fatty acids genus bacillus: example branched-chain preference' · fatty acids of the...

BACTRIUOLOICAL RIVIEWS, June 1977, p. 391-418Copyright © 1977 American Society for Microbiology

Vol. 41, No. 2Printed in U.S.A.

Fatty Acids of the Genus Bacillus: an Example ofBranched-Chain Preference'

TOSHI KANEDAAlberta Research Council, Edmonton, Alberta, Canada T6G 2C2

INTRODUCTION ............................................................. 391FATTY ACID ANALYSIS..................................................... 392Thin-Layer Chromatography and Liquid Chromatography ...................... 393

Gas-Liquid Chromatography.................................................. 393Separation of fatty acids.................................................... 393Identification by retention characteristics ...... ............................. 394

Mass Spectrometry .......................................................... 395FATTY ACID COMPOSITION OF BACILLUS SPECIES ....................... 396

BIOSYNTHESIS OF FATTY ACIDS ........................................... 396

Saturated Fatty Acids ........................................................ 396

Branched-chain acids...................................................... 396Straight-chain acids........................................................ 400Cyclohexyl acids ........................................................... 401

Unsaturated Fatty Acids...................................................... 401Stereospecific Synthesis of Anteiso Fatty Acids ...... .......................... 401Branched, Short-Chain Fatty Acids ......... .................................. 401

FACTORS AFFECTING FATTY ACID PATTERN AND CONTENT .... ......... 402Pattern of Saturated Fatty Acids .......... ................................... 402

Relative activity of chain initiators ........ ................................. 402Factors related to chain initiators ........ .................................. 403Factors related to chain extender ......... .................................. 405

Proportion of Unsaturated Acids.............................................. 405

Fatty Acid Content .......................................................... 405FUNCTION OF BRANCHED-CHAIN FATTY ACIDS ..... ...................... 406Branched-Chain Patty Acids................................................. 406Phospholilpids ............................................................. 408

GROUPING OF BACILLUS SPECIES BASED ON FATTY ACID PATTERNS .. 408GENERAL BACTERIAL TAXONOMY AND FATTY ACID TYPES .... ......... 409

Gramn-Positive Bacteria....................................................... 409Gram-Negative Bacteria ..................................................... 410

EVOLUTION OF FATTY ACID SYSTEMS .................................... 410

CONCLUDING REMARKS.................................................... 411LITERATURE CITED................ 412

INTRODUCTIONFatty acids occur in nearly all living orga-

nisms as the important predominant constitu-ents of lipids. (Rare exceptions are several spe-cies of extremely halophilic bacteria in whichno fatty acid ester groups occur and the lipidscontain predominantly ether linkages [143].)They are usually straight carbon chains, withor without unsaturation; palmitic, stearic,oleic, linoleic, and linolenic acids are most com-mon. In some organisms, however, some sub-stituted fatty acids occur, a long-known exam-ple being the single- or multi-methyl-substi-tuted acids, such as tuberculostearic, phthenoic,and mycolic acids, in Mycobacterium tuberculo-sis (10).In 1960, the major occurrence of the termi-

' Contribution no. 794 from the Alberta Research Coun-cil, Edmonton, Alberta, Canada.

nally methyl-branched fatty acids, 13-methyl-tetradecanoic (iso-C15) and 15-methylhexadeca-noic (iso-C,7), in Bacillus subtilis (natto) (138),and 12-methyltetradecanoic (anteiso-C,5) acidin a species of Sarcina (4), was first reported.After these reports, extensive studies con-ducted in my laboratory led to the discovery ofthree additional branched-chain fatty acids; 12-methyltridecanoic (iso-C14), 14-methylpentade-canoic (iso-C16), and 14-methylhexadecanoic(anteiso-C,7) acids in the lipids of B. subtilis(76). These branched-chain acids are grouped inthree series, based on their biosynthetic rela-tionships (28, 77, 79, 80, 92, 102, 119) (Fig. 1).The definitions of iso and anteiso permit only

iso methyl-substituted fatty acids (an iso ethylwould be an anteiso methyl of one-higher chainnumber), but would allow both methyl andethyl as anteiso substitutions. Only the methylanteiso substituent has so far been found in

391

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392 KANEDA

Anteiso series

CH3-CH2-CH-CH2 (CH2). COOH*

CH3

Odd-numbered iso series

CH3-CH-CH2-CH2 (CH2)n-COOH*I '

CH3Even-numbered iso series

CH3-CH3-CH2- (CH2)n COOH*

CH3* n = 9 or 11

FIG. 1. Three series of branched-chain fatty acids.

nature, although bacteria can synthesize ethylanteiso compounds when appropriate precur-sors are provided.Some 48 species of the genus Bacillus are

recognized (23). Of these, 22 species have beenexamined, all of which were found to containiso and anteiso saturated fatty acids as majoracid components of lipids (60 to 90% of the totalfatty acids). Although data available are lessextensive, iso and anteiso fatty acids predomi-nate also in eight genera of gram-positive bac-teria other than Bacillus and in four genera ofgram-negative bacteria, and occur in somehigher forms (a few plant pathogenic fungi,sheep wool wax [degras], and the skin wax ofhumans). Thus the occurrence of branched-chain fatty acids is not as rare as once thought,and their study in the important genusBacillusis probably significant to the biochemistry ofother organisms.One of the points to be made in this review is

that the enzyme system synthesizing iso andanteiso fatty acids is one oftwo types ofde novofatty acid synthetases occurring in nature. Theother type is, of course, palmitic acid synthe-tase, which occurs widely in both lower andhigher forms. Furthermore, the biosynthesis ofiso and anteiso fatty acids is closely linked withthe biosynthesis of branched-chain aminoacids, and these fatty acids may have played animportant role at an early stage ofthe evolutionof life.Of all the genera in which iso and anteiso

acids occur, Bacillus has probably been mostthoroughly studied. Thus this review considersprimarily the genus Bacillus, but findings withother genera are also included to broaden thediscussion wherever appropriate. Special em-phasis is given to the biosynthesis, control ofsynthesis, and evolutionary significance of isoand anteiso fatty acids. In addition, their bio-logical functions as components of phospholip-ids are included. For a more thorough treat-

ment of bacterial lipids in general, the reader isreferred to the many reviews and books thatare available (10, 55, 73, 103, 123, 145).

FATTY ACID ANALYSISAny study of branched-chain fatty acids de-

pends on careful analytical work to separatethe various branched-chain compounds fromother closely related fatty acids and to obtainunambiguous identification and accurate quan-titative measurements on the separated frac-tions. A detailed discussion of analytical pro-cedures is not the primary interest of this re-view. However, many papers have been pub-lished in which the identification of fatty acidsis inadequately handled. Under these circum-stances, it is appropriate to discuss criticallythe analytical approaches commonly used andto call attention to their limitations, in an at-tempt to improve this situation.

Identification of fatty acids from biologicalsources is often carried out solely by gas-liquidchromatography. Two sets of retention-timedata relative to a standard are commonly ob-tained, one from a polar (selective, such asethyleneglycoladipate) and one from a nonpolar(nonselective, such as SE-30) column. This ap-proach is generally adequate, although tenta-tive, for simple mixtures of usual fatty acidscomposed of straight chains. However, a num-ber of unusual fatty acids occur in bacteria, andsome of these give retention times identical ornearly identical to those of certain other com-mon acids. The fractionation of fatty acids intoclasses before gas chromatography is a usefulaid to identification and is in some cases essen-tial to avoid errors.

Furthermore, separation during gas-liquidchromatography depends only on the physicalcharacteristics of a given substance, and directevidence for a specific chemical structure mustbe provided by some other means. Mass spec-trometry is best suited to this purpose.A standard procedure for the adequate iden-

tification of bacterial fatty acids includes threetechniques used in the sequence: thin-layerchromatography for class separation, gas-liquidchromatography for individual separation andtentative identification, and mass spectrometryfor structural identification. High-pressure liq-uid chromatography is a rapidly developingtechnique that has been used with other bacte-rial lipid systems to replace the thin-layer chro-matographic class separation and, to some ex-tent, the gas-liquid chromatographic individualseparation. Its ultimate usefulness remains tobe seen. Some recent developments of thesevarious techniques are considered below. For amore general treatment of lipid analysis, the

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FATTY ACIDS OF THE GENUS BACILLUS 393

reader is referred to recent books (33, 75, 96,108).

Thin-Layer Chromatography and LiquidChromatography

As the methodology of lipid analysis by thin-layer chromatography reaches a state of matu-rity, recent developments in this field remainlargely improvements of established tech-niques. A well-established procedure for theseparation of the three major bacterial fattyacid classes, saturated, unsaturated, and hy-droxylated, makes use of thin-layer supportsimpregnated with silver nitrate or borate. Arecent review covers this area (59). The multi-development procedure for separation of lipids(64, 71) and polyunsaturated acids (11) is asignificant improvement over previous meth-ods. Visualization of chromatograms has beenimproved by the use of a malachite green sprayreagent for lipids (149) and a reagent contain-ing molybdate for phospholipids (157). Fluoro-metric analysis of lipids after spraying chro-matograms with rhodamine 6G is useful inquantitative estimation (122).The new technique of high-pressure (high-

speed) liquid chromatography has been success-fully applied to this problem in conjunctionwith highly sensitive detectors, and the subjecthas recently been reviewed (35). A 1-m columnwith reverse-phase support separates a mixtureof C18 acids and of C20 acids on the basis ofdegree of unsaturation within 15 min (129).Geometric isomers (cis-trans), difficult to sepa-rate by gas-liquid chromatography unless theacids are expoxidized (44), are rather easilyseparated on high-pressure columns in a shorttime (139). The ultraviolet detector is currentlythe most popular. Fatty acids have been ana-lyzed after conversion to such ultraviolet-ab-sorbing derivatives as benzyl ester (133), 2-naphthacyl ester (34), and phenacyl esters (20,42), and after individual calibration, quantita-tive results can be obtained. An interestingdevelopment is the use of a gradient elutiontechnique to improve the resolution of lipidsand to simplify the operation. With this tech-nique, 28 lipid components were separated in 3h (147). The potential of high-pressure liquidchromatography in lipid analysis is enormous.

Gas-Liquid ChromatographyThe fatty acids most commonly found in the

lipids of bacteria are composed of saturated andmonounsaturated straight chains with 14, 16,and 18 carbons. The gas-liquid chromato-graphic separation of these fatty acids, usuallyas their methyl esters, is easily done on any of

the appropriate short (6-foot [ca. 1.9-ms])packed columns.The two major classes of bacterial fatty acids,

saturated and unsaturated, can be separated bytreating a fatty acid sample with bromine be-fore chromatography. The brominated productsof unsaturated fatty acids have much longerretention times and are easily separated fromthe saturated homologues (84).Most of the fatty acids occurring in the lipids

of all Bacillus species, however, are of thebranched iso and anteiso series. The separationof the iso acid from the anteiso with the samenumber ofcarbons is rather difficult and cannotbe accomplished by the short column. Further-more, the identification ofbranched-chain acidsrequires thorough investigation to determinethe carbon skeleton. These specific problemsare considered here. A more general account offatty acid analysis by gas-liquid chromatogra-phy is available in recent books (2, 100>.

