JOURNAL OF BACTERIOLOGYVol. 88, No. 2, p. 425-432 Augu8t, 1964Copyright @ 1964 American Society for Microbiology

Printed in U.S.A.

LIPIDS OF SARCINA LUTEAI. FATTY ACID COMPOSITION OF THE EXTRACTABLE LIPIDS

CHARLES K. HUSTON AND PHILLIP W. ALBRO

Physical Defense Division, U.S. Army Biological Laboratories, Fort Detrick, Frederick, Marylandl

Received for publication 13 April 1964

ABSTRACT

HUSTON, CHARLES K. (U.S. Army BiologicalLaboratories, Fort Detrick, Frederick, Md.), ANDPHILLIP W. ALBRO. Lipids of Sarcina lutea. 1.Fatty acid composition of the extractable lipids.J. Bacteriol. 88:425-432. 1964.-The extractablelipids of Sarcina lutea were separated into severalfractions by a combination of column and thin-layer chromatography. Qualitative and quantita-tive characterization of the fatty acid content ofthese lipid fractions was accomplished by meansof gas-liquid chromatography and infrared anal-yses. Of the total extract, the lipids consisted of2.1%o free fatty acids, 51.0% glycerides, and 22.7%ocomplex lipids; they had a fatty acid content witha complete spectrum of carbon numbers from C8to C22 . The fatty acids included a large componentof branched-acids in addition to the normalstraight-chain acids. The branched-acids, com-prising 40% of the fatty acids analyzed, consti-tuted a homologous series of iso-acids from C12to C19. Two 18-carbon unsaturates were foundcis-9-octadecenoate and cis-11-octadecenoate. Arelatively high percentage (20.5%) of the extracta-ble material from S. lutea was found to be hydro-carbon. This material was not further character-ized.

Bacterial lipids, in general, differ substantiallyfrom those of higher life forms in such respectsas the absence of sterols, phospholipids low innitrogen and high in carbohydrate, the presenceof large proportions of free fatty acids, and thepresence of certain fatty acids not ordinarilyfound in other life forms. The presence of theseunusual fatty acids and their possible relation tobacterial pathogenicity has stimulated much re-search.A review of the literature on bacterial fatty

acids was made by Porter (1946) and more re-cently by Asselineau and Lederer (1960) and byO'Leary (1962). Although investigators havereported the fatty acid composition of some bac-terial lipid fractions (Gray, 1962; Macleod and

Brown, 1963), little information is available onthe distribution of fatty acids among specificbacterial lipid classes.

Sarcina lutea, a gram-positive, aerobic (facul-tatively anaerobic), nonmotile, pigment-produc-ing micrococcus, has been found in air, soil, andwater all over the earth (Gregory, 1961). Theonly reported analysis of the lipids of Sarcina isthat by Akashi and Saito (1960), who studiedthe fatty acid composition of the acetone-solubleand insoluble fractions.

This communication reports on the distribu-tion of fatty acids among the various generallipid classes in the extractable lipids of S. lutea.For the purposes of this study, onlyr the C8 toC22 fatty acids are discussed.

MATERIALS AND METHODS

Culture conditions. S. lutea (ATCC 533) wascultured at 25 C for 48 hr in continuously aeratedTrypticase Soy Broth (pH 7) found to containless than 0.02% lipid. The cells were harvestedby centrifugation, washed free of medium with0.1 M aqueous KCl solution, and lyophilized toconstant weight.

Extraction. Lyophilized cells (10 g) were shakenat room temperature with 150-ml portions ofsolvent according to the extraction scheme out-lined in Fig. 1. Extracts 1 through 4 were com-bined and evaporated to dryness in vacuo; theresidue was taken up in chloroform-methanol, 2 :1(v/v). This solution was washed according to themethod of Folch, Lees, and Sloan-Stanley (1957)to remove nonlipid contaminants. The chloro-form portion was evaporated to dryness in vacuoand weighed.The remaining cell residue was examined for

nonextractable lipids by subjecting it to a 2-hrreflux with 2 N aqueous KOH. The resulting ma-terial was acidified, and a chloroform extract ofthis was examined separately.

Shaking the cells first with acetone permitted425

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Lyophilized Cells*I+ Acetone

(1 hr)

Extract 1

Residue

+ CHCl3-CH30H2: 1 (v/v)

(2 hr)I~~~~~~~~~~~~~~~~~~~~~

Residue

+ CHC13-CH30H1:1 (v/v)

(1 hr)

Residue+ 2 N aqueous KOH

reflux(2 hr)

Residue

+ CHCl3-CH30H2:1 (v/v)

(2 hr)I

Extract 2

Extract 3

Extract 4

Solution

+ Acid

+ CHC13

Water-solublepart

CHCl3-solublepart

FIG. 1. Sarcina lutea extraction scheme. Extracts 1 through 4 were combined, washed, and examined as

the "total extract." The chloroform-soluble part of the base-hydrolyzed residue was examined as the "basehydrolysate."

a more rapid and complete extraction by theother solvent systems. All extractions were car-

ried out at room temperature to prevent altera-tions to complex lipid structures (Marinetti,1962), and as rapidly as possible to avoid pro-

longed exposure to methanol (Lough, Felinski,and Garton, 1962).

