication of this high- est boiling fraction gave a low melting (28-29°) crystalline acid of the composition C19H36O2, which was given the name 4000 3000 2000 J \ I Wave numbers in cm-i 1500 1200 1000 900 800 700 T 7 8 9 10 11 Wave length in microns Fig. 1.2. Infrared absorption spectra of cyclopropane fatty acids. (1) "Phytomonic" acid from A. tumefaciens. (2) Lactobacillic acid from L. casei. LACTOBACILLIC ACID TABLE 1.1 Comparison of Main X-ray Spacings of Lactobacillic Acid from L. arabinosus and from A, tumefaciens L. arabinosus A. tumefaciens Main Short Spacings 4.65 M * 4.35 M 4.07 M- 3.7^. Wt 3.58 W+ 3.42 W- 4.67 St 4.38 M 4.30 S 4.07 W+ 3.81 W 3.61 W 3.43 W- Long Spacing 41.0 41.4 * M = medium t S = strong 1 W = weak lactobacillic acid in view of its first isolation from a lacto- bacillus. Lactobacillic acid from L. arabinosus and L. casei is identical as concerns melting point, infrared absorption spectrum, and x-ray diffraction pattern. Lactobacillic acid is also identical with phytomonic acid (11), a compound previously isolated from A. tumefaciens whose true chemical nature had not been recognized by earlier investigators (13- 17). The matching infrared absorption spectra (Fig. 1.2), x-ray diffraction patterns (Table 1.1), and melting points offer unequivocal evidence for identity. Lactobacillic acid appears to be a major constituent of the bacterial phospho- lipids (18). FATTY ACID METABOLISM IN MICROORGANISMS CH2 H3C-(CH2)x-C H 1 H H, , PtO, (I) C— (CH2)v— COOH CH2 (H) CH3 H3C— (CH2):c— CH2 CH2— (CH2);^— COOH Solid acid. m.p. eS'-eS.S" * + y = 14 H3C— (CH2)x— C^ H CH2— (CH2)y— COOH CHa (HI) H3C— (CH2)x— CH2 ^ C— (CH2)v— COOH H ^ Liquid fraction, m.p. 13'-14' Fig. 1.3. Hydrogenolysis products of lactobacillic acid. LACTOBACILLIC ACID 2. STRUCTURE OF LACTOBACILLIC ACID The behavior of lactobacillic acid (8, 9) which provides the key to its chemical constitution is illustrated on Fig. 1.3. The acid is stable toward oxidizing agents but undergoes hydrogenolysis in presence of platinum and hydrogen with absorption of one mole of hydrogen. The resulting mixture of hydrogenation products contains nonadecanoic acid plus a product also containing 19 carbon atoms which melts at 13 to 14°, and on the basis of C-methyl determinations, possesses a branched carbon-chain. The latter material consists of a mixture of branched chain acids later recog- nized as DL-11- and DL-12-methyloctadecanoic acids. These materials arise from the simultaneous hydrogenolysis of the carbon-carbon bonds 2 and 3 in the lactobacillic acid mole- cule. Treatment with hydrogen bromide in glacial acetic acid converts lactobacillic acid into a mixture of mono- bromononadecanoic acids (see Chapter 2, section 4). The chemical behavior of lactobacillic acid, i.e., its stabil- ity toward oxidation and its lability toward hydrogen bro- mide and hydrogenolysis, coupled with the fact that hydro- genolysis produces three acids— one of which contains a straight carbon chain (nonadecanoic acid)— pointed to the presence in lactobacillic acid of a cyclopropane ring. Sta- bility toward oxidation and lability to hydrogenolysis and hydrogen bromide are characteristic properties of this par- ticular ring system. The infrared absorption spectrum (Fig. 1.4, curve 1) supports the cyclopropane nature of lactobacil- lic acid. The spectrum exhibits the characteristic cyclopro- pane absorption maximum at 9.8 ^ (19), which disappears on hydrogenation (curve 2). FATTY ACID METABOLISM IN MICROORGANISMS T 5.0 7.0 9.0 Wave length in microns Fig. 1.4. Infrared absorption spectra of lactobacillic acid and one of its hydrogenation products. (1) Lactobacillic acid from L. arahinosus. (2) Liquid hydrogenation product. All of the properties cited fit formula I (Fig. 1.3) as a likely structure for lactobacillic acid with the position of the cyclopropane ring unassigned. LABORATORY SYNTHESIS OF LONG-CHAIN FATTY ACIDS CONTAINING THE CYCLOPROPANE RING Although the position of the methylene bridge cannot be established by the reactions described in Chapter 1, sec- tion 2, structures for lactobacillic acid with the ring located LACTOBACILLIC ACID in the 9,10 and 11,12 positions were considered likely be- cause of their relation to the monoethenoid fatty acids (9- octadecenoic and 11-octadecenoic acids) which occur in bac- terial lipids. If we consider the stereochemistry of this type of molecule, it is apparent that each position isomeric cyclopropane fatty acid can occur in four stereoisomeric forms, two of which belong to the cis, the other to the trans series. These forms are illustrated for the 9,10-compounds in Fig. 1.5. In order to have available model compounds for compari- trans (CH2)7 COOH (CH2)7 COOH -^ Asymmetric carbon atoms Fig. 1.5. The stereochemistry of 9,10 methyleneoctadecanoic acid. 10 FATTY ACID METABOLISM IN MICROORGANISMS son with lactobacillic acid, four long-chain fatty acids con- taining the cyclopropane ring— namely, racemic cis and trans 9,10-methyleneoctadecanoic acids and racemic cis and trans 1 1,12-methyleneoctadecanoic acids— were synthesized. The synthetic route to the trans series is illustrated on Fig. 1.6. Trrtn5-cyclopropane-l,2-dicarboxyclic acid (I) was selected as a logical starting point for preparation of acids of this series (20), since it is important to have available models of established stereo-structure for comparison with lactobacillic acid. The monomethyl ester chloride (II) of (I) is con- densed with sodio ethyl acetoacetate, and the resulting crude diketo ester (III) on exposure to sodium methoxide in methanol gives methyl ^rfln5-y-keto-a:,/3-methyleneadipate (IV) (21). Alkylation of (IV) with 77-butyl iodide (V, x = 5) followed by saponification and decarboxylation gives trans- 4-keto-2,3-methylenenonanoic acid (VI, x = 5), which is con- verted into — LACTOBACILLIC ACID 13 X \ ■ ' o o o // ■M Ec] a 5 C3 w' T > QJ 1 y \ w >> ^ X V Cu ■s ^ / ••« o 5 1 >- hH 1 ffi o t/3 + w 1 OJ 1^ 1 < CM -. V i' § F-H 1 J. J 5- b -s / • . / CO ^, 'X ^ *ffi sc ffi o O 1 1 1 1 CO m K H c 14 FATTY ACID METABOLISM IN MICROORGANISMS 400C ) 1 20 00 Wave 1500 1 1 1 1 bers in cmri 1000 ill. BOO 1 700 1 lUU ■^ J^ ^ n A , <—^\ y^^ 80 1 — 1 h /' "i\ / V 1 60 J \^ ) 40 \ \ ' 20 \l 100 ^ (^ '^^ n 80 f ( f ^\ \ r \ "2 60 \ / \ J 40 Ai J 20 \ \ 100 ■v^ r "^ n r\ 80 f «^. / '\ \ r J A 3 60 j / \ / 40 \\ / 20 \ 0 7 9 11 13 Wave length in microns 15 Fig. 1.7. Infrared absorption spectra of cyclopropane fatty acids. (1) DL-ira?j5-ll,12-Methyleneoctadecanoic acid. (2) DL-^ran5-9,10- Methyleneoctadecanoic acid. (3) Lactobacillic acid from L. casei. foetida, is subjected to controlled catalytic hydrogenation (20, 22). The cis configuration for dihydrosterculic acid appeared likely, since low temperature catalytic hydrogena- tion of double bonds favors cis addition of hydrogen. The chemical structure of sterculic acid and location of the three-membered ring is based on the reactions which are illustrated on Fig. 1.8 (22). Since cis-trans isomers in the series of long-chain cyclopro- pane fatty acids differ little in the very physical properties fcD u S o V O O 03 03 •^ .5 U 5£° LD t^ ■,— I T— O rO r--' r--' CN CN r-~' cn CO CO CO rO m CO O O Oj O ^_ (^) c^3 r--' r^ un r-~ LO oo oo oo 00 oo oo I I I I I I O rj- CN O -^^ O O O VD xf vd Tj-' cx) 00 00 OO 00 OO 0 CS -I" 0 CS s 9 be ^' .S ^ O Cn -r- CO '^ CO O ^ 13"'^ ^ CO T-^ --^ CO CO t— 1 a,> ^ Tt ^ ^ -^ ^ t£) •I .SO Tl O o O O lO O (M O "^ I^ Cn CO CM Tj- CO CO CO rO I I I I I I O r- ^O LO sO o oo CN CO o oo T-H C\l CO CO c<~, CO CO T3 G C a. o U _u ^ rs 'C oj o u cd 'o fC CJ 'o c .y 'o o c ct3 CJ (U G rt ^-^ QJ "T^^ -0 t^ s a; >> _G o ^^ CJ to Cn |i ^ cC -^ o c _t^ .'-5 >> s^ ^ y -d ■? V V c^ v=; -1 ►J J iJ 1—^' Q Q Q Q Q 15 16 FATTY ACID METABOLISM IN MICROORGANISMS ffi o T / o=o o o I (M o I ffi cj- Xi ffi CJ- I 00 in « g O O c^ o 8 I I g I \ / LACTOBACILLIC ACID 17 which are useful for purification purposes, methods for the synthesis of the cis cyclopropane fatty acids had to be de- vised which precluded the formation of mixtures of stereo- isomers. Thus, an intermediate of unquestionable cis con- figuration had to be prepared which would lend itself to conversion into the desired long-chain cyclopropane fatty acids without any risk of cis-trans inversion during the process. Cyclopropane-<:/5-l,2-diacetic acid, a compound not previ- ously described, seemed ideally suited for this purpose, since methods are available for its conversion into the desired long-chain acids, and since the separation of its carboxyl groups from the centers of asymmetry eliminates the possibility for cis-trans inversions. Our route (23) to cyclopropane-aVl,2-diacetic acid (Fig. 1.9) involves reaction of equimolar proportions of cyclohexa-l,4-diene (I) with dibromocarbene according to the method of Doering (24) to give the intermediate (II) which is readily converted by oxidation into (III). Cyclopropane-d5-l,2-diacetic acid is obtained in good yields when (III) is subjected to hydro- genolysis over a Raney nickel catalyst in presence of potas- sium hydroxide. The method of synthesis employed and the fact that cyclopropane-<:/5-l,2-diacetic acid, identical with the acid prepared by the above described scheme, can be obtained from cyclopropane-c?5-l,2-dicarboxyIic acid (25) leaves no doubt regarding the stereochemical configuration of this compound. For conversion into long-chain fatty acids, the carboxyl ends of cyclopropane-c/5-l,2-diacetic acid are elongated through suitable manipulations (26, 27). For construction of the hydrocarbon end (Fig. 1.10), the diacetic acid (I) is converted into the monomethyl ester, and the acid chloride of the latter (II) is reacted with a suitable alkylcadmium reagent to form the keto acid (III), which is 18 FATTY ACID METABOLISM IN MICROORGANISMS reduced with hydrazine to give an intermediate of structure (IV). Elongation of the carboxyl end of intermediate (IV) (Fig. 1.11) is effected according to a scheme which was developed by Stallberg-Stenhagen (21). Identity of the synthetic com- pound (x and y = 7) with dihydrosterculic acid unequivo- cally establishes the structure of this compound as Di.