Separation of fatty acids. The resolution ofapair of iso-anteiso acids with the same numberof carbons requires at least 12,000 theoreticalplates. This efficiency can be achieved by anyone of three types of columns currently in use:packed, support-coated open tubular (SCOT),and wall-coated open tubular (Golay), if appro-priate operating conditions are used. However,conventional packed columns are most conven-ient and are particularly suited to quantitativework. Columns with a wide variety of gas-liq-uid chromatographic characteristics can easilybe prepared in an average laboratory, and thesample-handling capacity is large enough topermit introduction of an accurate amount(more than 10 1A) of sample. A well-establishedprocedure includes the use of a 20-foot (ca. 6-m)column packed with ethyleneglycol adipate pol-ymer (7%) coated on Chromosorb W, operatedisothermally at 1900C (24, 79, 167).

Alternatively, the separation of the iso-an-teiso pair can be achieved by a 50-foot (ca. 15-m)SCOT column coated with the same liquidphase and operated at 1700C (88). The SCOTcolumn has an advantage over the packed typein that it requires less time to complete a runand is effective in separating fatty acids ofhigher carbon number than is possible withpacked columns. Although the sample-carryingcapacity of a SCOT column is much smallerthan that of the packed type, it is large enoughto accommodate a few microliters of sample(which, of course, is largely solvent). The majordisadvantage of SCOT columns lies in theirhigher cost and shorter useful life-span.Although Golay columns (conventional capil-

lary columns) seem to be an attractive alterna-tive, the extremely limited sample-carrying ca-

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pacity creates many problems: the detectormust be frequently cleaned to minimize noise,and a sample splitter must be used to introducea small portion (a few percent) of the injectedsample. A serious problem arises, since thesmall portion of the sample analyzed may notrepresent the composition of the whole sampleinjected because ofa probably nonuniform split-ting with respect to individual components ofthe injected sample. Thus Golay columns areused exclusively for identification purposes andnot for quantitative analysis. Recently, the useof glass capillary tubing instead of stainless-steel tubing has attracted wide interest, andthis area is currently developing very rapidly(17, 140, 156). Glass capillary tubing offers anumber of advantages over the stainless-steeltype: the inert inner wall minimizes thermaldecomposition of labile compounds (158), andthe wall surface can be acid etched to increasethe surface area, with a consequent increase ofsample-carrying capacity (5). Glass is far lesscostly than stainless steel, and glass capillariescan be prepared in the laboratory ifproper toolsare provided. With these advantages, wide useof glass capillary columns in effecting difficultresolutions is expected in the near future.

Identification by retention characteristics.Although retention time is the quantity mostcommonly measured in gas-liquid chromatog-raphy, net retention volume (measured fromair peak and adjusted for pressure drop) is thebasic parameter (see Table 2.1 ofreference 100).The retention characteristics of fatty acidmethyl esters are generally expressed in any ofthree ways: (i) relative retention time (or vol-ume) (usually relative to methyl stearate); (ii)retention index (Kovats index); or (iii) equiva-lent chain length (ECL) (or carbon number)(114). Relative retention is based on the com-parison with a single reference compound, andthe actual value is sensitive to small errors inthe retention of the reference compound and tosmall changes in the column composition andproperties, particularly for compounds well re-

moved from the reference. The other two sys-tems are superior because they each depend oncomparison with a homologous series (100), us-ing a logarithmic relationship between net re-

tention volume and carbon number in the se-ries (which conveniently becomes a linear rela-tionship in programmed temperature gas chro-matography [621). The retention index is basedon two normal alkanes that are adjacent to thesample peak (a substance eluting midway on alog scale betwen n-Cl6 and n-C,7 alkanes wouldhave a retention index of 1,650), whereas theECL is based on the series of normal alkanoic

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fatty acid methyl esters (a fatty acid methylester eluting midway on a log scale betweenmethyl palmitate and methyl margarate [n-C,7] would have an ECL of 16.50). The ECL isused extensively in identification of fatty acids.Usually the ECLs of an unknown fatty acidester as measured on two columns, such asEGA and SE-30, are used for identification.The ECL of an isomeric or a substituted

fatty acid ester is almost never the even num-ber of the corresponding normal fatty acid es-ter. Generally speaking, branching leads toshorter retention times (shorter ECL). Someexamples ofECL values are given in Table 1 forfatty acid methyl esters of B. cereus (84).

In this table some regularities are observed.The ECL is determined not only by the natureof the substitution but also by its location alongthe alkanoic chain (e.g., anteiso compoundshave ECLs 0.25 lower than the parents; isocompounds have ECLs 0.45 lower). The intro-duction of a polar function, such as an unsatu-ration or a hydroxyl group, leads to longerECLs on the polar column (0.32 for A10-n-C,, onEGA) and to shorter ECLs on the nonpolarcolumn (-0.2 for A10-n-C,,; on SE-30). Table 2gives additional ECLs used in identifying bac-terial fatty acids. A more detailed treatment of

TABLE 1. Gas-liquid chromatographic equivalentchain length (ECL) of methyl esters of fatty acids

from lipids ofBacillus cereus

ECLMethyl ester

EGAa SE-30b10-Methylhendecanoic (iso-C12) 11.62 11.6n-Dodecanoic (n-C12) 12.00 12.0l1-Methyldodecanoic (iso-C13) 12.58 12.610-Methyldodecanoic (anteiso-C13) 12.72 12.7n-Tridecanoic (n-C13) 13.00 13.012-Methyltridecanoic (iso-C14) 13.60 13.6n-Tetradecanoic (n-C14) 14.00 14.013-Methyltetradecanoic (iso-C,2) 14.60 14.612-Methyltetradecanoic (anteiso-C15) 14.74 14.7n-Pentadecanoic (n-C15) 15.00 15.014-Methylpentadeca- (iso-C16) 15.57 15.6

noicn-Hexadecanoic (n-C16) 16.00 16.015-Methylhexadecanoic (iso-C,7) 16.58 16.614-Methylhexadecanoic (antei8o-C,7) 16.73 16.7n-Heptadecanoic (n-Cl7) 17.00 17.0cis-A1O-14-Methylpenta- (iso-C1*") 15.90 15.4

decenoiccis-A'0-n-Hexadecenoic (n-C1,6,&) 16.32 15.8cis-A'O-15-Methylhexad- (iso-C71) 16.88 16.4

ecenoiccis-A1O-14-Methylhexad- (anteiso-C,711") 16.97 16.5

ecenoic

a A 20-foot (ca. 6-m) ethyleneglycol adipate polymer (7%)column.

° A 5-foot (ca. 1.5-m) SE-30 (2.5%) column. ECLs arerounded to significant figures since the column used isshort. For details, see reference 84.


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TABLE 2. Equivalent chain length (ECL) of methyl esters of C,5 acid isomers

ECLChemical structure of esters

EGAa SE-30&CH3-CH2-CH2-CH2-CH,-CH2-(CH2)8-COOCH3 15.00 15.00

CH3-CH-CH2-CH2-CH2-(CH2)8-COOCH3 14.62 14.63ICH3

CH3-CH2-CH-CH2-CH2-(CH2)s-COOCH3 14.76 14.72

CH3

CH3-CH2-CH2-CH-CH2-(CH2)8-COOCH3 14.65 14.55IOH3

CH3

CH3-C-CH2-CH2-(CH2)8-COOCH3 14.10 14.08

CH3

CH3

CH3-GH2---CH2-(CH2)8-COOCH3 14.40 14.38

OH3

CH3-CH2-CH-CH2-(CH2)8-COOCH3 14.68 14.68

02H5a Values are taken from reference 88.Unpublished data (T. Kaneda) determined under the conditions described in reference 84.

ECL-based approaches is discussed in a review(2).One important consideration, often over-

looked, is that the columns usually used in thissort of work are easily overloaded. Even slightoverloading by a component causes a shift to ahigher retention value, particularly on polarcolumns. This may also affect a closely follow-ing peak so that the retention values observedfor both would be slightly too high. Overloadedpeaks frequently show greater asymmetry thansmaller peaks. To overcome this problem, re-tention should be measured by lowering thesample size until the retention volume is inde-pendent of sample size.

Mass SpectrometryThe identity of the mass spectra of a com-

pound and an authentic sample is, perhaps, thebest single evidence for their chemical identity.The recent introduction of combined gas-liquidchromatograph-mass spectrometer systemsby several commerical companies has elimi-nated a tedious and time-consuming purifica-

tion procedure by preparative gas-liquid chro-matography to prepare a sufficient quantity ofpure material for mass spectrometry. In somecases there are minor but significant differ-ences in the mass spectra ofthe same compoundmade by different instruments (81). It is desira-ble to make a direct comparison of a compoundand its authentic sample on the same massspectrometer.The mass spectra of normal and iso-series

fatty acid methyl esters have rather similarpatterns (1). However, the other branched es-ters, containing a tertiary or quaternary car-bon, give characteristic fragmentation patternsand can be identified rather easily (88, 151,167). The use of fatty methyl ethers instead ofmethyl esters allows a more conclusive identifi-cation of branches (94).The location of unsaturation along the car-

bon chain of unsaturated fatty acids is tradi-tionally determined by KIO4-KMnO4 oxidationof the acid followed by gas-liquid chromatogra-phy of the products (52, 150). The procedure istime-consuming and requires individual fatty

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acid fractions isolated by preparative gas chro-matography. Many attempts to replace thisprocedure by mass spectrometric procedureshave been made. The use of the trimethylsilylderivatives of the corresponding hydroxy estershas resulted in some success (130). Recently,two promising procedures have been developed:one includes the conversion of the double bondto a methoxy derivative (113), and the otherincludes the use of pyrrolidides without de-stroying the double bond (8). The latter proce-dure is particularly attractive and is also usefulin the mass spectrometric identification ofbranched and other fatty acids by simplifyingmass spectrometric fragmentation patterns.

Similarly, simple patterns can be obtainedfrom benzyl derivatives which are also usefulfor liquid chromatography, since they havestrong ultraviolet absorption to enhance thedetector sensitivity (133).