Spot tests with Rhodamine 6G indicated thatno further lipid was extracted with chloroform-

methanol (2:1, v/v), after two shakings. Thefinal extraction with chloroform-methanol (1:1,v/v), did, however, yield a small additionalquantity of lipid as well as appreciable amountsof nonlipid.

Silicic acid column chromatography. The ex-

tracted lipids were separated according to classand purified on three columns prepared as fol-lows. Column I consisted of a glass tube (1.7 by

Residue

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150 cm) packed to a depth of 120 cm with ahexane slurry of activated Mallinckrodt silicicacid (30 g, 100/120 mesh) and Hyflo Super-Cel(15 g). The column was washed with 100-ml por-tions of acetone, ethyl ether, and n-hexane, inthat order. Columns II and III consisted of glasstubes (0.8 by 30 cm). Column II was packedwith 15 g of 160/200 mesh activated silicic acidand column III with 15 g of 100/140 mesh acti-vated silicic acid. Both columns II and III werewashed with 50-ml portions of acetone, ethylether, and n-hexane, and, in addition, column IIIreceived a final wash with 50 ml of chloroform.The lipid extract was separated into general

classes on column I by using a flow rate of 8ml/min. A crude hydrocarbon fraction was elutedwith 150 ml of n-hexane-benzene (49:1, v/v).The simple lipids were eluted next with 300 mlof chloroform, but the complex lipids required300 ml of chloroform-methanol (1:1, v/v), fol-lowed by 100 ml of methanol, for their completeremoval from the column. Each fraction wasevaporated to dryness in vacuo, weighed, andredissolved in chloroform pending further treat-ment. Overall recovery of lipid from this andsucceeding columns averaged 99.5%.The crude hydrocarbon fraction was further

resolved on column II. A pure hydrocarbonfraction was eluted with 100 ml of n-hexane.Nonhydrocarbon carry-over was then elutedwith 50 ml of ethyl ether and added to the simplelipid fraction.

Simple lipid impurities were separated fromthe complex lipids on column III by eluting themwith 125 ml of chloroform. This eluted materialwas added to the simple lipid fraction. The com-plex lipids were finally eluted with 200 ml ofchloroform-methanol (1:2, v/v). The combinedsimple lipid fraction was further fractionatedinto a free fatty acid fraction and a glyceridefraction on a KOH-treated silicic acid column,as described by McCarthy and Duthie (1962).

Thin-layer chromatography (TLC). The variouslipid fractions were monitored on thin layers(250 to 275 ,u) of Silica Gel G (Stahl) on standardglass plates (20 by 20 cm). All plates were spreadwith a Desaga-Brinkman nonadjustable appli-cator, activated for 2 hr at 110 C, and developedby the ascending method in unlined tanks.

All column cuts were examined for lipid classesby developing the plates in n-hexane-ethyl ether-glacial acetic acid (90:10:1, v/v/v; Malins and

Mangold, 1960). The hydrocarbon fraction fromcolumn II was developed in n-hexane-benzene(90:10, v/v). The complex lipids from column IIwere resolved in a system of chloroform-metha-nol-4.3 N ammonium hydroxide (17:7: 1, v/v/v).The simple lipid fraction was resolved into glyc-eride types by development according to Brownand Johnston (1962).Methyl ester preparations were examined for

the presence of hydroxyl- or epoxy-esters bychromatographing them on Silica Gel G impreg-nated with silver nitrate (Morris, 1962). Themethyl esters were also resolved into saturatedand unsaturated fractions by preparative TLCof the acetoxymercuri-methoxy derivatives (Man-gold and Kammereck, 1961). Two fractions ofthese derivatives were eluted from the Silica Geland hydrolyzed to the original esters for furtheranalysis.

Spots on the variously developed plates weremade visible with (i) 0.001% aqueous Rhodamine6G, (ii) unsaturated chromic-sulfuric acid withcharring, (iii) iodine vapors, (iv) 0.4% ninhydrinin water-saturated n-butanol, and (v) a molyb-date reagent (Skidmore and Entenman, 1962).

Gas-liquid chromatography (GLC). Fatty acidmethyl esters were prepared for GLC by refluxingthe lipid material for 2 hr in methanol containing0.5% (by weight) concentrated sulfuric acid and5% (by weight) 2,2-dimethoxypropane as a waterscavenger. After stopping the reaction with water,the esters were extracted with gas-chromato-graphically pure n-hexane.