-cis- 9,10-methyleneoctadecanoic acid. DL-c/5-ll,12-Methylene- octadecanoic acid (DL-lactobacillic acid) is synthesized from cyclopropane diacetic acid by the same procedures. Addition of iodomethyl zinc iodide across the double bond of olefins affords cis cyclopropane derivatives in high yield (28). This reaction has served to synthesize dihydro- CH2 CH2 (H) Oxidation "v" Ni, H,,OH- f Br Br HOOC— CH2 CH2— COOH HOOC— CH2 CH2— COOH (IV) (III) Fig. 1.9. Stereospecific synthesis o£ c/5-cyclopropane-l,2-diacetic acid. LACTOBACILLIC ACID 19 H H HOOC— CH2 CH2— COOH (I) H H H3C — (CH2)^_2— Cd/2 + CI— C— CH2 CH2— COOCH3 (II) H H H3C— (CH2);,_2~C — CH2 CH2— COOH (in) H H H3C— (CH2);c CH2— COOH (IV) Intermediate for dihydrosterculic acid x = 7 Intermediate for DL-lactobacillic acid x = 5 Fig. 1.10. Elongation of hydrocarbon end. 20 FATTY ACID METABOLISM IN MICROORGANISMS o=u 8 K A r" ~ \/\ -1 \/ H >-i > \/ ' — B > «N * B 1 -^ 1 1 II II ^ ;^ II II IS C3 .22 ss N 0 4-» •12 • pH cd >^ u tlH ^ ^ ■^ o 1 r ? cc ►^ IH 53 a 4iJ ^ ■§ b 00 (3 Cj «4H c 0 -5h ^ >% "^ jM > V 3 cr W <: •S --. I I ^+^+TTTT CM N_^CO ^-^--_^-^'^^-~_-- ^_; +J G (V i M (d u s r- o c; > 03 _u -o > 5 i^ •S2 .^ O S cj S ^^^^ 21 bo O -Q "G 5- O C^ ^ hJ •^ U ci M nj ^ ^ •^ •^ u RJ .^ -T3 G cd c«^ 3 G ^' *u O .1-1 c3 03 CJ ■t-> G U 'o S *o G G c^ K3 CJ o (U O (U -a 73 -o c^ K3 (U CJ CO CJ o ?^ o flj jG . ^ ^ Jv. C^ ►J J -a S Q P ^ ::: s > - G * o 22 FATTY ACID METABOLISM IN MICROORGANISMS sterculic acid (28) and dl-c/j-9, 10-methylenehexadecanoic acid (29) from oleic and palmitoleic acids, respectively. In Table 1.3, certain physical properties of both the cis and trans forms of synthetic racemic 9,10- and 11,12-methyl- eneoctadecanoic acids are compared with corresponding properties of lactobacillic and dihydrosterculic acids, dl- aVll,12-Methyleneoctadecanoic acid is similar to, but not identical with, lactobacillic acid. The infrared absorption spectra of the synthetic cis acids match those of lactobacillic and dihydrosterculic acids. 4. X-RAY STUDIES OF CYCLOPROPANE FATTY ACIDS Extensive x-ray crystallographic studies by Jeffrey et al. (30-32), using the above mentioned four synthetic racemic cyclopropane fatty acids, i.e., dl-c/^- and trans-9,l0-methyl- eneoctadecanoic and DL-m- and ^?flrz5-ll,12-methyleneocta- decanoic acids, established the detailed stereochemistry of these molecules and contributed materially toward solution of the structure of lactobacillic acid. Two dimensional Fourier projections of DL-<:/5-ll,12-methyleneoctadecanoic acid (left) and DL-^rfln5-9,10-methyleneoctadecanoic acid (right) (Fig. 1.12) illustrate the striking difference in the shape of the cis and trans series of acids. The outstanding feature of the stereochemistry of the cis acids is the boomer- ang-like shape of the molecules with the bend about the cyclopropane ring. This is in marked contrast to the gen- erally straight chain geometry of the trans acid. The ar- rangement of the molecules follows the head to head and tail to tail pattern, which is a characteristic feature of the crystal structure of long-chain fatty acids. Molecules of the LACTOBACILLIC ACID 23 D and L configuration are hydrogen bonded into dimers through the centers of symmetry. The trans cyclopropane ring fits compactly into the regular arrangement of the methylene groups along the chain. The unit cell dimensions of the dl-cw-9,10- and 11,12- acids and of the DL-^rrt7z^-9,10- and 11,12-acids, respectively, are identical within the limits of the experimental proce- dures. The crystal lattices are nearly identical (isomor- phous) because of the closely related arrangement of geo- metrically similar structural subunits. Single crystal and powder x-ray diffraction data show that dihydrosterculic and DL-ci\j-9,10-methyleneoctadecanoic acid are identical (30). The crystal structure analysis of lactobacillic acid (33) carried to the stage of locating the carbon and oxygen atoms in one projection with sufficient precision to establish the general stereochemistry is shown on Fig. 1.13. Taken in conjunction with the chemical evidence (see Chapter 2, section 4) and the crystal structure analysis of the cis-\\,\2,- methyleneoctadecanoic acid racemate (32) the electron density map provides conclusive evidence that lactobacillic acid is D or L czVl 1,12-methyleneoctadecanoic acid. Indi- vidual molecules in the racemate and the naturally occur- ring species appear to have the same characteristic boom- erang-like shape with the bend about the cis substituted cyclopropane ring, but the packing of individual molecules within the crystal lattice of the natural acid is significantly different from that which occurs in the synthetic racemate. The packing is less compact when all the molecules have the same sense as exemplified by the lower melting point and density (29° versus 37°; 0.97 g./cm.^ versus 1.005 g./cm.s) of lactobacillic acid compared to DL-a5-ll,12-methyleneocta- decanoic acid. In place of the centro symmetrically related left- and right-handed molecules which form the hydrogen- 24 FATTY ACID METABOLISM IN MICROORGANISMS Fig. 1.12. Electron density maps of cyclopropane fatty acids. Left: DL-d5-ll,12-Methyleneoctadecanoic acid. Right: DL-trans-9,lO-Methyl- eneoctadecanoic acid. LACTOBACILLIC ACID 25 Fig. 1.12 continued. 26 FATTY ACID METABOLISM IN MICROORGANISMS Fig. 1,13. Electron density map of lactobacillic acid. LACTOBACILLIC ACID 27 bonded dimer in the racemate, an infinite sheet system of hydrogen bonds appears to link the carboxyl groups in the lactobacilhc acid crystal (Fig. 1.13). It is well-known that long-chain monocarboxylic acids ex- hibit trimorphism in both the odd and even series. In efforts to obtain crystals suitable for x-ray intensity measure- ments, a variety of solvents and conditions was employed, but in no instance was there any evidence for more than one crystal form of the cyclopropane acids. The acids crystallized from the melt give powder photographs which are of the same structure as those of the single crystals grown from solution. The cyclopropane ring appears to restrict the number of "economical" modes of packing the long chains into the crystal lattice. Although synthesis of lactobacillic acid has not been achieved, there can be little doubt regarding the position of the cyclopropane ring and its cis configuration. 5. OCCURRENCE OF LACTOBACILLIC ACID AND ITS HOMOLOGS IN BACTERIA Based on studies with radioactive tracers, OTeary (34) suggested the presence of a C17 cyclopropane fatty acid in the lipids of an Escherichia coli mutant, and Dauchy and Asselineau (35) isolated an acid of this type from this or- ganism. From an unidentified laboratory strain of E. coli, Kaneshiro and Marr (29) isolated a similar or possibly identical acid and provided convincing chemical evidence for the cyclopropane nature and the Ci5-9,10-location of the methylene bridge. Similar findings were reported by Innes Chalk and Kodicek (36). It is of interest to note that in addition to this cyclopropane acid, which accounts for some 28 FATTY ACID METABOLISM IN MICROORGANISMS 20% of the fatty acids, the organism also contains lacto- bacillic acid. Clostridium hutyricum, according to Bloch et al. (37), contains a family of cyclopropane fatty acids with 13, 15, 17, and 19 carbon atoms, respectively. Evi- dence for the presence in microorganisms of position iso- meric methyleneoctadecanoic acids is presented in Chapter 3, section 3. 6. MICROBIOLOGICAL ACTIVITY OF CYCLOPROPANE FATTY ACIDS The marked biotin-sparing activity of oleic acid in the nutrition of a variety of microorganisms, originally described by Williams and Fieger (1), was confirmed and extended by a number of investigators (2-7). These studies have shown that long-chain mono- and polyunsaturated fatty acids of both the cis and trans series have this biological property. Saturated fatty acids are inactive but may exert a synergistic effect when supplied to the organisms in con- junction with unsaturated acids (5). The cis isomers of a number of position and stereoisomeric monoethenoid octadecanoic acids exhibit practically the same biotin-spar- ing activity for L. arahinosus regardless of the position of the double bond. With exception of elaidic acid, which possesses a high degree of biotin-sparing potency, the trans isomers are less active than the corresponding cis forms, and the activity decreases in a stepwise manner, as the double bond is shifted from the center of the chain toward either the methyl or the carboxyl end (38). We find (39) that certain cyclopropane fatty acids share with the unsaturated fatty acids the ability to bring about microbial growth in presence of suboptimal quantities of biotin (Table 1.3). LACTOBACILLIC ACID 29 Lactobacillus arabinosus and L. casei respond to unsaturated as well as cyclopropane fatty acids. With L. casei, the activity of the cyclopropane acids almost equals that of the unsaturated acids. The position (9,10 or 11,12) and stereo- chemistry {cis or trans) of the cyclopropane ring appears to exert little effect on the microbiological activity. Lactobacil- lus delbrueckii is more fastidious than L. arabinosus and L. casei; it responds well to oleic, ce^-vaccenic, and elaidic acids, but grows poorly on ^raw5-vaccenic acid. DL-cf5-9,10- Methyleneoctadecanoic and lactobacillic acid are growth promoting; DL-^rfln5-9,10-methyleneoctadecanoic acid exhib- its a low order of activity and DL-^ran5-ll,12-methyleneocta- decanoic acid is inactive. Lactobacillus acidophilus exhib- its a marked preference for the cis isomers of the various fatty acids. It is stimulated by ce.s-vaccenic, lactobacillic and DL-cw-9,10-methyleneoctadecanoic acids, but fails to re- spond to the corresponding trans isomers, regardless of whether they are derived from unsaturated or cyclopropane fatty acids. Shifting of the ring from the 9,10- to the 11,12- position does not influence microbiological potency. The observation that L. acidophilus grows in presence of lacto- bacillic and DL-<:?5-9,10-methyleneoctadecanoic acid, but fails to respond to the trans isomers, provides biological support for the assigned cis configuration of lactobacillic acid. Clos- tridium butyricum responds to oleic, czVvaccenic, linoleic, and linolenic acids but is not stimulated significantly by the other compounds. This result is unexpected, since the or- ganisms contain both Cu and C19 cyclopropane fatty acids (37). Based on this experimental evidence, it must be con- cluded that certain long-chain cyclopropane fatty acids share with long-chain unsaturated fatty acids the ability to pro- 30 FATTY ACID METABOLISM IN MICROORGANISMS mote growth of certain microorganisms in the presence of suboptimal amounts of biotin. REFERENCES 1. Williams, V. R., and E. A. Fieger, /. Biol. Chem., 166, 335 (1946). 