FATTY ACID COMPOSITION OFBACILLUS SPECIES

The fatty acid compositions of bacteria, in-cluding Bacillus, are significantly differentfrom those of higher organisms in having nopolyunsaturated fatty acids (fatty acids withmore than one unsaturation). (A notable excep-tion is Mycobacterium phlei, in which unu-sually long-chain polyunsaturated acids makeup about 5% of the total free acids. Hexatria-conta, 4,8,12,16,20-pentaenoic acid is the maincomponent [9].) The various species ofBacillusdo, however, have some additional unusual fea-tures as described below.The predominance of terminally methyl-

branched iso and anteiso fatty acids having 12to 17 carbons is a characteristic observed in allspecies ofBacillus studied (82, 84, 85, 163, 168).Table 3 shows fatty acid compositions of 16species of Bacillus.The normal fatty acids such as myristic and

palmitic, the most common fatty acids in themajority of organisms, are generally minor con-stituents in the genus Bacillus. Another note-worthy feature is that, with the exception oftwo groups, B. anthracis, B. cereus, and B.thuringiensis (which are taxonomically closelyrelated), and B. insolitus, B. psychrophilus,and B. globisporus (which are all psychro-philes), Bacillus species produces little, if any,unsaturated fatty acids (82, 84, 85).An interesting occurrence is a rare acido-

philic, thermophilic Bacillus species in whichthe major occurrence of cyclohexyl fatty acidstogether with the iso and anteiso series hasbeen observed (40, 127).

BIOSYNTHESIS OF FATTY ACIDSSaturated Fatty Acids

Branched-chain acids. In the majority of or-ganisms, de novo synthesis of fatty acids isachieved by the repeated condensation ofmalo-nyl-coenzyme A (CoA) with acetyl-CoA and iscatalyzed by palmitic acid synthetases. Al-though the physical and chemical characteris-tics of the synthetases vary with the source ofthe enzymes, in all cases palmitic acid is thedominant end product (105, 148, 160, 161). Theoverall reaction can be expressed by the follow-ing equation:acetyl-CoA + 7 malonyl-CoA

+ 14 NADPH + 14H+ -- (1)

palmitic acid + 7CO2 + 14 NADP+

where NADPH and NADP+ are reduced andoxidized nicotinamide adenine dinucleotidephosphate, respectively.The fatty acid synthetases of B. subtilis (28,

92) and B. cereus (119) are different from pal-mitic acid synthetase (159, 161). The chain ini-tiators are branched, short-chain acyl-CoA es-ters rather than acetyl-CoA, namely, isobu-tyryl, isovaleryl, and 2-methylbutyryl, whenthe organisms are grown on any of the commonmedia. As with most organisms, malonyl-CoAand NADPH serve as the chain extender andthe hydrogen donor, respectively. In this case,the product fatty acids are iso-C,4 and -C16, iSO-C15 and -C17, and anteiso-C,5 and -C17 acids.The chemical structures ofbranched-chain sub-strates and fatty acid products are shown inFig. 2. The overall reaction can be expressed bythe following representative equation:

isobutyryl-CoA + 6 malonyl-CoA

+ 12 NADPH + 12H+ (2)-* iso-C,6 acid + 6CO2 + 12 NADP+

The fatty acid synthetases of both B. subtilisand B. cereus can be divided into two fractions:an enzyme and a heat-stable factor. Eitherfraction from one organism can be substitutedfor the similar fraction in the system of theother. Cross-experiments with the four frac-tions show that both the B. subtilis enzyme andits heat-stable factor dominate over the frac-tions of B. cereus and tend to give a fatty acidpattern with respect to chain length similar tothat found in growing cells ofB. subtilis wheneither one of the B. subtilis fractions is usedwith the complementary fraction of B. cereus(119). Thus, a fatty acid pattern similar to that

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VOL. 41, 1977 FATTY ACIDS OF THE GENUS BACILLUS 397

TABLE 3. Fatty acid compositions of species of Bacillus

Organism Fatty acid composition(%)

Branched chain chain

Species Strain Even Nor- OthersTtl Anteiso Odd-iso Even-iso Eve malTotal mal

013 C1, 017 C13 015 017 014 016 014 016

B. alveia B-32B-84

B. brevis B-33B-34

B. circulans B-28B-29

B. licheniformis B-49B-50

B. macerans B-40B-41

B. megoterium B-15B-78

B. polymyxa C-42-3

B. pumilus B-12B-13

B. subtilis B-4B-770

B. larvaeb B-2605B-2610

B. lentimorbus B-2522B-2530

B. popilliae B-2309B-2519

B. anthracisc R-F.PHL

B. cereus B-17B-19B-82

B. thuringiensis W.S.B-2172

B. acidocaldariuse Agnano

95 0 38 17 0 1585 0 41 4 0 29

75 0 36 4 0 2592 0 50 8 0 28

82 0 35 5 0 2666 0 26 9 0 18

93 0 31 19 0 2889 0 29 16 0 27

88 0 30 10 0 3090 0 30 17 0 26

90 0 60 7 0 1688 0 52 3 0 27

70 0 41 19 0 0.3

67 0 27 8 0 1879 0 26 11 0 25

95 0 33 10 0 1495 0 40 0 13

85 0 39 3 0 770 0 57 2 0 5

55 0 45 2 0 657 0 41 7 0 2

70 0 57 2 0 577 0 62 2 0 5

84 0.7 16 6 1 1981 0 10 8 1 31

86 2 16 7 4 2682 1 10 6 4 2373 2 9 7 4 20

80 3 15 5 4 2581 3 11 2 8 21

36 0 1 13 0 1

15 2 88 0 3

6 0 53 3 9

7 7 88 1 4

14 0 314 0.3 4

13 1 514 0.2 4

0 5 21 4 1

0 1 9

7 2 514 0.4 3

15 4 1113 2 9

7 0.8 82 2 3

20 0.650 1

2 2 31 4 4

4 6 1410 3 12

6 7 169 6 217 16 9

5 3 56 9 8

15 0 6

Organisms of this group were grown on glucose medium at pH 7.0, 37°C (82).Organisms of this group were grown on glucose-yeast extract medium at pH 7.0, 30°0 (85).

c Organisms of this group were grown on glucose medium at pH 7.0, 37°0 (84).d Represents the sum of unsaturated acids.' Grown on glucose-yeast extract medium at pH 4.0, 6000 (39).' Represents the sum of cyclohexyl fatty acids.

in growing cells of B. cereus is obtained only chain length of fatty acids synthesized. When a

when the combination ofB. ceieus enzyme and higher concentration of malonyl-CoA (200 ,uM)its own heat-stable factor is used. is provided to the fatty acid synthetase of B.The concentration of malonyl-CoA affects the subtilis, the chain length of fatty acids synthe-

0 52 13

4 211 6

7 124 30

0 71 11

1 110.3 10

2 83 9

3 27

4 302 19

0 60 5

1 124 19

5 271 33

4 195 10

3 62 9

2 73 103 19

4 53 2

0 3

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FATTY ACIDS OF THE GENUS BACILLUS

sized was increased significantly (119). UnlikeEscherichia coli synthetase (132), this high con-centration does not inhibit the synthesis.

Isobutyryl-CoA, isovaleryl-CoA, and 2-meth-ylbutyryl-CoA are provided to the system by a-ketoisovalerate dehydrogenase (120) from a-ke-toisovalerate, a-ketoisocaproate, and L-a-keto-,8-methylvalerate, which in turn are derivedeither from de novo synthesis or an exogenoussupply of valine, leucine, and isoleucine, re-spectively. The pathway is expressed by reac-tion 3:

a-Ketoisovalerate + NAD+ +

CoA-SH -* isobutyryl-CoA (3)+ C02 + NADH + H+

where SH is sulfhiydryl. In addition to isottyric, isovaleric, and 2-methylbutyric acids,number of other C3- to -C6 fatty acids, withwithout branching, can serve as chain inittors to yield related, artificially induced falacids (77, 79, 80, 87, 88). The details are dcussed in the next section. In this case, thEshort-chain fatty acids are presumed to be ccverted to their acyl-CoA esters by reactiorbefore incorporation:

R-C-OH + CoA-SH energy donor

R-CS-CoA11

Reaction products of B. subtilis synthet-were identified as free acids (92) as in the caofE. coli synthetase or pigeon liver syntheti(21). However, in the case of yeast synthetait was the acyl-CoA ester (105). It is hig]likely that in B. subtilis the final product, facid, is derived from the fatty acid-acyl carrprotein (ACP) derivative by its enzymatic Idrolysis because a similar mechanism Mfound in the case ofE. coli (12). Free fatty aproducts can be converted to their CoA estbyB. megaterium acyl-CoA synthetase (109).The following sequence of reactions is pos

lated for the elongation of branched-chiprimers. It is largely adopted from the weestablished sequence for the synthesisstraight-chain fatty acids by E. coli syste(159).R-CS-CoA + ACP-SH 2

11

R-CS-ACP + CoA-SH11V

COOH

CH2

CS-CoA + ACP-SH r±11

COOH

CH2 + CoA-SH

CS-ACP11u

COOH/

R-CS-ACP + CH211\O CS-ACP

11

R-C-CH2--CS-ACP + C02 + ACP-SH11 11uo u

ls R-C-0H2-CS-ACP + NADPH + H+ ±a)n- 11 114 0 0

R-CH-CH2-CS-ACP + NADP+11

OH 0

OH 0R-CH=CH-CS-ACP + H20

ase IIase 0ase R-CH=CH-CS-ACP + NADPH + H+se, 11hly 0ree R-CH2-CH2---CS-ACP + NADP+ier IIIhy-

(6)

(7)

(8)

(9)

(10)

ras where R is (CH3)2 OCH-, (CH3)2*CH * CH2-,cid CH2(C2H5) * CH-.Irs The sequence of reactions 5 to 10 elongates,

by two carbons, the chain length of branched-t chain acyl-CoA ester synthesized by either re-'in action 3 or reaction 4. This process is repeated'll- further four or five times to yield ACP deriva-of tives of branched-chain fatty acids with 14 to 17ms carbons.