All chromatograms were obtained with anF & M model 500 gas chromatograph with amodel 1609 flame ionization detector (F & MScientific Corp., Avondale, Pa.). Helium, at anoutlet flow rate of 88 ml/min, was used as thecarrier gas, and all columns were of coiled coppertubing (outer diameter, 0.25 in.). The followingcolumn packings and conditions were used: (i)silicone gum rubber (GE SE-30), 5% on 100/120mesh gas chrom P; the column (2 ft) was tem-perature-programmed from 150 to 288 C at 15 Cper min; (ii) Apiezon L (ApL), 10% on 50/60mesh Anakrom ABS; the column (4 ft) was main-tained isothermally at 210 C; (iii) ethylene glycolsuccinate (EGS), 10% on 120/140 mesh gaschrom P; the column (8 ft) was maintained iso-thermally at 150 or 180 C; (iv) ethylene glycolglutarate (EGGlu), 10% on 70/80 mesh AnakromABS; the column (8 ft) was maintained isother-

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mally at 180 C; (v) cyclohexanedimethanol suc-cinate (CMDS), 11% on 80/100 mesh Chromo-sorb W; the column (8 ft) was maintainedisothermally at 185 C.

Infrared analysis. Infrared spectra of the vari-ous lipid fractions and methyl ester samples weremade from thin films on KBr pellets by use of arecording infrared spectrophotometer (The Per-kin-Elmer Corp., Norwalk, Conn.; model 21).

RESuLTS

Total lipid composition. Lyophilized cells of S.lutea were found to contain on the average 1.30%solvent-extractable lipid. An additional 0.25%lipid material could be obtained by base hy-drolysis of the cell residue.The extractable material fractionated on the

TABLE 1. Fatty acid composition ofSarcina lutea lipids

RelativeComposition

Fatty acid carbon Relativechain* rtientionydolchantime Total extract ysate

8:0-10:0 0.06+ 5.2 36.5B 0.14 2.2 5.4

12:0 0.19 8.1 7.2B 0.21 2.6 0.4

13:0 0.24 1.8 3.013:1 0.27 - 0.2

BR-14:Ot 0.30 0.9 -14:0 0.33 5.2 1.1C 0.37 26.4 19.1

15:0 0.42 1.5 TracetBR-16:0 0.50 3.2 0.9

16:0 0.60 10.1 8.0D 0.67 0.9 -

16:1 0.70 2.7 0.916:2 0.83 - 0.2E 0.93 2.6 5.6

18:0 1.00 5.7 5.0F 1.18 4.0 0.2

19:0 1.32 2.5 Trace18:2 1.50 - 0.220:0 1.74 1.9 0.221:1 2.29 2.622:0 3.00 0.9 0.220:4 3.40 3.4 0.1

* Number of carbon atomsdouble bonds.

t BR = branched.

t Trace = less than 0.1%.

in acid:number of

TABLE 2. Fatty acid composition of major lipidclasses in Sarcina lutea

CompositionFatty acid carbon chain'

Simple lipidsb Complex lipids

8:0-10:0 8.6 1.6A 5.4 Tracec

12:0 9.4 3.5B 2.8 2.1

13:0 5.2 1.413:1 -

BR-14:0d 0.8 1.014:0 2.4 6.1C 13.0 33.9

15:0 2.8 TraceBR-16:0 2.6 3.7

16:0 8.8 12.3D 0.4 1.3

16:1 0.7 3.116:2E 2.8 1.0

18:0 5.4 9.1F 2.6 8.5

19:0 4.4 1.618:2 -20:0 1.2 2.121:1 2.6 2.722:0 9.0 2.720:4 4.0

a Number of carbon atoms in acid:number ofdouble bonds.

b Free fatty acids plus glycerides.c Trace = less than 0.1%.d BR = branched.

silicic acid columns was fQund to consist of 20.5%hydrocarbon material, 53.1% simple lipid (2.1%free fatty acids and 51.0% glycerides), and 22.7%complex lipid.TLC indicated the presence of hydrocarbons,

free fatty acids, mono-, di-, and triglycerides, anda complex mixture of highly polar phosphorus-containing compounds. The complex lipids con-tained both ninhydrin-positive and -negativecomponents. There was no evidence for the pres-ence of fatty alcohols, sterols, sterol esters, waxes,or sphingomyelins.