2. Hofmann, K., and A. E. Axelrod, Arch. Biochem., 14, 482 (1947). 3. Axelrod, A. E., K. Hofmann, and B. F. Daubert, /• Biol. Chem., 169, 761 (1947). 4. Williams, W. L., H. P. Broquist, and E. E. Snell, /. Biol. Chem., 170, 619 (1947). 5. Axelrod, A. E., M. Mitz, and K. Hofmann, /. Biol. Chem., 175, 265 (1948). 6. Shull, G. M., and W. H. Peterson, Arch. Biochem., 18, 69 (1948). 7. Shull, G. M., R. W. Thoma, and \\\ H. Peterson, Arch. Biochem., 20, 227 (1949). 8. Hofmann, K., and R. A. Lucas, /. Am. Chem. Soc, 72, 4328 (1950). 9. Hofmann, K., R. A. Lucas, and S. AL Sax, J. Biol. Chem., 195, 473 (1952). 10. Hofmann, K., and S. M. Sax, /. Biol. Chem., 205, 55 (1953). 11. Hofmann, K., and F. Tausig, /. Biol. Chem., 213, 425 (1955). 12. Hofmann, K., and F. Tausig, /. Biol. Chem., 213, 415 (1955). 13. Chargaff, E., and M. Levine, /. Biol. Chem., 124, 195 (1938). 14. Geiger, \V. B., Jr., and R. J. Anderson, /. Biol. Chem., 129, 519 (1939). 15. Velick, S. F., and R. J. Anderson, /. Biol. Chem., 152, 523 (1944). 16. Velick, S. F., /. Biol. Chem., 152, 533 (1944). 17. Velick, S. F., /. Biol. Chem., 156, 101 (1944). 18. Kaneshiro, T., and A. G. Marr, /. Lipid Research, 3, 184 (1962). 19. Derfer, J. M., E. E. Pickett, and C. E. Boord, /. Am. Chem. Soc, 71, 2482 (1949). 20. Hofmann, K., O. Jucker, W^ R. Miller, A. C. Young, Jr., and F. Tausig, /. Am. Chem. Soc, 76, 1799 (1954). 21. Stallberg-Stenhagen, S., Archiv. Kemi. Mineral GeoL, A22, No. 19, 11 (1946). 22. Nunn, J. R., ;. Chem. Soc, 313 (1952). LACTOBACILLIC ACID 31 23. Hofmann, K., S. F. Orochena, S. M. Sax, and G. A. Jeffrey, /. Am. Chem. Soc, 81, 992 (1959). 24. Doering, W. von E., and A. K. Hoffmann, /. Am. Chem. Soc, 76, 6162 (1954). 25. Vogel, E., K. H. Ott, and K. Gajer, Ann. Chem., 644, 172 (1961). 26. Hofmann, K., S. F. Orochena, and C. W. Yoho, /. Am. Chem. Soc, 79, 3608 (1957). 27. Hofmann, K., and C. W. Yoho, J. Am. Chem. Soc, 81, 3356 (1959). 28. Simmons, H. E., and R. D. Smith, /. Am. Chem. Soc, 81, 4256 (1959). 29. Kaneshiro, T., and A. G. Man, /. Biol. Chem., 236, 2615 (1961). 30. Brotherton, T., and G. A. Jeffrey, /. Am. Chem. Soc, 79, 5132 (1957). 31. Brotherton, T., B. Craven, and G. A. Jeffrey, Acta Cryst., 11, 546 (1958). 32. Craven, B., and G. A. Jeffrey, Acta Cryst., 12, 754 (1959). 33. Craven, B., and G. A. Jeffrey, /. Am. Chem. Soc, 82, 3858 (1960). 34. O'Leary, W. M., /. Bact., 78, 709 (1959). 35. Dauchy, S., and J. Asselineau, Compt. Rend. Acad. Sci., Paris, 250, 2635 (1960). 36. Innes Chalk, K. J., and E. Kodicek, Biochem. Biophys. Acta, 50, 579(1961). 37. Goldfine, H., and K. Bloch, J. Biol. Chem., 236, 2596 (1961). 38. Cheng, A. L. S., S. M. Greenberg, H. J. Deuel, Jr., and D. Melnick, /. Biol. Chem., 192, 611 (1951). 39. Hofmann, K., and C. Panos, /. Biol. Chem., 210, 687 (1954). CHAPTER BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 1. ESTIMATION OF THE FATTY ACID COMPOSITION OF BACTERIAL UPIDS A method for the quantitative estimation of individual fatty acids in small samples of bacterial lipids was required to permit insight into the metabolic interplay among various fatty acids in bacterial metabolism. In 1950, Bolding (1) devised a chromatographic micro- technique for determining the composition of mixtures of straight-chain saturated fatty acids. This procedure was modified in our laboratory (2) and applied to the simul- taneous determination of monoethenoid, saturated, and branched chain fatty acids as they occur in the lipids of certain microorganisms. The method involves (1) isolation of the fatty acids from the acid autoclaved cells, (2) hydrox- ylation of the fatty acid mixture with performic acid, (3) separation of the resulting mixture of hydroxylated fatty acids by reversed phase chromatography on rubber columns, and (4) microbiological determination of lactobacillic acid 32 BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 33 in the stearic-lactobacillic acid fraction derived from chro- matography. Acetone-water mixtures of increasing acetone content serve as eluant for the various fatty acids. The initial hydroxylation step converts the monoethenoid fatty acids, mainly cis-vaccenic acid, into the corresponding di- hydroxy derivatives. In contrast to the unsaturated acids, which cannot be cleanly separated from the saturates by rubber chromatography, the dihydroxy acids evolve from the column with the eluants of the lowest acetone content and are thus readily separable from the saturated acids and lactobacillic acid. This "dihydroxy" fraction is a measure of the proportion of unsaturated fatty acids both Ciq and Cis in the bacterial lipids. A typical chromatogram ob- tained with a synthetic mixture of fatty acids relating frac- tion number to the volume of 0.0 LV sodium hydroxide required to titrate each fraction to the phenolphthalein endpoint is illustrated in Fig. 2.1. In this experiment the recovery of individual fatty acids ranged from 81 to 110% of theory. Lactobacillic and stearic acids evolve from the column as a single inseparable peak. The lactobacillic acid content of this fraction is readily determined by microbio- logical assay with L. delbrueckii. A typical curve relating lactobacillic acid content of the medium and cell growth is illustrated on Fig. 2.2. The studies which are summarized on Table 2.1 demonstrate conclusively that addition of stearic acid to the lactobacillic acid samples does not inter- fere with the biological assay. Verification of this point was of considerable importance since saturated fatty acids may increase synergistically the growth promoting potency of certain unsaturated fatty acids (3). Gas-liquid chromatography of the methyl esters of bac- terial fatty acids (4, 5, 6) has largely replaced the older procedure, since it offers significant advantages in speed. 34 FATTY ACID METABOLISM IN MICROORGANISMS Fraction number Fig. 2.1. Chromatographic pattern of a synthetic fatty acid mixture (47.8 mg.), composed of: oleic acid, 17.6 mg. (81%); capric acid, 1.6 mg. (103%); lauric acid, 2.2 mg. (110%); myristic acid, 2.5 mg. (96%); palmitic acid, 13.0 mg. (98%); lactobacillic acid, 10.9 mg. (97%). The figures in parentheses represent recoveries from the column. Solvents were changed at the positions indicated by the dotted lines. 180 — 1 1 1 ^.— L-,- 160 140 |l20 |l00 - ^^•"^ - S 80 ^ 60 : y^ — 40 -/ — 20 n ^ , 1 1 1 1 20 40 60 80 Micrograms per tube 100 120 Fig. 2.2. Lactobacillic acid growth curve determined with L. del- brueckii. BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 35 TABLE 2.1 Microbiological Determination of Lactobacillic Acid in the Presence of Stearic Acid Material Assayed Lactobacillic Acid Found Recovery Stearic Acid Inactive mg. mg. % S (0.0) + L (3.6) S (1.0) + L (5.7) S (1.8) +L(2.8) S (2.5) + L (2.5) S (4.0) + L (1.0) 3.6; 3.9 5.3; 5.3 2.9; 2.9 2.6; 2.7 1.0; 0.9 100; 110 93; 93 105; 105 106; 109 100; 90 convenience, accuracy, and the amount of material re- quired. However, the fundamental observations which led to recognition of the biosynthetic route to lactobacillic acid were based on the use of reversed phase chromatography on rubber as outlined in this section. 