With respect to its physicochemical nature,the branched-chain fatty acid synthetase fromeither B. subtilis orB. cereus is quite similar toE. coli synthetase: both are the so-called "solu-

(5) ble systems" which cannot be sedimented byultracentrifugation (92) and which require ACPor heat-stable factor (28, 92, 119). (The involve-

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ment of ACP in fatty acid synthesis by B. sub-tilis synthetase has recently been establishedin this laboratory [unpublished data].)The major difference between branched-

chain fatty acid synthetase and straight-chainfatty acid synthetase lies in their specificitytoward the chain initiator (reaction 5) catalyzedby acyl-CoA-ACP transacylase. The formerprefers acyl-CoA esters with four to six carbons,whereas the latter uses acyl-CoA esters withtwo to three carbons. This barrier can be re-moved if the appropriate acyl-ACP derivative,instead of its acyl-CoA ester, is provided tobypass reaction 5 (28). Thus, with respect to thechain-extension process, there is no differencebetween branched-chain acid synthetase andstraight-chain acid synthetase. Figure 3 sum-marizes the synthetic pathways catalyzed bythe two synthetases. It should be noted thatpalmitic acid, for example, can be synthesizedby branched-chain acid synthetase ifeither ace-tyl-ACP or butyryl-CoA is provided as chaininitiator. There is an exception in that the sys-tem of rat adipose tissue, which is responsiblefor palmitic acid synthesis, can synthesize thebranched-chain fatty acids from the relatedbranched short-chain acyl-CoA esters (65). Pre-sumably, the transacylase in this system isnonspecific toward the chain length of the acyl-CoA ester substrates.Although de novo synthesis of long-chain

fatty acids in B. subtilis appears to be straight-forward and consistent with the scheme estab-

lished for other systems, there are some obser-vations that remain to be explained.The dehydrogenase catalyzing reaction 3 is a

multienzyme complex with a molecular weightof millions which can be sedimented by ultra-centrifugation (120). When the dehydrogenasewas removed from a preparation of B. subtilisfatty acid synthetase by ultracentrifugation, itssynthetic activity remained unchanged (92). Itis possible that the very small amount of dehy-drogenase activity remaining in the superna-tant fraction of the synthetase preparation maybe sufficient to provide chain initiator for thefatty acid synthesis because the activity of thedehydrogenase in the preparation was thou-sands of times greater than that of fatty acidsynthetase. Further evidence to support the in-volvement of reaction 3 is essential before thepresent scheme (Fig. 3) is completely estab-lished.

Straight-chain acids. The obvious questionto ask regarding Bacillus is how the common,normal fatty acids, namely, myristic and pal-mitic, although they are minor acids, are syn-thesized in the organisms. Branched-chainfatty acid synthetase from B. subtilis is capableof synthesizing these straight-chain fatty acidsif acetyl-ACP, not acetyl-CoA, is provided tothe system (28). Thus acetyl-CoA-ACP trans-acetylase is absent from the organism. An al-ternative synthesis for acetyl-ACP must be con-sidered. Recently the acetyl-ACP-malonyl-ACP condensing enzyme from E. coli has beenshown to catalyze the following reaction (58):

Branched- chain acidsynthetase

C3-toC -Acyl CoAesters

A

Straight-chain acidsynthetase

C2 -orC3-Acyl CoAester

B

CiitaCe -Acids

FIG. 3. Schematic pathways of fatty acid synthe-sis by two synthetases. Arrow A at the upper portionis catalyzed by acyl-CoA-ACP transacylase, whereasarrow B is catalyzed by acetyl-CoA-ACP transacyl-ase.

COOH/

CH2 -* CH3-CS-ACP + CO2\ ~~~~~~~~~~~~~~~~~~11CS-ACP 011v

(11)

where CS is thioester. Similarly, in Bacillusspecies, malonyl-ACP synthesized by reaction 6may be converted to acetyl-ACP by the enzymethat catalyzes reaction 11. Subsequently theacetyl-ACP is used to synthesize straight-chainfatty acids. This postulated pathway is consist-ent with the observation that in biotin-requir-ing strains ofB. subtilis (79) andB. cereus (70),the proportion of straight-chain fatty acids in-creased as a function ofamount of biotin addedto the culture media. The biotin added wouldincrease the synthesis of acetyl-CoA carboxyl-ase, a biotin enzyme (159), with a resultingincrease ofmalonyl-CoA synthesis, thus acetyl-ACP.

Alternatively, a butyryl derivative, such asits CoA ester, may be a precursor for straight-

C2-toC -Acyl ACPe sters

; Chainextension

C1,-to C18-AC Pesters

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chain fatty acids in Bacillus. A possible path-way involves the synthesis of butyryl-CoA bythe reduction of acetoacetyl-CoA. However, inhighly aerobic organisms, such as Bacillus,this reaction is less likely to occur.

Cyclohexyl acids. w-Cyclohexyl acids, al-though rare in nature, occur in tobacco leaves(115). B. acidocaldarius is so far the onlyknown microorganism containing these acids,w-cyclohexylundecanoic and w-cyclohexyltride-canoic, as major acid constituents. The cyclo-hexyl moiety of the acids has been shown to besynthesized through shikimic acid (40, 127), butthe detailed biosynthetic pathway is unknown.

Unsaturated Fatty AcidsIn bacteria, unsaturated fatty acids are syn-

thesized either by an aerobic pathway involv-ing the direct desaturation of saturated fattyacids or by an anaerobic pathway involving theelongation of the medium-chain unsaturatedacids, A3-Co and -C,2, to synthesize All-C,8 andA9-C,8 acids, respectively (45). The unsaturatedfatty acids found in Bacillus species are synthe-sized by the aerobic pathway.Most common monounsaturated fatty acids

in nature are A9-isomers. In certain Bacillusspecies, unusual monounsaturated fatty acidssuch as A5- (B. licheniformis, B. megaterium,B. pumilus, B. subtilis), A8_, and A10- (B.brevis, B. cereus, B. licheniformis, B. marcer-ans, B. stearothermophilus) isomers are alsofound (38, 48, 51).

Polyunsaturated fatty acids are, in general,absent in Bacillus species. However, ifone usesa very sensitive assay method, they can bedetected in some species. For instance, when B.licheniformis was grown at 20TC in the pres-ence of [1-'4C]palmitate, synthesis of 5,10-hex-adecadienoic acid could be detected (49). Thediene probably results from the activity of twoseparate enzymes, one ofwhich is active towardthe 5-position, the other active toward the 10-position. Each of the enzymes would act pri-marily upon palmitic acid, but would also showactivity toward the monounsaturated productof the other enzyme.Unsaturated fatty acids could be reduced to

the related saturated fatty acids. InB. cereus, atemperature-dependent reduction of oleic acidto stearic was observed (97).

Stereospecific Synthesis of Anteiso FattyAcids

The anteiso-C15 and -C,7 acids from B. sub-tilis have been shown to be of the L(+) seriesregardless ofthe culture medium used (80). ["1-series," originally printed in reference 80,

should read "d-series," which is the L(+) series.]When any one of these: a-ketobutyrate, i-threo-nine, L-a-keto-,8-methylvalerate, L-isoleucine,n-a-keto-,8-methylvalerate, L-alloisoleucine, ora-methylbutyrate, is added to the culture me-dium, an increase in the synthesis of anteisoacids is observed. On the basis of this effect,and from observations with the cell-free system(28, 92, 119), the following pathways are postu-lated for the stereospecific synthesis of anteisofatty acids (Fig. 4) (80).From glucose, L-(+)-anteiso fatty acids are

synthesized by the sequence of steps a, b, c, andd. When any one of L-threonine, L-isoleucine,and L-alloisoleucine, but not their 1)-isomers, isprovided, it is deaminated to the respective a-keto acid by the stereospecific step e, f, or h. Aninduced formation of branched-chain L-aminoacid dehydrogenase has been observed in B.subtilis (135). Step i is included since step c,catalyzed by a-ketoisovalerate dehydrogenase(120), is specific to the L-isomer. Step g is spe-cific to the 1-acid. Thus, enrichment of the L-acid in the culture medium was observed whenthe organism was grown in the presence of D,L-a-methylbutyrate (80).

Branched Short-Chain Fatty AcidsB. subtilis is capable of synthesizing such

branched, short-chain fatty acids as isobutyric,isovaleric, and 2-methylbutyric acids from therelated a-keto acids. The synthesis is carriedout by a combination of two enzymes, a-keto-isovalerate dehydrogenase (reaction 3) and anacyl-CoA hydrolase, both of which are detectedin cell-free extracts of B. subtilis (120). Bothisobutyric and isovaleric acids were detected inthe amino acid-rich culture medium in whichB. subtilis had grown (146).The branched, short-chain fatty acids, how-

ever, were not detected when an amino acid-poor culture medium was used (76). These fattyacids are presumed to be consumed as they areformed in the cells because ofthe limited supplyof the precursor amino acids valine, leucine,and isoleucine. It should be noted that thesebranched, short-chain fatty acids are inhibitorsfor the growth of B. subtilis species (167). Insome rumen bacteria, large amounts of thebranched, short-chain fatty acids were synthe-sized from the related amino acids and escapedinto the culture medium. Conversely, thesefatty acids were utilized to synthesize theamino acid in the cell (7).Although the elucidation of synthetic path-

ways is progressing nicely, very little work hasbeen done regarding the degradation of fattyacids. The evidence presently available comes

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L-Threonineor

1- Homoserine

1- Isoleucine

L-12-

CoA

ic and

L-14-Methylhexadecanoic Acids

FIG. 4. Stereospecific synthesis of anteiso fatty acids in B. subtilis.

primarily from work designed to investigatesome other area. In the resting cells ofB. cer-eus, none of myristic, palmitic, and stearicacids was converted to its lower homologues(38). In the growing cells ofB. subtilis, none ofthe n-C6 to n-C,0 acids, when added to the cul-ture medium, caused an increase in the amountof either even- or odd-numbered normal acids(88), though if any of the added acids weredegraded to yield n-C4 or n-C5 acid, such anincrease should have been observed. These re-sults suggest that in Bacillus, degradation offatty acids by either the a- or /3-oxidationscheme is insignificant.

FACTORS AFFECTING FATTY ACID PAT-TERN AND CONTENT

Pattern of Saturated Fatty AcidsMost living organisms synthesize only one

series of fatty acids, the normal series(straight-chain fatty acids with or without un-saturation and/or functional groups such as hy-droxyl, cyclopropyl, and ketonic functions). Onthe other hand, certain bacteria, includingmembers of the genus Bacillus, as discussedearlier, synthesize the three branched series offatty acids as major acids, in addition to thenormal series which occurs as minor acids. Inthese bacteria, the relative proportions of thefour series of fatty acids can vary, dependingupon genetic characteristics and physiologicaland environmental conditions.

The schematic pathway of branched-chainfatty acid synthesis shown in Fig. 5 is formu-lated on the basis of the fatty acid compositionsof 22 species of Bacillus and the fatty acid bio-synthesis studied with B. subtilis and B. cer-eus, as discussed earlier. The normal series isexcluded from the figure because its proportionis minor and, as seen later, usually unvarying.Factors affecting the relative proportions of thebranched fatty acid series may be grouped intothree categories: (i) the relative activity of thethree chain initiators toward fatty acid synthe-tase at step c; (ii) the relative availability ofchain initiators by the three pathways, endoge-nous (path A,-B) and exogenous (path A2-Band path D); and (iii) the amount of chain ex-tender ("C2 donor" at step c). Factor (i) is char-acteristic of the fatty acid synthetase and isfixed for a given microorganism. Factors (ii)and (iii) are variable, depending upon physio-logical and culture conditions.