2. CHEMICAL NATURE OF THE MONOETHENOID FATTY ACIDS IN MICROORGANISMS Oleic acid has been reported to be a constituent of the lipids of many microorganisms. Its presence has been in- ferred from the isolation of stearic acid on hydrogenation, and from iodine numbers obtained prior to hydrogenation. Careful structure analyses of the monoethenoid octadecanoic acid from L. arabinosus and L. casei led to its identification as cw-vaccenic acid (m-11-octadecenoic acid) (7, 8). The melting points of r/^-vaccenic acid and oleic acid and a number of their derivatives exhibit a striking similarity 36 FATTY ACID METABOLISM IN MICROORGANISMS (Table 2.2); consequently, great care must be exercised in the identification of monoethenoid octadecanoic acids of bacterial origin. Characterization of such acids may be achieved by (1) conversion into the respective dihydroxy derivatives, (2) oxidative cleavage of the latter and identifica- tion of the fragments, and (3) preparation of a number of derivatives and comparison with corresponding derivatives of established identity. Since the x-ray diffraction patterns of the dihydroxy derivatives of isomeric octadecenoic acids exhibit characteristic differences (9), this property also is useful in establishing the position of the double bond. Gas-liquid chromatography allows identification of the oxidative split products derived from position isomeric monounsaturated fatty acids containing 16 and 18 carbon atoms, respectively, as they occur in bacterial lipids and makes possible the simultaneous identification of the double- bond position in both classes of acids on a micro scale (4, 10). By this procedure, it was shown conclusively that in addi- tion to its presence in L. arabinosus, where it was discovered, c?5-vaccenic acid is widely distributed in bacteria. It occurs as the sole monoethenoid octadecanoic acid in L. casei and mixtures of czVvaccenic acid and of oleic acid are present in the lipids of a Streptococcus species, in E. coli (6) (70% cw-vaccenic, 30% oleic acid), in A. tiimejaciens (10) (90% aVvaccenic, 10% oleic acid), in Azotohacter agilis (10) (80% c/^-vaccenic, 20% oleic acid), and in C. hutyricum (63% cis- vaccenic, 37% oleic acid). Palmitoleic acid (<:zV9,10-hexa- decenoic acid) is contained in the lipids of E. coli (6), L. planarum, A. tumefaciens, and A. agilis (10). Isomers of this acid differing in the location of the double bond are present in a Streptococcus species (11) (c/5-ll,12-hexadeca- noic acid) and in C. butyricum. The C^q unsaturated frac- ^ o ^ CM q in o + 1 o 00 1 1 oo 00 in i od m 1 §■ lO en en U b c3 |i u o ?Vm .^ -= cNj o en q cNj q en en o o 1^ -^ T-l CN 00 O Tt Tj- I I I I I I M; Tf q q q en CN ■^ 00 in vd cq -.-. CN r^ NO "^ Tf q 00 T:f \q q q eO en in O o6 Tf Ui— 1 CN r~- ^o in t:)- ° in GO vD -^ o o CN r^' T}-' 00* r~-' (N T— 1 (^ !>• in in Tj- en -"^ o \d i^' '^* T-H CN 00 O Tj- T}- I I I I I I "^ CN o o o in U CN CN oo in \0 CN t-1 CN r~- vo "^ '^ 'u Tf (N 00 00 en in < 1 3 o" 1 en CN 1 1 q so 1 1 00 en 1 en c^ Cn in so >- u ^^ >< (U o, ^ S^ § ^ ">, ^ •S -S -o """ '>. w .a C C Si -Q (U p3 y o ^ -s j:: .^ S gi 5 S (^ ^ o S ii 37 38 FATTY ACID METABOLISM IN MICROORGANISMS tion of the latter organism consists of a mixture of cis-9,10- hexadecenoic and czV7,8-hexadecenoic acids (12, 13). Sev- eral strains of tubercle bacillus (4) and Mycobacterium phlei contain oleic acid as the sole monounsaturated Cig com- ponent. Polyunsaturated fatty acids do not occur in these various microorganisms (14). 3. FATTY ACID INTERCONVERSIONS IN LACTOBACILLI The first clue pertaining to a close metabolic relation between c?Vvaccenic and lactobacillic acids came from the results (15) recorded on Table 2.3 which illustrate the effect of variations in the growth medium on the fatty acid spec- trum of L. delbrueckii. Since this organism requires Tween 40 (sorbitan monopalmitate) for maximal growth in pres- ence of unsaturated fatty acids (16), information on the effect of Tween 40 on the fatty acid spectrum is important. Com- parison of the biotin cells (culture I) with the biotin plus Tween 40 cells (culture II) shows the total fatty acid con- tent of the latter to be almost twice that of the former. Inspection of the fatty acid spectrum reveals a markedly higher palmitic acid content of the Tween cells, but an increase in the (Ci^ plus C19) and in the Qg fraction is also observed. The "dihydroxy" fraction is lower than that of the controls. Adsorption to the cell surface of palmitic acid, derived from the Tween, may explain the high pal- mitic acid content of the culture II cells, but the increase in other fatty acids is hardly explicable along these lines. It appears likely that, in addition to providing the organ- isms with a source of palmitic acid, the Tween stimulates fatty acid biosynthesis. h ^ a; > o _, «j 0 Oh "S ^ 'u > < '+J >- 3 r3 U Uh « '1^ eo 'oj O M U H P3 i^ "6 c .2 -< 2 •^ H Ik :2 « a o "S . U 0 p. s o o ^ . ii bJD -^ -^ m m -o ^ u 2 i oi 00 U U o] 00 cn \o m CO U-^ OO Cv] LO o U U i^ in o oo cvi CM -^ ^ CO ^ LO CN 1^ Tj- rt oi CN en T-I CN '^ T-H un so 2 LO CO CM t^ U d --! -h' d >> X . . . . X; O u-i C^ -^ ^ •X ^ Tit -^ CM CM LO oo CN en — I - - - . g en .2 c ^ m o ^• 3. I^ r-H T-H ^ d cS d> V V V u 00 en vo o vo T-i en en CM CM 3 3 ^ ^ 39 "•"', o p (U rt « o Ci oo n 'ij o ai ^^ >;> o cd biD ii ■t: '^ 22 o 3 g ^ a V. P. V O U <*H ^'O ° O ^ bo *" C '^ ^^- .2 S IS o a o b/D o ^ > H qj O l-c 3 biD bO° £ bb •^ =^ o en 3 O 3 qj X5 i^ 'a bC $3 en 03 40 FATTY ACID METABOLISM IN MICROORGANISMS The biotin content of the cells which are grown on the various fatty acids (cultures III to V) is significantly lower than that of the cells grown in the presence of biotin (cul- tures I and II). Thus, the growth-promoting potency of unsaturated or cyclopropane fatty acids is not explicable in terms of their serving as precursors for biotin biosynthesis. Trace amounts of biotin (below the limits amenable to quantitative determination) are always present in the fatty acid grown cells; however, the metabolic role of these trace levels of biotin is difficult to evaluate. Substitution of biotin by cw-vaccenic acid (culture IV), although not affecting significantly the "dihydroxy" acid level, practically doubles the Ci9 acid content of the bacteria. Most remarkable is the composition of the fatty acid mixture of the lactobacillic acid grown cells (culture V). The lipids of these cells are completely devoid of unsaturated fatty acids, as reflected by the absence of "dihydroxy" fatty acids. As is to be expected, the (Ci8 plus C19) and the C^g content of these lipids is markedly higher than in the biotin cells. Lactobacillic acid grown cells of L. arahinosus and L. casei (17) (Table 2.4) are also free of unsaturated fatty acids. Biotin grown cells of these organisms contain a sizable "dihydroxy" fraction. Lactobacillic acid appears to possess the ability to substi- tute for c?\y-vaccenic acid in the metabolism of all three or- ganisms. We concluded from these findings (15) that "the biosynthesis of lactobacillic acid may involve the addition of a 'Ci' fragment to the double bond of ciVvaccenic acid." Experiments with labeled c^Vvaccenic acid and labeled one- carbon donors, to be discussed in Chapter 2, section 5, validate our hypothesis. .SS 0 O s 0 < S o o> r- CO 00 M S-i o 'o u r-' CN fNi ^ <^-i CN CO Ti '^ w bb bb 'iJ ^ >< 00 _ m ^^ CNJ O C3 U h tin < ."2 o 00 en CN en m* csi cN a; c^ HI o c bJD CM u o c en o 'Hh _ bb c JO ^ '-M *u ^" .2 ^ =n in tj "^ o£^ '-^" o bb a; .• =5, 'Zl bo_ 5 6. 3 "-^ ^ gjl S Oh P ■^ 2 •- o ^ g 2 g 41 42 FATTY ACID METABOLISM IN MICROORGANISMS 4. POSITION OF THE CYCLOPROPANE RING IN LACTOBACILLIC ACID AND SEPARATION OF CARBON ATOM 19 FROM THE MOLECULE As has been mentioned previously (Chapter 1, section 2), lactobacillic acid reacts readily with hydrogen bromide with formation of a mixture of monobromononadecanoic acids which arise from addition of the elements of hydrogen bromide to the cyclopropane ring. Dihydrosterculic, dl- ^r<2n5-9,10-methylene- and DL-^?fl/75-ll,12-methyleneoctadec- anoic acids exhibit the same behavior (18). Four structural possibilities, A, B, C, and D (Fig. 2.3), must be considered for the ring opening products. However, structures C and D appear to represent the most likely ones. The ring opened compounds derived from the two synthetic trans acids, from lactobacillic acid and from dihydrosterculic acid separately were dehydrobrominated by exposure to boiling collidine. In this manner each bromo acid was converted into a mixture of olefinic acids which may contain some or all of the components shown on Fig. 2.3. The crude dehydrohalogenation products from each acid were then oxidized by the Lemieux procedure (19) and the mixture of oxidation products separated into a neutral and an acidic fraction. The latter was analyzed for its various components by chromatography on buffered silica-gel col- umns (20). The experimental conditions employed for analysis were so selected that the dibr^sic acids could be sharply separated, whereas monocarboxylic acids evolved at the very beginning of the column development as a single unresolved peak. The nature of the dibasic oxidation products provided important insight into the position of the cyclopropane ring in the original acid. As is apparent from inspection of the patterns shown on Fig. 2.4, the BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 43 a, o i-< a, o Sh to ^3 b ^ 44 FATTY ACID METABOLISM IN MICROORGANISMS 1 \ — \ \ \ \ r ^'.■.■.■.^^■.^v.v.v.v.v^v.■...■.■.v.v.■.■a •-.^■^-r-'^-Tn -p -t""p'!i«i^%*s.. m •«:}; CM O 00 UD IT) ^ ^ — ; O o HOI a: IO'O }0 sj9i!i!ii!iA| o ,^ CO ffi- O 3 PQ g ^ ^ JD K E O ZJ c 3 5l^ PU ^ O C3 u. p ^ CM K O CQ PQ CTi'^CMpoOvO'^CMO ^r-.^^OQO OCT) HOHA/" TO'OP SJ8J!|!||!|A| e 2^ BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 45 CTi^CNOOOUD'^CMO HOMAT I0"0 io SJ8}!|!||!1A| uo^N mo ^0 sjidvmm 46 FATTY ACID METABOLISM IN MICROORGANISMS dibasic acids derived from the degradation of trans-9,\0- methyleneoctadecanoic acid (mainly azelaic acid plus sub- eric acid) differ markedly from those resulting from degrada- tion of irfl?75-ll,12-methyleneoctadecanoic acid (undecanedi- oic acid plus sebacic acid). The acids corresponding to each chromatographic peak were isolated and their identity veri- fied by melting point, by mixed melting point with an authentic sample, and by x-ray diffraction measurements. The finding that the dibasic acid pattern derived from the degradation of dihydrosterculic acid matches that obtained from ^rflnj-9,10-methyleneoctadecanoic acid; whereas that derived from degradation of lactobacillic acid duplicates essentially that obtained from ^ra775-ll,12-methyleneoctadec- anoic acid establishes the position of the cyclopropane ring in these acids. The experiences with this type of degradation (Fig. 2.5), indicate that the major route follows the pathways marked by heavy lines. In addition to providing information regarding the ring position, the above mentioned degradative scheme allows the selective removal of the methylene bridge carbon atom from the rest of the carbon chain. Oxidation with hypo- iodite of the total mixture of neutral and acidic products derived from the oxidative degradation yields iodoform. Only two of the many plausible oxidative fragments, namely compounds I and II, can be precursors of this compound. Since the ketone methyl groups of these products originate from the methylene bridge carbon of the original com- pound, the iodoform carbon is likewise derived from this source. The implications of this in the elucidation of the biosynthesis of lactobacillic acid will be discussed in the following section. BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 47 X o I 1 1 O CO 1 •2 K S o K ^ o o c/3 1 1 T 1 ^ ^ N .2 5 / > ^ J B ^ 1 / 1 ^ II -K ^/ / ^ ffi "o -o ^^ \ J G \ O \ 1 8 \ o II 1 1 ^ o 1 ^^ c a ^ 4 g 1 o r^ > ^ o 48 FATTY ACID METABOLISM IN MICROORGANISMS Cri.3 CH3 I I H3C— (CH2)x— CO OC— (CHs)^;— COOH (I) (ID The gas-liquid chromatographic identification of the methyl ketones resulting from the chromic trioxide oxida- tion of the products of hydrogenolysis (Fig. 1.3; structure III) provides another means for locating the position of the methylene bridge in cyclopropane fatty acids. Thus, the formation of a mixture of 2-nonanone and 2-octanone on oxidation of the hydrogenolysis products derived from a methylenehexadecanoic acid from an E. coli species locates the methylene bridge between positions 9 and 10 (6). These experimental results corroborate fully the assumption, stated in Chapter 1, section 2, that the hydrogenolysis of cyclopropane fatty acids brings about formation of an in- separable mixture of methyl branched fatty acids plus that straight chain fatty acid containing the same number of car- bon atoms as the original compound. 5. CYCLOPROPANE RING BIOSYNTHESIS Studies on fatty acid interconversions in lactic acid or- ganisms which were presented in Chapter 2, section 3, sug- gested a close metabolic relation between aVvaccenic acid on the one hand and lactobacillic acid on the other. • Experiments to be discussed in this section demonstrate that the biosynthesis of lactobacillic acid in L. arahinosus proceeds in the manner illustrated in Fig. 2.6. A one- carbon fragment, through as yet unelucidated mechanisms, is added across the double bond of aVvaccenic acid which provides the source for the entire carbon chain of the BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 49 H H ;< H3C— (CH2)5 — ^ ^» — (CH2)9— COOH (I) H3C— (CH2)5 — C ■ C — (CH2)9— COOH H H Fig. 2.6, Biosynthetic route to lactobacillic acid. lactobacillic acid molecule. The experimental evidence for this rather novel biochemical reaction stems from studies on the distribution of radioactivity in individual fatty acids isolated from L. arabinosus grown on such various radio- active precursors as aVvaccenic acid-l-C^^, methionine methyl-C^^, and formate-C^^, respectively (21, 22). In ex- periments in which the bacteria grow on media low^ in biotin in presence of carboxyl labeled cf^-vaccenic acid (6.72 X 106 c.p.m.), 1.26 X 10^ c.p.m. or 18.7% of the radio- activity is associated with the cells. The distribution of label among the various fractions (Table 2.5) shows that 99.2% of this radioactivity is located in the fatty acid frac- tion; the other cellular constituents are practically inactive. The major proportion of activity is located in the unsatu- rated ("dihydroxy") fatty acid and lactobacillic acid frac- tions; capric, lauric, myristic, and palmitic acids and the nonsaponifiable material show negligible labeling (Table 2.6). The specific activity of the "dihydroxy" (1.10 X 10^ 9i s O It 0 « :^ « ©o o u ^5 6< ^ 5 6 ^ X XX i "^ CM h ^ d X XX ^ li X h ^ u^ O T-< ^ q (N h CN d 00 (N CN d oo XX g o in H eg CN fN d 00 fN d cs o o o _ •<— T-H i; 1 s t2 4 X NO XX g u (N O (N 03 -§ >.CC c s ^ o .o 1^, fN Cv] (Nj o Ln 00 o" o d o csi o cn' Q "^ 'o s t^ t^ t^ r- r^ t^ o o o o o o T— ' T— 1 T— 1 T— 1 1— 1 T— 1 ^ xxxxxx : s so O CN •^ (N LO ■ ■^ in en Tj- T-H o d <6 <6 <6 <6 d> d^ 6 1 s-> en o 00 q q 00 Lo o3 |g in d d 1— 1 o en r-I T- Tj- en CN 3 <^ 1 >. Distribi tion o Radio 24A 69.9 94.0 "o t~ t^ s o o x'*T"^"*T"*~x : s o ; ; ; ; in ■ CIh ^ -i cS'^Cj f*^. C^fNOCOOT}-^- r''^ -3 'o o^ in d d d ^' T^ cn' 'w'^^^_^,-H "^enoo o ?3^ .y o o O O nj :^ -B -§ U t2 bo G E ^ .5 -° ii > Is CO c 51 52 FATTY ACID METABOLISM IN MICROORGANISMS c.p.m./mmole) and that of the lactobacillic acid fraction (1.25 X 10^ c.p.m./mmole), which corresponds to 82 and 92% respectively of that of the added cw-vaccenic acid (spe- cific activity 1.35x10^ c.p.m./mmole), demonstrates con- clusively that lactobacillic acid is indeed formed from m-vaccenic acid. The results show further that there is little degradation or redistribution oi cis-\a.ccemc acid car- boxyl carbon under the conditions employed in these ex- periments. In the studies with one-carbon ( jnors methionine-C^^ (2.69 X 107 c.p.m.) or sodium formate-C^* (7.57 X 10^ c.p.m.) were added with 40 mg. of nonlabeled-c/^-vaccenic acid per liter of medium. Cells grown in 3.5 liters of medium were analyzed. In the experiments with cell suspensions, bac- terial cells grown in 1 liter of medium, fortified with 40 mg. of nonradioactive cis-\a.ccenic acid, were harvested and washed with 0.85% sodium chloride solution. The bacteria were then suspended in 10 volumes of 0.03M potassium di- hydrogen phosphate (pH 6.8) containing 1% of glucose and sodium formate-C^^ (1.20 X 10^ c.p.m.) and incubated at 37° for 24 hours with occasional shaking, Methionine-methyl-C^^ is effectively incorporated into the bacterial cells which contain 3.15 X 10^ c.p.m. or 33.4% of the added radioactivity (Table 2.5). In contrast to the observations with c?Vvaccenic acid, 69.8% of the label is located in the nonlipid fractions, the remaining radioac- tivity being present in the mixed fatty acids (Table 2.5). The bulk of the radioactivity (96.5%) in the mixed fatty acids is located in the lactobacillic acid; the other fatty acids exhibit a very low degree of labeling (Table 2.6). Incorpo- ration of formate carbon into the bacterial cells is low under both sets of experimental conditions. Only 3.5% of the added radioactivity is incorporated into the growing BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 53 cells; the incorporation into the resting cells is 9.6%. The mixed fatty acids show little labeling in contrast to other cellular constituents which contain between 98.3 and 99.7% of the radioactivity (Table 2.5). The label present in the mixed fatty acids is distributed in the main between the "dihydroxy" and lactobacillic acid fractions (Table 2.6). Samples of biosynthetically labeled lactobacillic acid from the methionine and formate experiments were de- graded in the man jr described, and the specific activity of the resulting ioddlbrm was recorded. As has been men- tioned previously, in this scheme of degradation the iodo- form carbon is derived exclusively from the methylene bridge carbon of the lactobacillic acid molecule. The ob- servation (Table 2.7) that the specific activity of the iodo- form is 84% that of the lactobacillic acid in the methionine experiment and 80% that of lactobacillic acid in the formate experiment demonstrates conclusively that the methylene bridge carbon of the lactobacillic acid molecule is derived from one-carbon fragments. OTeary (23, 24) working with L. arahinosus also demon- strated incorporation of c?5-vaccenic acid-l-C^* and of me- TABLE 2.7 Radioactivity of lodoform-C^^ Obtained from Biosynthetic C^^ Lactobacillic Acid Methionine- C^^-Formate Methyl-C^^ Experiment Experiment dis./min./mmole dis./min./mmole Lactobacillic acid 3464 22350 Iodoform 2767 * 18787 * * The iodoform was purified to constant radioactivity. 54 FATTY ACID METABOLISM IN MICROORGANISMS thionine-methyl-C^* into the lactobacillic acid molecule, but he did not elucidate the exact position of the label. In their use of gas chromatography for separation of fatty acids, Innes Chalk and Kodicek (25) observed incorporation of methionine-methyl-C^* into lactobacillic acid with E. coli and L. casei. In L. casei all the label was located in the lactobacillic acid, which is the sole cyclopropane fatty acid present. In E. coli which contains both lactobacillic acid and its lower homolog c/5-9,10-methylenehexadecanoic acid, the label was distributed between these acids in the pro- portion 42 to 58%. Incorporation of methionine-methyl into cf5-9,10-methyleneoctadecanoic acid had been demon- strated earlier (24). Although the exact position of the label in the lower homolog of lactobacillic acid has not been established by degradation, it appears very likely that it is located in the methylene bridge carbon. Addition of unlabeled formate to the growth medium of L. casei does not affect incorporation of methionine-methyl-C^^ into the lactobacillic acid molecule. Thus, the methyl group of methionine is probably not incorporated into lactobacillic acid via oxidation to "active formate" (25). The biosynthesis of 10-methylstearic acid from oleic acid, which represents a major metabolic reaction of the latter acid in Mycobacterium phlei (14), appears to be related intimately to lactobacillic acid formation. However, there is no net change in the oxidation state in lactobacillic acid biosynthesis— in contrast to 10-methylstearic acid formation, which must involve a reductive step. The methyl group of methionine serves as the source for the extra carbon in both processes. Addition of one-carbon fragments to double bonds pro- vides a general biochemical mechanism for formation of branched-chain compounds not only in the long-chain fatty BIOSYNTHESIS OF CYCLOPROPANE FATTY ACIDS 55 acid series but also with sterols. The methyl group in posi- tion 28 of ergosterol derives from methionine methyl (26, 27) and is probably introduced via related routes. REFERENCES 1. Boldingh, J., Rec. Trav. Chim. Pays Bas, 69, 247 (1950). 