Relative activity of chain initiators. Therelative activity of the three branched a-ketoacid substrates used as the source ofchain initi-ators to the B. subtilis fatty acid synthetase is5:2:1 in the order a-keto-,(-methylvalerate, a-ketoisocaproate, and a-ketoisovalerate,whereas toward B. cereus synthetase it is 4:3:1(92, 119). The relative proportions of the threebranched series of fatty acids in the lipids ofB.subtilis and B. cereus grown on glucose are 57,25, and 13% of the total acids and 19, 40, and20% of the total acids in the order anteiso, odd-

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FATTY ACIDS OF THE GENUS BACILLUS

Glucose

4fPyruvatea-Ketobutyrote(Endogenous precursors)

\A1 AX/

B

C |IC2 donor"

IsoleucineValineLeucine(Exogenous precursors)

2 -MethylbutyroteDo oIsobutyrateIsovalerote(Exogenous substrates)

[i3nchad-hinatt3!ocidilFIG. 5. Schematic pathways involved in the biosynthesis of branched-chain fatty acids.

iso, and even-iso series, respectively (82). Thus,in the case ofB. subtilis the relative activity ofthree a-keto acid substrates is directly relatedto the relative proportions of the threebranched series offatty acids synthesized in thecell. In B. ceteu8, however, this is not so, sug-gesting that other factors must play an impor-tant role in determining the relative propor-tions of fatty acids. Probably the endogenoussupply of a-ketolsocapoate in the organism islarger than that of &-kto-f3-methylvalerate, sothat the synthes of odd-Iso series in compari-son with that of anteiso series is greatly in-creased.

Factors related to chain Initiators. Precur-sors of chain initiators for fatty acid synthesisare supplied from two sources, exogenous andendogenotus. The exogenous supply dependsupon the composition of the culture medium,whereas the endogenous supply, which is deter-mined by the enzyme activities involved andavailable intermediates, is affected by physio-logical conditions and genetic characteristics ofthe organism. Thes factors all contribute todetermining the relative availability of chaininitiators and, In turn, the relative proportionsof fatty acids synthesized.The addition of the precursor of one of the

natural chain initiators (anteiso-C5, iso-C5, andiso-C4 primers) to a glucose medium increasedgreatly the synthesis of fatty acids related tothe add substrate. For example, isoleucineincreased the synthesis of anteiso fatty acids,

and their proportion was increased up to 98% ofthe total fatty acids from the control value of57% (79). Similar increases, though to a lesserdegree, were observed on the addition of valineand leucine. A similar conclusion was obtainedin the case of an unidentified thermophilic Ba-cillus species (37).

Substrates that increased one of threebranched fatty acid series and two normal fattyacid series are listed in Table 4. Increases in theanteiso series by a-ketobutyrate and its meta-bolic precursors indicate the close link be-tween the biosynthesis of branched-chain fattyacids and that ofbranched-chain amino acids inB. subtilis. This is further supported by theincorporation of uniformly labeled valine intoboth even-numbered and odd-numbered iso se-ries (78).The increases of normal fatty acid series by

certain straight-chain substrates shown in Ta-ble 4 indicate that the pathway for the synthe-sis of branched-chain acids shown in Fig. 5 alsoaccepts straight-chain compounds (aminoacids, keto acids, and short-chain fatty acids),as long as their chain length falls within acertain range (C3 to Ci).When appropriate branched, short-chain acid

substrates, not related to the endogenous chaininitiators, were added to the culture medium,B. subtilis synthesized new branched, long-chain fatty acids by chain elongation of theadded substrates (88). For example, even-num-bered fatty acids (anteiso-C,4 and -C16) were

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TABLE 4. Short-chain substrates that enhance synthesis of specific fatty acids by growing cells of Bacillussubtilisa

Even iso acids Odd iso acids Odd anteiso acid Even normal- Odd normal-acidsSubstrate (n-C14 and -C06) (iso-C15 and -C07) (anteiso-Cu5 and -C17) acid- (n-C,1 and -C07)

(14%)b (21%) (57%) (n-C,,and-CI6) (<1%)

Amino acid L-Valine L-Leucine L-Isoleucine L-Norvaline L-Norvaline, L-a-aminobutyrate

a-Keto acid a-Ketoisovalerate a-Ketoisocaproate L-a-Keto-3-methyl- a-Ketovalerate a-Ketobutyratevalerate

Short-chain Isobutyrate Isovalerate D-a-Methylbutyrate n-Butyrate Propionate, n-val-acid erate

Others L-Threonine L-ThreonineL-Homoserine

a-Ketobutyrate

a Data from references 79 and 80.bPercentage of total fatty acids in each series in the cells grown on the standard medium. The percentage of each series

is increased by the addition of substrates listed on the same column.

synthesized from added 3-methylpentanoate.Corresponding results were obtained with 2-ethylbutyric, 2,2-dimethylbutyric, 3,3-dime-thylbutyric, 2-methylpentanoic, and 4-methyl-pentanoic acids; only in the last case are theresulting C14 and C,6 acids found naturally.The relative proportions of these four series

of fatty acids in B. subtilis and B. cereus varywith growth phase. In both species, the anteisoseries was most variable and is more prevalentin cells in the early logarithmic phase than inthe late logarithmic or stationary phase. Thesituation is reversed in the even- and odd-num-bered iso series. The abundance of the normalseries remains essentially unchanged through-out all growth phases (79, 87, 154).

Sporulation and germination are characteris-tic features of the genus Bacillus, taking placeunder specific environmental conditions. Foreither process, lower homologues of iso acidsappear to be essential. InB. thuringiensis, ace-tate is incorporated only into the iso-C13, iso-C14, and iso-C15 acids, presumably as the chainextender (121). In B. megaterium, the propor-tion of branched C15 acids decreased during theprocess, whereas the proportions of a branchedC14 acid, presumably an iso acid, increased (15).In Bacillus stearothermophilus, grown at 450C,the proportion of odd-numbered iso series invegetative cells was higher than in spores,whereas the proportions of anteiso and even-numbered iso series between the two formswere no different (168).By increasing the growth temperature of B.

cereus from 21 to 37°C, the proportions of odd-numbered iso series in the phospholipids wereincreased, whereas the proportions of anteisoseries were decreased. Both normal and even-

numbered iso series remained essentially un-changed (91). In B. stearothermophilus, the in-crease in culture temperature caused a largeincrease in the normal series, with, of course, aconcomitant decrease in the amount of theother series (168). The significance of this tem-perature-dependent alteration of fatty acid pat-tern is considered in the section "Function ofbranched-chain fatty acids."The growth of B. subtilis (ATCC 7059) was

completely dependent on valine, partially de-pendent on leucine, and independent of isoleu-cine (79). The relative abundance of the threebranehed-acid series related to these aminoacids in the organisms grown on an amino acid-poor medium was anteiso, odd-iso, and even-isoin decreasing order. The endogenous supply ofthe amino acids has a direct bearing on therelative abundance of the related threebranched-acid series.The relationship is more clearly shown by

experiments using amino acid auxotrophs ofB.subtilis (Table 5). In an isoleucine-requiringmutant (CA-273), the abundance of anteiso se-ries was reduced to 78% of that found in theparent strain. Similarly, in a leucine-requiringmutant (CA-186), the abundance of odd-iso se-ries was 62% of that found in the parent strain.In both cases, the reduction was observed onlyin the C05 acid, not in the C07 acid. No changewas observed in the even-iso series.When strains AC-186 and AC-273 were

grown together in a flask, they populated theculture equally. The fatty acid pattern of thismixed culture was, however, quite differentfrom the fatty acid pattern calculated as anaverage of the individual patterns of the orga-nisms (Table 5, last column). The proportion of

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TABLE 5. Alteration offatty acid pattern in mutantsofBacillus subtilis due to defect in leucine and

isoleucine synthesisaFatty acid distribution

(%)

Fatty acid Strain Strain StrainParent CA-186 CA-273 CA-186 +strain (leucine-) (isoleu- strain CA-

(ecnl cine-) 273

anteiso-C11 55 65.2 50.8708 9 376 |50.7 3512|anteiso-C,7 lO.lj 20 .1 13.1j 11.2

isO-C15 17 25.1 9.0 15.7 22.9 35.1 20° 30.6iso-C17 7.9J 6.7J 12.2] ioij

isso-C', 047}5.6 08 5.9 1 5.4 6.617.5iSO-C16 4.7] siS 3.8 10.9]

n-C14 001 4.0 .071 7 0 777 ° 0 5.6n-Ca6 41054.07f 7 79. 5.6

a Original data from reference 79.

the even-iso series was extremely high, sug-

gesting that during growth the two strains af-fect each other's metabolic activities. A possibleexplanation is that in the mixed culture, thesupply of a-keto-,8-methylvalerate and a-keto-isocaproate by their de novo synthesis is one-

half that of the parent strain, whereas that ofa-ketoisovalerate is the full strength. Thus, thesynthesis of branched-chain acids related to a-

ketoisovalerate is significantly enhanced.Factors related to chain extender. Malonyl-

CoA is the chain extender in de novo synthesisof fatty acid for all of the known systems. Thus,the effect of malonyl-CoA and its precursor on

fatty acid composition is of interest.The addition of malonic acid to the culture

medium did not alter the fatty acid compositionin the lipids of B. subtilis (T. Kaneda, unpub-lished observation). This may be due to the lackof a transporting system and/or the lack of a

malonyl-CoA synthetase.Malonyl-CoA is synthesized by the carboxy-

lation of acetyl-CoA. The reaction is catalyzedby acetyl-CoA carboxylase, a biotin enzyme(161). In biotin-requiring strains of B. subtilis(79) and B. cereus (70), fatty acid content in thecells increased as a function of amounts of bio-tin added to the culture medium. Furthermore,the proportion of the normal series was, in eachcase, significantly increased over that found inthe parent strain.

Proportion of Unsaturated AcidsIn B. cereus, the proportions of A5- and A10-

acids are apparently temperature dependent.Unsaturated fatty acids made up 9% ofthe totalfatty acids when the organism was grown at

3500 and were predominantly the AW0-isomer.However, at 2000, the amount of unsaturatedfatty acids was increased to 27% of the totalfatty acids. Furthermore, a significant propor-tion of new acids, A5-isomers, were synthesizedin addition to A1O-isomers (91).

In B. megaterium, a A5-desaturating enzymewas induced by growing or incubating the cellsat 200C, but it was deactivated rapidly at 30'C.Thus, its activity was observed only when as-sayed at 200C, regardless of growth or preincu-bation temperature (31, 48). Similarly, in B.subtilis, B. pumilus, B. licheniformis, and B.alvei, the A5-desaturating activity measured byincubation at 20TC for 4 h was much higherthan when incubation was at 300C. A tempera-ture-dependent synthesis of n-A5-C16 acid wasalso observed in a thermophilic Bacillus species(36). In B. brevis, palmitic acid was desatu-rated at the 8-, 9-, or 10-position and the de-saturating activities measured by incubationat 20 and 3000 were not significantly different.