2. Hofmann, K., C. Y. Hsiao, D. B. Henis, and C. Panos, /. Biol. Chem., 217, 49 (1955). 3. Axelrod, A. E., M. Mitz, and K. Hofmann, /. Biol. Chem., 175, 265 (1948). 4. Cason, J., and P. Tavs, /. Biol. Chem., 234, 1401 (1959). 5. Goldfine, H., and K. Bloch, /. Biol. Chem., 236, 2596 (1961). 6. Kaneshiro, T., and A. G. Marr, /. Biol. Chem., 236, 2615 (1961). 7. Hofmann, K., R. A. Lucas, and S. M. Sax, /. Biol. Chem., 195, 473 (1952). 8. Hofmann, K., and S. M. Sax, /. Biol. Chem., 205, 55 (1953). 9. Liitton, E. S., W. F. Huber, A. J. Mabis, and C. B. Stewart, /. Am. Chem. Soc, 73, 5206 (1951). 10. Kaneshiro, T., and A. G. Marr, J. Lipid Research, 3, 184 (1962). 11. Hofmann, K., and F. Tausig, /. Biol. Chem., 213, 415 (1955). 12. Bloch, K., P. Baronowsky, H. Goldfine, W. J. Lennarz, R. Light, A. T. Norris, and G. Scheuerbrandt, Federation Proc, 20, 921 (1961). 13. Scheuerbrandt, G., and K. Bloch, /. Biol. Chem., 237, 2064 (1962). 14. Lennarz, W. J., G. Scheuerbrandt, and K. Bloch, /. Biol. Chem., 237, 664 (1962). 15. Hofmann, K., D. B. Henis, and C. Panos, /. Biol. Chem., 228, 349 (1957). 16. Kitay, E., and E. E. Snell, /. Bact., 60, 49 (1950). 17. Hofmann, K., W. M. O'Leary, C. W. Yoho, and T. Y. Liu, /. Biol. Chem., 234, 1672 (1959). 18. Hofmann, K., G. J. Marco, and G. A. Jeffrey, /. Am. Chem. Soc., 80,5717(1958). 19. Lemieux, R. U., and E. von Rudloff, Can. J. Chem., 33, 1701 (1955). 20. Klenk, E., and W. Bongard, Z. Physiol. Chem., 290, 181 (1952). 56 FATTY ACID METABOLISM IN MICROORGANISMS 21. Hofmann, K., and T. Y. Liu, Biochim. Biophys. Acta, 37, 364 (1960). 22. Liu, T. Y., and K. Hofmann, Biochemistry, 1, 189 (1962). j 23. O'Leary, W. M., /. Bact., 11, 367 (1959). 1 24. O'Leary, W. M., /. Bact., 78, 709 (1959). 25. Innes Chalk, K. J., and E. Kodicek, Biochim. Biophys. Acta, 50, j 579(1961). 1 26. Danielsson, H., and K. Bloch, /. Am. Chem. Soc, 79, 500 (1957). j 27. Alexander, G. J., and E, Schwenk, /. Am. Chem. Soc, 79, 4554 (1957). CHAPTER BIOSYNTHESIS OF MONOUNSATURATED FATTY ACIDS BY MICROORGANISMS 1. GROWTH STUDIES Since c?j-vaccenic and lactobacillic acids are important constituents of the lipids of many microorganisms, and since these two acids are closely linked metabolically, we explored the biosynthetic routes to c/j-vaccenic acid. As a point of departure, it was assumed (1, 2) that the biosynthesis of un- saturated fatty acids either may take place through desatura- tion of saturated long-chain fatty acids or may involve elongation of the carbon chain of an already unsaturated (or potentially unsaturated) precursor. Suspensions of L. arabinosus, L. casei, and L, delbrueckii show no dehydrogenase activity toward stearic, palmitic, lauric, and myristic acids under a variety of experimental conditions (2). Since it was possible that the fatty acids are incapable of entering the cell, a series of experiments were conducted with ruptured cell preparations with simi- larly negative^ results. These observations in conjunction 57 58 FATTY ACID METABOLISM IN MICROORGANISMS with the failure of saturated fatty acids to exert the typical sparing action of unsaturated fatty acids on the biotin re- quirements of various lactic acid organisms (Chapter 1, sec- tion 6) eliminated direct desaturation of long-chain satu- rated fatty acids as the pathway to unsaturated fatty acids. As it appeared more likely that the organisms introduce the unsaturation into a smaller molecule, which would then undergo elongation of its carbon chain through successive 2-carbon additions, we synthesized three unsaturated fatty acids of the general structure shown on Fig. 3.1 (where n represents 1, 3, and 5) and tested their ability to support growth of L. arahinosus, L. casei, and L. delbrueckii in presence of suboptimal amounts of biotin. Starting with ciVvaccenic acid (n = 9) and including palmitoleic acid (n = 7) a homologous series of five unsaturated acids differ- H3C — (CH2)5 (CH2)„— COOH C C[ U H n Acid 9 ds -vaccenic 7 palmitoleic 5 cis-7, 8-tetradecenoic 3 cis-5, 6-dodecenoic 1 cis-3, 4-decenoic Fig. 3.1. Chemical structure of homologous series of unsaturated fatty acids. MONOUNSATURATED FATTY ACIDS 59 250 200 150 palmitoleic acid plus Tween 40, o cjs-vaccenic acid plus Tween 100 0.5 Fig. 3.2. Fatty acid growth curves for L. arabinosus. Abscissa, /^moles of fatty acid per tube. Ordinate, Klett readings. ing from one another by successive 2-carbon shortening of the chain was available for study. The position of the double bond with respect to the methyl group was kept constant in this series of acids. The ability of the organisms studied to utilize unsaturated cis acids possessing carbon chains shorter than cw-vaccenic acid (Figs. 3.2, 3.3, and 3.4) lends support to the hypothesis that unsaturated fatty acid biosynthesis may involve elonga- tion of the carbon chain of a shorter chain unsaturated precursor. However, the short-chain acids may substitute 60 FATTY ACID METABOLISM IN MICROORGANISMS 300 0.1 0.2 0.3 0.4 0.5 0.6 Fig. 3.3. Fatty acid growth curves for L. casei. Abscissa, ^moles of fatty acid per tube. Ordinate, Klett readings. functionally for c/^-vaccenic acid without undergoing chain elongation. C/5-vaccenic and palmitoleic acids exhibit ap- proximately equal growth-promoting activity in all three or- ganisms. The ciW-tetradecenoic acid is somewhat less effective than the Cie and Cis acids. The c/5-5-dodecenoic acid exhibits a low order of activity, and the c/5-3-decenoic acid is inactive. In view of Wakil's demonstration of the role of biotin in saturated fatty acid biosynthesis (3), it is of interest to note that aV7-tetradecenoic and cw-5-dodecen- oic acids lose their ability to support growth for L. arabino- sus when the bacteria used to inoculate the assay tubes are subjected to extensive washing with 0.85% sodium chloride. Biotin in amounts which failed to stimulate growth in ab- MONOUNSATURATED FATTY ACIDS 61 sence of the fatty acids (0.05 m/^g./tube) restored the growth- promoting potency of the fatty acids. This phenomenon was not observed with the other organisms, but more reliable growth curves were obtained when the media were fortified with 0.01 m/xg. of biotin. The growth-promoting activity of c/5-vaccenic, palmito- leic, and d5-7,8-tetradecenoic acid for L. casei and L. delbriieckii is markedly increased by addition of Tween 40 to the culture medium. With L. arahinosus, the Tween 40 increased the stimulatory effects of c/5-vaccenic and pal- mitoleic acids but suppressed the growth effect of <:/5-7-tetra- decenoic acid. The part played by the Tween is obscure but appears to 250 - 1 1 palmitoleic acid plus Tween 40; > 1 1 1 i j/^^.^cis-^iccmc acid V jcr"^ plus Tween 40; - 200 - /^ cjs-7-tetradecenoic /^ acid plus Tween 40; - 150 — /" as-5-dodecenoic acid.^^ — 100 - / A^^ - 50 / v^ A-'^'^Y^^ds-y-tetradecenoic acid; - 0 Y^ 1 1 1 1 1 1 0.1 0.2 0.3 0.4 0.5 0.6 Fig. 3.4. Fatty acid growth curves for L. delbrueckii. Abscissa, /iinoles of fatty ,acid per tube. Ordinate, Klett readings. 62 FATTY ACID METABOLISM IN MICROORGANISMS be more complex than to serve merely as a source of satu- rated fatty acids. Lactobacilli fail to grow on media low in biotin which are supplemented with unsaturated fatty acids and contain a saturated fatty acid in place of the Tween. C?5-3,4-niethylenedecanoic acid, the cyclopropane analog of aVS-decenoic acid fails to promote growth of the or- ganisms. The synthetic precursor acetylenic acids of the unsaturated acids, i.e., 3-decynoic, 5-dodecynoic, 7-tetrade- cynoic and 11-octadecynoic acids also lack growth promoting ability. 2. STUDIES WITH LABELED COMPOUNDS That long-chain saturated fatty acids are incorporated into the lipids of L. planarum and C. hutyricum is evident from the results which are summarized in Table 3.1. Stearic acid labeled with C^^ is incorporated into the bacterial lipids, but the label is located almost exclusively in the saturated acid fraction. Labeling of the unsaturated fatty acids is insignificant. TABLE 3.1 Lack of Desaturation of Stearate in Anaerobic Bacteria C^^ stearate added G^** incorporated Total saturated acids Total unsaturated acids Adapted from Bloch et al., Federation Proceedings, 20, 921 (1961). L. planarum C. hutyricum c.p.m. X 10-3 c.p. m. X 10- 1700 2000 720 500 700 490 <20 20 MONOUNSATURATED FATTY ACIDS 63 TABLE 3.2 Incorporation of Saturated Acids into Long-Chain (Ci6 + Cis) Acids of C. butyricum C' Acid Percent of Incor- Percent of Incor- Added to porated C^^ in porated C^^ in Growth Medium * Saturated Acids Unsaturated Acids C2 60 40 Cs 75 25 Cio 80 20 C12 96 4 Ci4 96 4 C16 97 3 C18 99 1 * All the acids were labeled in the carboxyl group. Adapted from Bloch et al., Federation Proceedings, 20, 921 (1961). These experiments which corroborate previous findings (1, 2) show clearly that permeability factors are not the reason for the inability of saturated fatty acids to substitute for unsaturated fatty acids as promoters of bacterial growth in biotin deficient media. They demonstrate in addition that L. planarum and C. butyricum, just as L. arahinosus, L. casei, and L. delbrueckii, cannot bring about desatura- tion of long-chain fatty acids. Of particular significance is the observation (Table 3.2) that in contrast to saturated fatty acids containing 12, 14, 16, and 18 carbon atoms, octanoate-l-C^* and decanoate- l-C^^ are incorporated into both the saturated and the monounsaturated fatty acids of C. butyricum (4, 5). As can be expected, acetate also serves as a precursor of both types of fatty acids. The distribution of radioactivity in the