Ratios of unsaturated fatty acids to the chem-ically related saturated fatty acids in growingcells of B. thuringiensis and B. cereus indicatethat palmitic acid, a straight-chain C,0, is a farbetter substrate for the desaturating enzymethan any of the branched ones (84). Restingcells ofB. cereus desaturated normal fatty acidsto give A1O-isomers (38). The order of substrateactivity was n-C16 > n-C18 > n-C14. Interest-ingly, when n-C14 was added as substrate, mostof it was elongated to n-C16 and then desatu-rated.Upon addition of large amounts of biotin to

the culture medium, the proportion of normalseries in a biotin-requiring strain of B. cereuswas significantly increased (70). At the sametime, the proportion of unsaturated acids,mainly n-C16, was increased. This was due tothe availability of a large amount of the normalseries, which are preferred substrates for thedesaturating enzyme (38, 84).

Since the desaturating enzymes exhibit thispreference (38), it seems probable that any Ba-cillus species having significant amounts of un-saturated fatty acids is capable of synthesizingthe normal series in abundance. The major un-saturated acids are hexadecenoic acids. Thisappears to be applicable to several mesophilicspecies (51, 84, 91) and to three psychrophilicspecies of Bacillus (89).

Fatty Acid ContentVarious factors affecting the fatty acid pat-

tern of Bacillus species should also causechanges in the fatty acid content, but littledetailed work has been done on this aspect.

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Growth phase affects the fatty acid content ofB. subtilis (79). The cells in the stationaryphase store fatty acids in an amount far exceed-ing the amount required for growth. The fattyacid content of 3-h cells (an initial log phase)was only one-eighth that of 7-h cells (an earlystationary phase) on a cell-weight basis. Fur-ther incubation, however, resulted in a slightdecrease.The supply of chain initiators appears to be a

major factor limiting the amount of fatty acidssynthesized in B. subtilis. The addition of iso-leucine to the culture medium increased theamount of anteiso fatty acids up to 10 timestheir control content. Consequently, theamount of total fatty acids was increased to fivetimes that of the control value (79). Similarincreases, but to a lesser degree, were observedon the addition of valine and leucine.Although the supply of chain extender, malo-

nyl-CoA, is plentiful and not a limiting step infatty acid synthesis in B. subtilis under normalgrowth conditions, this factor can be rate limit-ing under specific conditions. The amount offatty acids per cell was reduced to 1/30 of thecontrol value in a biotin-requiring strain of B.subtilis when a limited amount of biotin wassupplied (79). This was presumably due to areduced synthesis of acetyl-CoA carboxylasecaused by biotin deficiency, because the enzymethat catalyzes the synthesis ofmalonyl-CoA is abiotin enzyme. In other organisms, this enzymehas been considered to be the rate-limiting stepof fatty acid synthesis and is activated by cit-rate and other tricarboxylic acid intermediates(161). This area has not been explored with anyBacillus species.

FUNCTION OF BRANCHED-CHAINFATTY ACIDS

Branched-Chain Fatty AcidsMost of the fatty acids in genus Bacillus

appear to occur in the membranes. The cyto-plasmic membrane of B. subtilis 168 is 16% byweight lipids, mainly phospholipids (75% of thetotal) (16). The properties of phospholipids de-pend on the distribution of fatty acids betweenthe 1- and 2-positions, the nature of the phos-phate derivative in the 3-position, and the phys-ical properties of the fatty acid components.Thus, the functions of branched-chain fattyacids are considered in these areas: (i) as sub-strates in phospholipid synthesis and (ii) ascomponents contributing to the fluidity of phos-pholipids. In addition, possible molecular struc-tural significances of branched-chain fattyacids are also included as (iii) conformationalcontributions. Some comments are made at the

end of this section on possible roles of phospho-lipids in organisms.The phospholipids of higher plants and ani-

mals exhibit a high degree of positional prefer-ence with regard to saturated and unsaturatedfatty acids. The 1-position is usually occupiedby saturated fatty acids and the 2-position bypolyunsaturated fatty acids. Monounsaturatedfatty acids are distributed between the two po-sitions but tend to be more common in the 2-position. Most of the fatty acids of most speciesof Bacillus are saturated. Some of these must,therefore, be located in the 2-position, behavingin this respect like the polyunsaturates ofhigher organisms. It was found that the 2-posi-tion is mainly occupied by the anteiso-C15 acidin B. subtilis and by the anteiso-C,5 and iso-C,5acids in B. cereus (90, 91). Table 6 shows thepositional preference of individual fatty acidstoward the 2-position of phosphatidylglycerol(90). Within a chosen series, the shorter thechain length, the higher the preference towardthe 2-position. Among the three acids with thesame carbon number, the preference towardsthe 2-position is in the order of anteiso > iso >normal. The tendency of iso-C15 and iso-C16acids to locate in the 2-position is almost equiv-alent to that of the straight-chain fatty acidshaving one carbon less. In B. cereus, the intro-duction of unsaturation into a given fatty acidaffects but little its positional preference inphospholipids (91).Branched-chain C04 and C,5 fatty acids are

required to fill the 2-position ofphospholipids inorder to achieve normal bacillary growth. Thegrowth of a mutant of B. subtilis defective inbranched-chain a-keto acid dehydrogenase wassupported only by certain branched or alicyclicshort-chain fatty acids, but not by any one ofthe straight-chain fatty acids tested (41, 167).B. subtilis appears to have a mechanism pre-venting synthesis of normal fatty acids beyonda limit. The largest proportion ofnormal series,which was achieved by addition of i-norvalineto the culture medium, was 60% of the total

TABLE 6. Positional preference of individual fattyacids towards the 2-position ofphospholipid from

Bacillus subtilisa

Total car- Percent located in the 2-position of phosphati-bons of dylglycerol

fatty acid Normal series Iso series Anteiso series

14 60 100 10015 17 68 9616 10 15 3917 0 3 3

aAfter reference 90.

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fatty acids, whereas branched-chain fatty acidsmay comprise up to 90% of the total (79). Theseobservations can be explained in relation tophospholipid synthesis. Propionic, butyric, andvaleric acids serve as primers in the synthesisof fatty acids in B. subtilis, to yield a pair of n-C15 and n-Cl7 acids or n-Cl4 and n-C1,6 acids (77,79). Two even-numbered acids, n-C14 and n-C16,are synthesized from a common n-C4 primer ina ratio of 1:10. When these are the sole fattyacids, as in the case of the mutant, the amountof n-C14 acid able to occupy the 2-position is toosmall to balance out the amount of n-C16 acid,which mainly takes the 1-position. In the case

of odd-numbered acids, n-C15 and n-C17, thesituation is worse since both acids prefer the 1-

position.The mechanism by which fatty acids are pref-

erentially incorporated into one of the two posi-tions of phospholipids in Bacillus species ispresently unknown. Phosphatidic acid plays a

central role in the biosynthesis ofphospholipidsin B. megaterium (128), as it does in E. coli (30,32,:93). The synthesis of phosphatidylethanola-mine and phosphatidylglycerol from phospha-tidic acid via the common cytidine diphosphateintermediate was demonstrated. The acylationof glycerol-3-phosphate in E. coli is carried outby an enzyme different from the one that cata-lyzes the acylation of lysophosphatidic acid(155). In the acylation of glycerol-3-phosphate,the enzyme directs palmitic acid to the 1-posi-tion and unsaturated fatty acids to the 2-posi-tion. In considering these facts, a mechanismsimilar to that ofE. coli may achieve the posi-tional preferential incorporation of saturatedfatty acid into the phospholipids in Bacillus.However, the difference in the fatty acid pat-terns of these phospholipids from B. subtilissuggests that the synthesis and metabolism ofthe phospholipids are complex (90).

Recently, the involvement of a fatty acid-ACP derivative in the synthesis ofphosphatidicacid has been demonstrated. The acylation ofglycerol-3-phosphate by an enzyme preparationfrom Clostridium butyricum was highly spe-

cific to the ACP derivative. The fatty acid-CoAester had little activity (56). The acyltransfer-ase ofE. coli, however, uses both the fatty acid-CoA ester (3, 98, 131, 155) and the fatty acid-ACP derivative (3, 155) as substrates. The pos-sible involvement of a fatty acid-ACP in phos-pholipid synthesis in Bacillus species remains

to be investigated.Maintenance of proper fluidity of membrane

lipids is important for the growth of microorga-nisms, and this is achieved by varying the fattyacid composition of the membrane lipids. Ac-

tually, the melting temperature of the compo-nent fatty acids is the factor contributing to thefluidity of the membrane lipids, which is char-acterized by temperatures of the phase transi-tion. A close relationship between the maxi-mum growth temperature and the temperatureof the phase transition of membrane lipids hasbeen observed inAcholeplasma laidlawii (109).This area has recently been reviewed (47). Themelting points ofbranched-chain fatty acids aregenerally lower than those of straight-chainfatty acids with the same number of carbons.Particularly, the melting points of the anteisoseries are 25 to 350C lower than those of thenormal series (Table 7). Since lowering the cul-ture temperature results in an increase in theamount of the anteiso series produced by B.cereus, B. subtilis, and B. stearothermophilus,it seems likely that these organisms use thebranched chains to maintain fluidity at coolertemperatures (27, 91, 168).

Melting points of the iso series, however, areonly slightly lower than those of the normalseries. This may be the reason why, in somethermophiles, large amounts of the iso seriesrather than the normal series are found as lipidconstituents (13).The possible functions of branched-chain

fatty acids that are not associated with phos-pholipids in Bacillus are unknown. It is highlyprobable that the methyl branchings make pos-sible a fit with hydrophobic groups of othermembrane components such as proteins andglycopeptides to form the structures requiredfor the function of cell membranes. An examplesuggesting this probable role has been reportedin the case of myxoviruses (19). The incorpora-tion of branched-chain fatty acids, marked byiso-C17 acid, into the viral envelope can alterthe structure of the envelope protein. Suchchanges may have an important role in theselection of fragments of viral genome andcould conceivably alter the genotype.

Recently, the involvement of the cholesterolester of the anteiso-C17 acid in peptide elonga-tion by the rat liver system has been demon-

TABLE 7. Melting point offatty acids

Total car- Normal series" Iso seriesa Anteiso series"bons

12 44.213 41.5 41.3 6.214 53.9 53.615 52.3 51.8 23.016 63.1 62.417 61.3 59.8 36.8

a After reference 108a.b After reference 164.

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strated (67). The ester appears to affect thefunction of the ribosomal A site and peptidyltransferase site and also that of the guanosinetriphosphate site and P site (68). This activityis specific with esters of anteiso fatty acids hav-ing 16 to 18 carbons. Neither straight-chainfatty acids nor iso fatty acids with this range ofcarbon chain were active (69).

In addition, the branched-chain fatty acids ingenus Bacillus may also be important as anefficient surface coating. The location ofmethyl-branched alkanoic fatty acids on mem-brane surfaces of higher organisms is observedin a few cases. Over 50% of the acids in wool fatare of the iso and anteiso series (164). Etherextracts of human skin contain 12% branched-chain fatty acids (74). Half the alkanes in theexternal lipids of tobacco leaves are of the isoand anteiso series, and these alkanes are syn-thesized from iso and anteiso fatty acids (81,83). In contrast, the internal lipids of thesehigher organisms are predominantly of the nor-mal series (86).

It appears that the conformational contribu-tion of branched-chain fatty acids to biologicalsystems is not limited to the microbial worldbut, rather, is universal. This is an area wheremuch remains to be explored.

PhospholipidsThe phospholipids that occur most commonly

in species of Bacillus are phosphatidylethanol-amine, phosphatidylglycerol, and diphosphati-dylglycerol (15, 25, 60, 90, 91, 124-126, 128).Phosphatidylcholine is generally absent (73).Some basic phospholipids are found in certainspecies of Bacillus; large amounts of lysylphos-phatidylglycerol are synthesized in cells of B.subtilis (126), and glucosaminylphosphatidyl-glycerol in B. megaterium (125), when theseorganisms are grown under acidic conditions.Ornithinylphosphatidylglycerol was found inlipids ofB. cereus (66). These basic phospholip-ids may function in the membranes to maintainthe pH. Lysylphosphatidylglycerol was shownto be synthesized by particular enzyme prepa-rations ofB. megaterium and B. cereus (57).The role of lipids in membrane transport sys-

tems of bacteria has drawn considerable inter-est and extensive study. However, except for E.coli (46), their direct involvement has not yetbeen demonstrated. In a glycerol-requiring mu-tant of B. subtilis, the simultaneous synthesisof phospholipids is not necessarily required forthe induction of the citrate transport system(166). With the depletion of glycerol, however,similar mutants ofB. subtilis suffer a reductionof various synthetic activities, including those

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which produce ribonucleic acid, deoxyribonu-cleic acid, protein, and phospholipid (104, 110-112). A specific role for each of the individuallipid classes was demonstrated by use of a phos-phatidylethanolamine-deficient mutant of B.subtilis. This mutant had enhanced osmoticfragility of protoplasts (14).

GROUPING OF BACILLUS SPECIESBASED ON FATTY ACID PATTERNSThe examination of 15 species (two strains of

each) ofBacillus reveals the fatty acid patternsto be strikingly uniform: (i) the two branchedseries (iso and anteiso) of the fatty acids ac-count for 55 to 95% of the total acids; and (ii)unsaturated fatty acids are generally absent orpresent only in very small amounts (76, 82, 83,85).The fatty acid pattern of a given species of

Bacillus can act as a "fingerprint" if the orga-nism is grown under culture conditions wherethe exogenous supply of the precursors of chaininitiators including the "naturals" (such asbranched-chain a-keto acids and branched-chain amino acids) and "artificials" (such asbranched short-chain fatty acids) is insiginifi-cant. To meet these conditions of total depend-ence on de novo synthesis, the organism shouldbe cultured on a simple medium such as onecontaining glucose, vitamins, and inorganicsalts. However, many species of Bacillus areincapable of growing on such a simple medium,and some additional nutrients must be added inlimited amounts sufficient to promote growthbut without significantly affecting the fattyacid pattern. A medium routinely used in mylaboratory is composed of glucose (1%), yeastextract (0.1%), and inorganic nutrients (79).When this is done, the fatty acid patterns ofthe various species of Bacillus can be dividedinto two groups: either the anteiso-C,5 or theiso-C,5 is most abundant among the fatty acids(79). The group, in which the anteiso-C,5 ismost abundant, includes most Bacillus species(B. subtilis group), whereas the other group, inwhich iso-C15 is most abundant, includes onlythree species (B. cereus group) which are taxo-nomically closely related.The B. cereus group has two other features

distinguishing it from the remaining species: (i)the range of chain length of the fatty acids iswider, containing C12 and C13 acids; and (ii)small but significant amounts of monounsatu-rated fatty acids (6 to 10% of the total) arepresent. However, the occurrence of iso-C,5 acidas the most abundant acid and the range ofunsaturation observed in the B. cereus groupmay be coincidental. They do not necessarily


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occur together. In support ofthis, recent reportshave shown that two species of extremely ther-mophilic Bacillus (163) and some species ofPro-pionibacterium (118) synthesize iso-C15 acidmost abundantly, but neither shorter-chainfatty acids (CI and C13) nor unsaturated fattyacids are synthesized in any significantamounts.

In the members of the B. cereus group, thepredominance of iso-C,5 acid is retained whenthe glucose medium is replaced by an aminoacid-rich medium such as Penassay broth(Difco) (84). In the B. subtilis group, however,the predominance of anteiso-C15 is severely af-fected by use of an amino acid-rich medium, inwhich case the proportion of the iso-C15 acidapproaches that of the anteiso-C15 acid. How-ever, B. polymyxa and three species of insectpathogens, B. larvae, B. lentimorbus, and B.popilliae, continue to synthesize anteiso-C15acid in extremely high proportions even whenan amino acid-rich medium is used (24, 82, 85).Although the occurrence of unsaturated fatty

acids in species ofBacillus is rare, some excep-tions are observed. Of the three psychrophilicspecies studies, all synthesize A5-monounsatu-rated fatty acids as major products (18 to 25% ofthe total) (83).

Figure 6 illustrates a summary of groupingsbased on fatty acid patterns of the genus Bacil-lus. Further subdivision is difficult. In a fewcases, however, this seems to be possible. Ingroup B, B. lentimorbus is distinguished fromB. larvae and B. popilliae (85). Similarly, thethree species in group E are distinguished fromone another (84). One exception is B. acidocal-

Unsaturated fatty acids

darius (group D), which has a unique fatty acidpattern distinguishable from any other speciesofBacillus. This grouping would be a useful aidin the taxonomy of genus Bacillus.The situation just described for the genus

Bacillus is in interesting contrast to variousgram-negative asporulating organisms such ascorroding bacilli and Bacteroides (72, 136),where even in the absence of carefully con-trolled growth conditions, specific fatty acidpatterns seem to be obtained for individualorganisms. This success is largely due to thefact that these sets are composed of two typesof bacteria, one with straight-chain fatty acidsynthetase and the other with branched-chainfatty acid synthetase. In many of these cases,the individual fatty acids, particularly branched-chain acids, have not yet been fully identified,but once this is done, the usefulness of thetechnique as a taxonomic tool will be greatlyincreased, because then the fatty acid profilescan be correlated with the specific biochemicalactivities of the organisms.

GENERAL BACTERIAL TAXONOMY ANDFATTY ACID TYPES

Gram-Positive BacteriaIn species ofBacillus (82, 84, 85, 89), Coryne-

bacterium (116), Listeria (29), Micrococcus(151), Nocardia (153), Propionibacterium (118),Sarcina (4), Staphylococcus (165), and Strepto-myces (63), which are all gram positive, thereoccurs a distinctive fatty acid pattern having apredominance of iso and anteiso fatty acids.Although the number of species examined in a

Range ofchainlength

Species

-+ anteiso-C,4 acid and(26-60%)

i80-C,5 acid (13-30%)

-4 Insignificant or very small anteiso-C,5 acid(<3%) (39462%)

Small proportion(7-12%)

-) Large proportion(17-28%)

-;iso-C,5 acid

-+ Cyclohexyl acids(59%)

0 iso-C,, acid(19-31%)

-o anteiso-C,5 acid

14-17 B. aluei, B. bevisA B. cimcuzdma, B. limniformnsB.mow, B. megariwn

B. pumilus, B. subtil

14-17 B B. I , B. lareB. lentfnobus, B. popiliae

14-17 C {B. abldcus, B.coioteralB. H

17-19 D B. wacidorza ius

12-17 E B. andtucis, B. mreusLB. thingienss

14-17 F B. id , B. ydsmozphilusB. globiaporw

FIG. 6. Grouping ofBacillus species based on fatty acid patterns. See Table 3 for references.

Predominant fatty acids

Bacillus

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given genus is rather limited, with the excep-tion of the genus Bacillus, the pattern seems tobe uniform within these genera.

Gram-Negative BacteriaAt one time, the predominant occurrence of

iso and anteiso fatty acids was considered to belimited to gram-positive bacteria (95). How-ever, further reports have shown that this isnot so, and some gram-negative bacteria,though not many, also exhibit this fatty acidpattern. An important difference betweengram-negative and gram-positive bacteria isthat among the former, the acid pattern varies.In Bacteroides, fatty acids ofB. ruminicola (99)and B. melaninogenicus (137) consist mainly ofbranched-chain fatty acids, presumably iso andanteiso series, whereas those ofB. amylophilusand B. succinogenes (J. E. Kunsman, Jr.,Ph.D. thesis, University of Maryland, CollegePark, 1966) are exclusively straight chain.Three strains ofRhizobium (53, 54), whose pre-dominant acids are centrally methylated orstraight chain with or without unsaturation,may synthesize up to 30% of iso and anteisofatty acids. Fatty acids of another Rhizobium,R. japonicum, are largely myristic, palmitic,octadecanoic, and Cjrcyclopropane acids (26).This complexity probably reflects a close associ-ation of these organisms with their respectivehosts and possibly an exchange of enzymes,related to these fatty acids, between them. InPsuedomonas, fatty acids ofP. maltophilia arepredominantly branched chain (iso series),whereas those of P. aeruginosa and P. cepaciaare all straight chain (117). Fatty acids ofMyxococcus xanthus are mostly branched (70%)(162).

EVOLUTION OF FATTY ACID SYSTEMSAlthough the amount of data on fatty acid

compositions of living organisms that has beenaccumulated is large and rapidly growinglarger, considering the number of life formsthat exist on earth, it is far from complete.Nevertheless, the available data are sufficientfor a general view of the distribution in natureof organisms with lipid systems based primar-ily on branched-chain fatty acids.The majority of such organisms are the bacte-

ria that have 1been already mentioned in thepreceding section. Thus, the lipid systemsbased on branched-chain fatty acids are a char-acteristic of procaryotes. Eucaryotes do nothave such lipid systems, with the exception of afew fungi (152).