hexadecenoicand octadecenoic acids provides the experi- 64 FATTY ACID METABOLISM IN MICROORGANISMS mental basis for a plausible mechanism of anaerobic unsatu- rated fatty acid biosynthesis (6). Of importance for appreciation of this mechanism is the fact that C. hutyricum contains two pairs of homologous unsaturated fatty acids, namely, 7,8-hexadecenoic-oleic and 9,10-hexadecenoic-cw-vaccenic acids. The acids in each pair differ from each other by one "C2" unit at the carboxyl end but the distance between the methyl group and double bond is identical, i.e., seven methylene groups in the oleic and five methylene groups in the c^Waccenic pair of acids. The hexadecenoic and octadecenoic acid fractions from the octanoate and decanoate grown organisms, respectively, were oxidized by the Lemieux procedure (7) and the ensuing dicarboxylic acids were separated by gas chromatography as the methyl esters. With octanoate-1-C^* as the precursor, only the C9 dicarboxylic acid from the hexadecanoate and the Cii dicarboxylic acid from the octadecanoate are radio- active. In experiments with decanoate-1-C^^ the label is lo- cated predominantly in the C7 dicarboxylic acid from the hexadecenoate fraction and the C9 dicarboxylic acid from the octadecenoic acids. These results show that the short- chain acids are indeed converted into the long-chain un- saturated acids by midtiple addition of "C2" units as predicted from our growth studies (2) and that direct double-bond interconversions do not occur. Based on the distribution of label in the unsaturated fatty acids, Scheuer- brandt et al. (6) suggested a scheme (Fig. 3.5) for the bio- synthesis of unsaturated fatty acids in anaerobes, which ap- pears to fit presently available experimental evidence. Octanoate (upper left) is postulated to undergo two different types of reaction with a "Co" unit to form either decanoate or via a hypothetical 3-hydroxydecanoic acid 3,4-decenoic acid. This latter acid, through multiple addi- MONOUNSATURATED FATTY ACIDS 65 ffi >^ K X o o X 8 ffi X 1 o o rj o 8 1 1 in X 1 ffi T 3 1 1/3 K 1 i o 1 X lO X 1 o X 1 X X 1 5 1 1 o 1 •o II o II o •8 t 1 II o ^ + X *o 1 L 3r 1 1 o 1 1 ffi 1 X g s .5 + X X o .5 X o ^ -C 1 1 -* 1 ^ ^ K X ^ f^ M i T o 1 o + ^ CO X 1 k X •S ^ o o o cti X o o *o I X o X o o I ^' I ffi + T o o X 2 g c 66 FATTY ACID METABOLISM IN MICROORGANISMS tions of "C2" units, is converted into palmitoleic acid and finally aWaccenic acid. Decanoate (upper right) may undergo the same types of chain elongation as octanoate with formation of 3,4-dodecen- oic acid via the hypothetical 3-hydroxydodecanoate. Chain elongation of 3,4-dodecenoic acid by subsequent "Co" addi- tions brings about formation of oleic acid via 7,8-hexadecen- oic acid. Concurrently, both octanoate and decanoate may undergo chain elongation with formation of palmitic and stearic acids. This biosynthetic route to monounsaturated fatty acids differs markedly from that present in yeast M. phlei and mammalian liver which involves dehydrogena- tion of palmitic or stearic acids via reactions requiring oxy- gen and TPNH (4). 3. METABOLISM OF MONOUNSATURATED FATTY ACIDS IN BACTERIA Lactobacilli cannot synthesize saturated or unsaturated fatty acids in absence of biotin, but cells grown on a supple- ment of oleic or cf^-vaccenic acid contain large proportions of saturated fatty acids, mainly palmitic acid. Since the media employed are supplemented with Tween 40 (sorbitan monopalmitate) the Tween may have provided the source for the palmitic acid. As has been mentioned (Chapter 2, section 3 and Chapter 3, section 1), the Tween is essential for growth of many organisms on biotin-low media supple- mented with unsaturated or cyclopropane fatty acids. Our own experiments (8) (Fig. 3.6) and those of our former collaborator O'Leary (9) with L. arabinosus cultured on ciVvaccinate-l-C^^ eliminate saturation of monounsatu- rated fatty acids as a route to saturated fatty acids in this MONOUNSATURATED FATTY ACIDS 67 2_0T X ;uBn|9 iLU ro/Ludo O O HO^NAriO'OPSJ9;!|!||!iAI -MI'S 68 FATTY ACID METABOLISM IN MICROORGANISMS organism. The results of Bloch et al. (4) (Table 3.3) with oleic acid grown L. planariim and M. phlei are in excellent agreement with our own findings. Thus, one must con- clude that these organisms are unable to convert unsaturated into saturated fatty acids. Their inability to effect the opposite reaction, i.e., desaturation of long-chain saturated fatty acids has been discussed. It appears that the major metabolic role of monounsatu- rated fatty acids in the bacteria is that of a precursor for the biosynthesis of cyclopropane or methyl branched fatty acids. The first clue in support of this now well-established fact was our observation (10) that L. delbrneckii, grown on biotin low media in presence of lactobacillic acid, fails to synthesize unsaturated fatty acids. These acids are present in high proportions in the lipids of biotin grown cells. We confirmed these observations with L. arahinosus and L. casei (2). The high degree of labeling of the cyclopropane acid fraction in L. planarum and of the 10-methylstearic acid fraction in M. phlei, when the organisms are cultured on oleic acid-l-C^^ (Table 3.3), shows that "methylation" TABLE 3.3 Transformation of 1-C^^ Oleic Acid by Microorganisms L. planarum M. phlei c.p.m. X 10-3 c.Yi.m. X 10" 0^4 Oleate added 760 1350 Total fatty acids 220 338 Pahnitic acid 1 20 Stearic acid 2 2 C^^ Cyclopropane acid 75 10-Methylstearic acid 200 Adapted from Bloch et al., Federation Proceedings, 20, 921 (1961). MONOUNSATURATED FATTY ACIDS 69 is a major metabolic fate of the unsaturated fatty acids. In organisms such as C. butyricum and E. coli, which con- tain both Ci6 and Cis unsaturated fatty acids, "methylation" takes place with formation of a mixture of C17 and C19 cyclopropane fatty acids (see Chapter 2, section 5). The chemical nature of the cyclopropane fatty acids, which arise when the microorganisms are cultured on oleic rather than on c?5-vaccenic acid, remains to be established— but two possibilities exist for oleic acid metabolism. Either the double bond undergoes isomerization with formation of czVvaccenic acid, which in turn is metabolized to lacto- bacillic acid, or the bacteria have the ability to "methylate" oleic acid with formation of one of the optical isomers of dihydrosterculic acid. O'Leary (9) suggested that L. arahin- osus brings about isomerization of added oleic acid with formation of czVvaccenic plus lactobacillic acids, but his experimental evidence in support of this unlikely course is not convincing. We find (10) that oleic acid grown L. delbrueckii produce a liquid saturated fatty acid fraction which exhibits 1.6 times the growth promoting activity of pure lactobacillic acid in L. delbrueckii assays. Since di- hydrosterculic acid, which contains the methylene bridge in the 9,10-position, is 1.4 times as active in the same assay as lactobacillic acid with the 11,12-methylene bridge, we concluded (10) "that oleic acid stimulates L. delbrueckii to produce a 'saturated' fatty acid exhibiting higher micro- biological activity than that of lactobacillic acid." Scheuerbrandt et al. (6) using C. butyricum demonstrated that this organism does not interconvert unsaturated fatty acids containing 5 methylene groups between the methyl terminus and the double bond into fatty acids having 7 methylene groups in that location. Thus, the available evidence suggests that the organisms are capable of bringing 70 FATTY ACID METABOLISM IN MICROORGANISMS about "methylation" of double bonds in long-chain fatty acids with double-bond locations five and seven methylene groups removed from the methyl end with formation of the corresponding cyclopropane fatty acids. However, iso- lation and careful chemical characterization of the "methyla- tion product" of oleic acid must be carried out in order to establish this point unequivocally. Mention was made in Chapter 1, section 6, of the experi- ments of Cheng et al. (11) who showed that a number of position isomeric octadecanoic acids possessing both cis and trans double bonds have the ability to stimulate growth of L. arahinosiis on suboptimal amounts of biotin. The mode of action of such acids is at present obscure. The possibility exists that they may be converted into the corresponding position isomeric cis or trans cyclopropane acids and that these may have the ability to serve as metabolic substitutes for lactobacillic acid. However, it seems unlikely that a family of compounds of such widely differing structure and belonging to different stereochemical series should be ca- pable of performing the same or similar functions in micro- bial metabolism. Similar arguments may be raised con- cerning the mode of action of the polyunsaturated fatty acids (linoleic, linolenic, and arachidonic), which are also capable of promoting growth of lactic acid organisms on biotin deficient media. Are these polyunsaturated acids un- dergoing "methylation" with formation of acids containing more than one cyclopropane ring? Studies on the metabolic fate of these various acids and a better understanding of the role of the cyclopropane fatty acids in microbial metabolism should aid in clarification of these questions. MONOUNSATURATED FATTY ACIDS 71 REFERENCES 1. O'Leary, W. M., and K. Hofmann, Federation Proc, 16, 228 (1957). 2. Hofmann, K., W. M. O'Leary, C. W. Yoho, and T. Y. Liu, /. Biol. Chem., 234, 1672 (1959). 3. Wakil, S. J., E. B. Titchener, and D. M. Gibson, Biochim. Biophys. Acta, 29, 225 (1958). 4. Bloch, K., P. E. Baionowsky, H. Goldfine, W. J. Lennarz, R. Light, A. T. Norris, and G. Scheuerbrandt, Federation Proc, 20, 921 (1961). 5. Goldfine, H., and K. Bloch, /. Biol. Chem., 236, 2596 (1961). 6. Scheuerbrandt, G., H. Goldfine, P. E. Baronowsky, and K. Bloch, /. Biol. Chem., 236, PC70 (1961). 7. Lemieux, R. U., and E. von Rudloff, Can. J. Chem., 33, 1701 (1955). 8. Liu, T. v., and K. Hofmann, Biochemistry, 1, 189 (1962). 9. O'Leary, W. M., /. Bact., 77, 367 (1959). 10. Hofmann, K., D. B. Henis, and C. Panos, /. Biol. Chem., 228, 349 (1957). 11. Cheng, A. L. S., S. M. Greenberg, H. J. Deuel, Jr., and D. Melnick, /. Biol. Chem., 192, 611 (1951). MARINE BIOLOGICAL LABORATORY LIBRARY WOODS HOLE, MASS. W. H. 0. I. INDEX Agrobacterium (phytomonas) tu- mejaciens, fatty acids of, 1 Azelaic acid, formation from trans- 9, 1 0-methyleneoctadecanoic acid, 44-46 Bacterial fatty acids, distillation curves of methyl esters, 2, 3 determination by gas-liquid chromatography, 33 estimation of, by rubber col- umn chromatography, 32 Bacterial lipids, "bound" form of, 1 Biotin, content of cells grown on fatty acids, 40 fatty acids as precursors for bio- synthesis, 40 Clostridium butyricum, inability to desaturate long-chain fatty acids, 62 Cyclopropane-cf5-l,2-diacetic acid, conversion into long-chain cy- clopropane fatty acids, 17-20 synthesis of, 17, 18 Cyclopropane fatty acids, biotin- like growth promoting activity of, 21 biotin-sparing action of, 28 chemical behavior of, 1 1 in Clostridium butyricum, 28 infrared absorption spectra oi, 14 some physical properties of, 15 stereochemistry of, 9 Decanoic acid, as precursor of sat- urated and unsaturated fatty acids of Clostridium butyri- cum, 63 cz5-3-Decenoic acid, as growth pro- moter for Lactobacillus arabi- nosus, 58-61 73 74 INDEX m-3-Decenoic acid, Cont. as growth promoter for Lacto- bacillus casei, 58-61 as growth promoter for Lacto- bacillus delbriieckii, 58-61 Dibasic acids, separation by chro- matography, 44, 45 Dihydrosterculic acid, see DL-di- 9,10-Methyleneoctadecanoic acid Dihydroxyoctadecanoic acids, X- ray diffraction patterns of, 36 cf5-5-Dodecenoic acid, as growth promoter for Lactobacillus arabinosus, 58-61 as growth promoter for Lacto- bacillus casei, 58-61 as growth promoter for Lacto- bacillus delbrueckii, 58-61 Elaidic acid, biotin-like growth promoting activity of, 21 biotin-sparing action of, 28 Ergosterol, methionine as source for carbon atom 28 in, 55 Formate-Ci4, incorporation into lactobacillic acid, 49-53 incorporation into Lactobacillus arabinosus cells, 52 incorporation into olefinic acids, 53 Gas-liquid chromatography of bac- terial fatty acid methyl esters, 33 d5-7,8-Hexadecenoic acid, labeling pattern in biosynthetic, 64 cij-7,8-Hexadecenoic acid, Cont. occurrence in Clostridium bu- tyric um, 38 c«-9,10-Hexadecenoic acid, label- ing pattern in biosynthetic, 64 Hydroxylation of unsaturated fatty acids, 33 Iodoform, formation from cyclo- propane fatty acids, 46 from labeled lactobacillic acid, 53 lodomethyl zinc iodide, in cyclo- propane ring synthesis, 18 reaction with olefins, 18 reaction with oleic acid, 18 reaction with palmitoleic acid, 18 Labeled precursors, incorporation into Lactobacillus arabinosus fatty acids, 49-54 Lactobacillic acid, ability to sub- stitute for d5-vaccenic acid in bacterial metabolism, 40 amide, 15 biosynthesis of, 40, 48, 49 biotin-like growth promoting ac- tivity of, 21 composition of, 4 cyclopropane ring in, 6, 7 density of, 23 dibasic acids from degradation of, 46 electron density map of, 26 hydrogen bonding in, 27 hydrogenolysis of, 6, 7 identity with phytomonic acid, 5 infrared spectrum of, 4, 7, 8, 14 INDEX 75 Lactobacillic acid, Cont. isolation from Lactobacillus arabinosus lipids, 4 location of cyclopropane ring in, 42-47 main X-ray spacings of, 5, 15 melting point of, 4, 15 metabolic relation to cw-vaccenic acid, 38, 39, 41 microbiological determination of, 33-35 nonadecanoic acid from, 6, 7 one-carbon fragment in biosyn- thesis of, 48 presence in bacterial phospho- lipids, 5 reaction with HBr, 7, 42, 43 stability to oxidation, 7 structure of, 6, 7, 23 cw-vaccenic acid in biosynthesis of, 40, 48, 49 DL-lactobacillic acid, see DL-cis- 11,12-Methyleneoctadecanoic acid Lactobacillus arabinosus, fatty acid composition of, 41 fatty acids of, 1 inability to convert m-vaccenic acid to palmitic acid, 67, 68 lack of fatty acid dehydrogenase in, 57 Lactobacillus casei, fatty acid com- position of, 41 fatty acids of, 1 lack of fatty acid dehydrogenase in, 57 Lactobacillus delbrueckii, fatty acid composition of, 39 Lactobacillus delbrueckii, Cont. lack of fatty acid dehydrogenase in, 57 Lactobacillus planarum, inability to convert oleic acid to stearic acid, 68 inability to desaturate long-chain fatty acids, 62 Mammalian liver, desaturation of fatty acids by, 66 Methionine-methyl-Ci4, as source of methylene bridge carbon of lactobacillic acid, 49-52 incorporation into d5-9,10-meth- ylenehexadecanoic acid by Escherichia coli, 54 incorporation into lactobacillic acid, 52 incorporation into Lactobacillus arabinosus cells, 52 incorporation into lactobacillic acid in Lactobacillus casei, 54 incorporation into lactobacillic acid in Escherichia coli, 54 Methyl ketones, formation from cyclopropane fatty acids, 46, 48 identification by gas-liquid chro- matography, 48 role in locating the position of cyclopropane ring in cyclopro- pane fatty acids, 48 "Methylation," of cw-vaccenic acid by microorganisms, 49-51 of oleic acid by microorganisms, 69 of various position isomeric oc- tadecenoic acids, 70 76 INDEX Ci5-9,10-Methylenehexadecanoic acid, location of cyclopropane ring in, 48 occurrence in Escherichia coli, 27 DL-c/5-9,10-Methylenehexadecanoic acid, synthesis of, 22 DLd5-9,10-Methyleneoctadecanoic acid, amide, 15 biotin-like growth promoting ac- tivity of, 21 ct5-configuration of, 14 dibasic acids from degradation of, 46 formation from sterculic acid, 14 identity with dihydrosterculic acid, 18 location of cyclopropane ring in, 42-47 reaction with HBr, 42, 43 some physical properties of, 15 synthesis of, 17-20 DL-fran5-9,10-Methyleneoctadeca- noic acid, amide, 15 biotin-like growth promoting ac- tivity of, 21 electron density map of, 25 infrared spectrum of, 14 location of cyclopropane ring in, 42-47 reaction with HBr, 42, 43 synthesis of, 10-13 DL-ci5-ll,12-Methyleneoctadecanoic acid, amide, 15 density of, 23 electron density map of, 24 synthesis of, 17-20 DL-fran5-ll,12-Methyleneoctadeca- noic acid, amide, 15 DL-rrarz5-ll,12-Methyleneocta- decanoic acid, Cont. biotin-like growth promoting ac- tivity of, 21 infrared spectrum of, 14 location of cyclopropane ring in, 42-47 reaction with HBr, 42, 43 synthesis of, 10-13 10-Methylstearic acid, biosynthesis from oleic acid, 54 biosynthesis in Mycobacterium phlei, 54 Monounsaturated fatty acids, as precursor of cyclopropane fatty acids, 68 "methylation" of, 68, 69 Mycobacterium phlei, desaturation of fatty acids by, 66 inability to convert oleic acid to stearic acid, 68 Octanoic acid, as precursor of sat- urated and unsaturated fatty acids in Clostridium butyri- cum, 63 Oleic acid, as precursor of 10- methylstearic acid in Mycobac- terium phlei, 54 biotin-like growth promoting ac- tivity of, 21 biotin-sparing action of, 28 distribution in bacteria, 36 labeling pattern in biosynthetic, 64 melting points of some deriva- tives, 37 "methylation" of, by Lactoba- cillus arabinosus, 69 INDEX 77 Oleic acid, Cont. "methylation" of, by Lactoba- cillus delbrueckii, 69 occurrence in microorganisms, 35 Oxygen, in fatty acid dehydrogena- tion, 66 Palmitoleic acid, as growth pro- moter for Lactobacillus arabi- riosus, 58-61 as growth promoter for Lacto- bacillus casei, 58-61 as growth promoter for Lacto- bacillus delbrueckii, 58-61 distribution in bacteria, 36 Polymorphism, absence of, in cy- clopropane fatty acids, 27 in long-chain monocarboxylic acids, 27 Polyunsaturated fatty acids, non- occurrence in microorganisms, 38 Rubber column chromatography, estimation of fatty acids with, 32 Saturated fatty acids, synergistic growth effects of, 28 Sebacic acid, formation from trans- 11,1 2-methyleneoctadecanoic acid, 44-46 Stearic acid, incorporation into bacterial lipids, 63 lack of desaturation by anaer- obes, 62 Sterculic acid, location of three- membered ring in, 16 Sterculic acid, Cont. structure of, 16 Streptococcus species, fatty acids of, 1 Suberic acid, formation from trans- 9,10-methyleneoctadecanoic acid, 44-46 c/5-7-Tetradecenoic acid, as growth promoter for Lactobacillus arabinosus, 58-61 as growth promoter for Lacto- bacillus casei, 58-61 as growth promoter for Lacto- bacillus delbrueckii, 58-61 Triphosphopyridine nucleotide (TPNH), role in fatty acid de- hydrogenation, 66 Tween 40 (sorbitan monopalmi- tate), as source of palmitic acid, 66 effect on bacterial growth, 38, 61, 66 Undecanedioic acid, formation from fran5-ll,12-methyleneoc- tadecanoic acid, 44-46 Unsaturated fatty acids, biotin- sparing action of, 28 scheme for biosynthesis of, 65 Unsaturated octadecanoic acids, characterization of, 36 determination of double-bond location in, 36 c/5-Vaccenic acid, as growth pro- moter for Lactobacillus arabi- nosus, 58-61 as growth promoter for Lacto- bacillus casei, 58-61 78 INDEX a'5- Vaccenic acid, Cont. as growth promoter for Lacto- bacillus delbrueckii, 58-61 biosynthetic route to, 57 biotin-like growth promoting ac- tivity of, 21 distribution in bacteria, 36 in Lactobacillus arabinosus, 36 in Lactobacillus casei, 36 labeling pattern in biosynthetic, 64 melting points of some deriva- tives, 37 cf5-Vaccenic acid, Cont. metabolic relation to lactobacil- lic acid, 38, 39, 41 c/5-\'accenic acid-l-Ci4, as pre- cursor of lactobacillic acid, 49- 51 incorporation into lactobacillic acid, 54 fran5-Vaccenic acid, biotin-like growth promoting activity of, 21 Yeast, desaturation of fatty acids by, 66 iMrMMM^M^^s^M^si^Szi^^Wi^^^^^^^^^T^^^^^M '0i&Mi^^^^^it^'¥Mw^M^^^^^^^^^^^^^ fi9^iWj^S^xM^i^^^^^^^'^^MM'^^^^^^^^^^^ — i^-i^.or; Si^PiiiSiiMilffliiiliiS^Wl