Convincing evidence exists to support the be-lief that unicellular, microscopic, anaerobic

procaryotes were among the earliest life formsto inhabit the earth (142). A critical step in theemergence of such an organism must be theformation of a membrane boundary, for only inthis way does the primitive organism isolateitself from the open environment. Although ourunderstanding of the early stages of micro-scopic life must remain highly speculative, it ismost likely that fatty acids, as lipid compo-nents, played a major part in the importantstep of forming membranes (134). Here, theevolutionary change of microbial fatty acidcomposition is considered. Special attention isgiven to the possible importance of branched-chain fatty acids in an early stage of evolution.The fatty acid composition of present-day life

varies widely. However, the general absence ofpolyunsaturated fatty acids in procaryotesclearly differentiates these from eucaryotes. Al-though the fatty acid composition of procar-yotes is itself variable, myristic, palmitic, andstearic acids always occur and may or may notbe supplemented by monounsaturated acids,iso acids, anteiso acids, cyclopropane acids, andhydroxy acids (95, 145).A proposed scheme of grouping organisms on

the basis ofthe biochemical origin ofthese fattyacid components is shown in Table 8. A distinc-tive feature of this scheme is the inclusion of asystem based on branched-chain fatty acids sep-arate from the straight-chain systems.Each synthetic system is composed of a de

novo synthetase with or without a modifyingenzyme. In some cases, a secondary modifyingenzyme, such as the cyclopropane-forming en-zyme, which is probably nonessential, partici-pates to modify the fatty acids synthesized bythe de novo synthetase.The de novo synthetase of type 1 differs sig-

nificantly from that of types 2 and 3. In type 1,the chain initiators are derivatives of fattyacids, usually branched, having four or fivecarbon atoms, whereas in the others the chaininitiators are acetic acid derivatives. Lipid sys-tems of type 1, "branched-acid type," as seen inthe genus Bacillus, do not require unsaturatedacids, although such acids do occur in some ofthese organisms. On the other hand, lipid sys-tems of type 2, "anaerobic type," must includeunsaturated fatty acids in order to functionproperly in supporting growth. Monounsatu-rated fatty acid-requiring mutants can be in-duced with organisms having this lipid type.Type 3, "aerobic type," is very similar to type 2,but unsaturated fatty acids are synthesized byan aerobic pathway (18, 45, 50). These threebasic lipid types represent essentially all thefatty acid patterns found in presently knownprocaryotes.

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TABLE 8. Proposed three types of lipid systems occurring in bacteria

Enzyme systems involved Characteristics

Lipid type De novo synthe- Essential modify- Natural chain ini- ratedacid Oxygen re- Bacteriatase ing enzyme tiator require- quirement

mentType 1 Branched-chain None Branched C4 and No No Bacilus subtilis

(branched) fatty acid syn- C, substrates Bacteroides rum-thetase inicola

Type 2 (anaero- Palmitic acid syn- Anaerobic unsat- C, substrate Yes No Clostridium butyr-bic) thetase urated acid icum

synthetase Escherichia coliType 3 (aero- Palmitic acid syn- Aerobic desatur- C, substrate Yes Yes Pseudomonas

bic) thetase ating enzyme aeruginosa

Branched C,, acids and unsaturated acids, asdiscussed in the section on function, have spe-cial functions, and the amount of these acidsmust be controlled to meet physiological re-quirements under given growth conditions.Type 1 includes a synthetase only, and thecomposition of fatty acids synthesized is largelydependent upon the relative availability ofbranched-chain initiators in the environment.Type 2 includes a synthetase and an anaerobicunsaturated acid-synthesizing system, al-though, as seen in the case ofE. coli, they sharemost of the enzymes involved (22). Thus thephysiologically important ratio of saturated tounsaturated acids can be altered rather easilyby controlling the activity of specific enzymes ofthe unsaturated acid-synthesizing system.Hence, type 2 is a more advanced system thantype 1. In the same sense, type 3 is the mostadvanced of all three because the synthesis ofunsaturated acids is separated, and controlledindependently, from that of saturated acids.The significance of this order of development

may be seen by considering the conditions ofprimitive Earth. It is believed that before theemergence of life, the atmosphere exhibited acomplete lack of molecular oxygen. Certain es-sential materials were present from nonbiologi-cal sources. The abiogenesis of important cellu-lar materials such as amino acids, purines, py-rimidines, carbohydrates, and lipids has beenwell established. Some premembranes or mem-brane-like materials were available to, andprobably utilized by, the first organisms (101,144).A drastic change in environmental condi-

tions occurred when oxygen-producing photo-synthetic organisms emerged and began intro-ducing oxygen into the atmosphere, therebyinducing the appearance of aerobic energy-yielding systems (43) and the appearance ofeucaryotes (61, 107).

Considering the procaryotic lipid types inthis light, the simplicity of type 1 suggests that

it may well be placed as the transitional phasebetween the primitive abiotic condition and abiologically controlled lipid system. The neces-sary precursors of synthesis, the branchedshort-chain acids, would be available abiologi-cally, and the preferential formation of theseacids from methane, a component of the pre-sumed primitve atmosphere, has been demon-strated (6).Type 2 represents a more advanced system,

probably developing from type 1. Type 3 makesits appearance at about the time molecular oxy-gen was becoming available, and represents afurther advance toward the emergence of theeucaryotic lipid (polyenoic) type. This proposedscheme is illustrated in Fig. 7.

It would be highly desirable to obtain directevidence supporting this scheme by determin-ing the composition of the fatty acid in lipids ofPrecambrian fossils. There are three knowngeological formations in the world preservingthe fossils of primitive microorganisms (141).Unfortunately, lipids are generally unstableduring long geological periods, and the quan-tity of the fossil sample available is too small toprovide sufficient material for analysis by thetechniques presently available. A vast im-provement in sensitivity of analytical devices isessential, but, considering the advances thathave taken place in the past decade or so, is nottotally to be ruled out.

CONCLUDING REMARKSThe occurrence of iso and anteiso fatty acids

in microorganisms is not as rare as once hadbeen believed; they are major components (60 to98%) of the fatty acids found within certaingenera. This pattern of occurrence has impor-tant implications in taxonomy and in establish-ing an evolutionary scheme for microorga-nisms.

In microorganisms, fatty acids occur mostlyin the form of phospholipids, which are mainlylocated on cell membranes. Branched-chain

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IEUCR YOTES

1.S xl10 YEARSAGO

AEROBICS

AND

ANAEROBICS

PROCARYOTES

H \SYSTEM

ANAEROBICS AEROBICS

AI

AERO31CS

SYSEM

N M

3.3 .x0 YEARS AGO

FIG. 7. Proposed evolutionary scheme of fatty acid system.

fatty acids play important roles in phospholipidsynthesis and in the functions ofmembranes.

Branched-chain fatty acids are rare in an-

imals, but a significant role of cholesterol esterof anteiso-C17 acid in peptide biosynthesis sug-

gests that the importance of these acids in an-

imals probably has not been fully appraised.Study of this whole area is still very incom-

plete, and further exploration is essential inorder to understand fully the biological func-tions of branched-chain fatty acids.

ACKNOWLEDGMENTSThis review was supported in part by grant MT-

1660 of the Medical Research Council, Ottawa, Can-ada.

I thank F. L. Jackson and R. N. McElhaney forcritical review of this manuscript and R. M. Elofson,Thelma Habgood, and H. W. Habgood for helpfuldiscussion during the course of our work outlined inthis review.

LITERATURE CITED1. Abrahamsson, S., S. Stallberg-Stenhagen, and

E. Stenhagen. 1960. The higher saturatedbranched-chain fatty acids, p. 35-58. In R. T.Holman and T. Malkin (ed.), Progress in thechemistry of fats and other lipids, vol. 7.Pergamon Press, New York.

2. Ackman, R. G. 1972. The analysis of fatty acids

and related materials by gas-liquid chroma-tography, p. 167-284. In R. T. Holman (ed.),Progress in the chemistry of fats and otherlipids, vol. 12. Pergamon Press, Oxford, NewYork, Toronto, Sydney, and Braunschweig.

3. Ailhaud, G. P., and P. R. Vagelos. 1966. Pal-mityl-acyl carrier protein as acyl donor forcomplex lipid biosynthesis in Escherichiacoli. J. Biol. Chem. 241:3866-3869.

4. Akashi, S., and K. Saito. 1960. A branchedsaturated C15 acid (sarcinic acid) from Sar-cina phospholipids and a similar acid fromseveral microbial lipids. J. Biochem. 47:222-229.

5. Alexander, G., G. Garz6, and G. Palyi. 1974.Method for preparing glass capillary col-umns for gas chromatography. J. Chroma-togr. 91:25-37.

6. Allen, W. V., and C. Ponnamperuma. 1967. Apossible prebiotic synthesis of monocarbox-ylic acids. Curr. Mod. Biol. 1:24-28.

7. Allison, M. J., and M. P. Bryant. 1963. Biosyn-thesis of branched-chain amino acids frombranched-chain fatty acids by rumen bacte-ria. Arch. Biochem. Biophys. 101:269-277.

8. Andersson, B. A., and R. T. Holman. 1974.Pyrrolidides for mass spectrometric determi-nation of the position of the double bond inmonounsaturated fatty acids. Lipids 9:185-190.

9. Asselineau, C., H. Montrozier, and J. C.Prome. 1969. Presence d'acides polyinsa-

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tur6s dans une bact6rie: isolement, a partirdes lipides de Mycobacterium phlei, d'acidehexatriacontapenta6ne-4, 8, 12, 16, 20 o'iqueet d'acides analogues. Eur. J. Biochem.10:580-584.

10. Asselineau, J., and E. Lederer. 1960. Chemis-try and metabolism of bacterial lipides, p.337-406. In K. Bloch (ed.), Lipide metabo-lism. John Wiley & Sons, Inc., New Yorkand London.

11. Bandyapadhyay, G. K., and J. Dutta. 1975.Separation of methyl esters of polyunsatu-rated fatty acids by argentation thin-layerchromatography. J. Chromatogr. 114:280-282.

12. Barnes, E. M., Jr., and S. J. Wakil. 1968. Stud-ies on the mechanism of fatty acid synthesis.XIX. Preparation and general properties ofpalmityl thioesterase. J. Biol. Chem.243:2955-2962.

13. Bauman, A. J., and P. G. Simmonds. 1969.Fatty acids and polar lipids of extremelythermophilic filamentous bacterial massesfrom two Yellowstone hot springs. J. Bacte-riol. 98:528-531.

14. Beebe, J. L. 1971. Isolation and characteriza-tion of a phosphatidylethanolamine-deficientmutant of Bacillus subtilis. J. Bacteriol.107:704-711.

15. Bertsch, L. L., P. P. M. Bonsen, and A. Korn-berg. 1969. Biochemical studies of bacterialsporulation and germination. XIV. Phospho-lipids in Bacillus megaterium. J. Bacteriol.98:75-81.

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