Fatty acids. Tables 1, 2, and 3 list the fattyacid composition as determined for the variouslipids of S. lutea. Peak areas, determined with aplanimeter, were obtained from chromatogramsusing the EGS column at 180 C. The area per

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cents are approximately equivalent to weightper cent for the range covered (Ettre and Kabot,1963). Although the EGS column did not resolveas many components as did the EGGlu column,the clean separation of those components thatwere resolved on EGS made quantitation lessarbitrary.The methyl ester peaks were identified on the

basis of their relative retention times as com-

pared with methyl stearate, their equivalentchain lengths from plots of carbon number versus

retention time for n-saturated methyl ester stand-ards, and their separation factors from n-satu-rates. In some cases, additional confirmation ofidentity was obtained from infrared spectra ofparticular components trapped from the columneffluent ahead of the detector. Monounsaturateswere distinguished from branched-chain satu-

TABLE 3. Fatty acid composition of Sarcinalutea simple lipids

CompositionFatty acid carbon chain*

Free fatty acids Glycerides

8:0-10:0 2.0 9.2A 0.3 5.9

12:0 1.5 10.0B 0.8 3.0

13:0 0.2 5.613:1 _

BR-14:Ot 0.1 0.914:0 14.9 2.3C 2.2 14.0

15:0 3.4 2.9BR-16:0 1.2 2.8

16:0 32.5 8.6D 26.0 Tracet

16:1 3.7 Trace16:2E 1.8 3.0

18:0 2.1 5.8F 0.9 2.8

19:0 0.1 4.718:220:0 0.7 1.321:1 0.5 2.822:0 0.2 9.720:4 1.0 4.3

* Number of carbon atoms in acid:number ofdouble bonds.

t BR = branched.t Trace - less than 0.1%.

o 2 4 6 8 10 12 14 16 II 20 2 24 26 2830 32 34 36

TIME {*UN)

FIG. 2. Gas-liquid chromatography on an ethyleneglycol succinate column at 180 C of the methyl estersof the fatty acids from Sarcina lutea. Chromatog-raphy conditions are described in Materials andMethods. The initial off-scale band is from the sol-vent. Designations used range from A to F, and cor-respond to the letters listed in Tables 1 to 3 and de-scribed in Results; whole numbers refer to methylesters of n-saturated acids of the same carbon num-ber; 16:1, methyl ester of monounsaturated C16 acid;BR-14 and BR-16, methyl esters of branched C14 andC16 acids. Peak C is shown at a 4X attenuation tokeep it on scale.

rates emerging at the same point from polyestercolumns by noting their reversed order on ApLand by comparing their degree of separation fromn-saturates on EGS at two different temperatures(Landowne and Lipsky, 1961). Figure 2 illustratesthe separation obtained on EGS at 180 C. Satu-rates in general were distinguished from unsatu-rates by comparison of the chromatograms ofthe two fractions obtained by TLC of the acetoxy-mercuri-methoxy derivatives. The position ofmethyl branches and the location and configura-tion of double bonds could, in some cases, betentatively determined from relative retentiontimes on EGGlu. An example of the separationpossible on EGGlu is shown in Fig. 3. Whenpossible, such identifications were verified bychromatographing known standards of the sus-pected ester constituents.Chromatograms obtained with the SE-30 col-

umn indicated that the complete spectrum ofcarbon numbers from C8 to C22 was present inthe various lipid fractions. The CMDS columnindicated that the bulk of the C8 to C10 acidsfrom the base hydrolysate were C8 acids.

Peaks A to F in the tables were found, withthe exception of peak D, to be resolved into morethan a single component on EGGlu. On the basisof the previously mentioned retention character-

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i6sisI

12 143 61-

0 2 4 6 * 10 12 14 16 1 20 22 24 26 2S

lIME(MIN)

FIG. 3. Gas-liquid chromatography on an ethyleneglycol glutarate column of the methyl esters of thefatty acids from Sarcina lutea. Chromatographyconditions are described in Materials and Methods.The initial off-scale band is from the solvent. Wholenumbers refer to methyl esters of n-saturated acids ofthe same carbon number; 12:1 and 16:1, methyl estersof monounsaturated C12 and C16 acids; CIS-9-18:1and CIS-11-18:1, methyl esters of cis-9-octadecenoicacid and cis-11-octadecenoic acid; BR-, methylesters of iso-branched acids of the same carbon num-ber; BR-161, methyl ester of anteiso-branched C15acid. The peak containing BR-1S, BR-151 and 15 isshown at a 4X attenuation to keep it on scale.

istics applied to the EGGlu column, tentativeidentifications of these peaks were as follows."A" was composed of n-hendecanoate and

10-methylhendecanoate, with the latter as themajor component."B" consisted of dodecenoate and 11-methyl-

dodecanoate, with the branched-acid again themnajor component.

"C," containing by far the largest percentage ofthe esters in the total extract, was resolved intothree components corresponding to tetradecen-oate and two branched-acids. The branched-acidsmade up the majority of "C" and correspondedto 13-methyltetradecanoate and 12-methyltet-radecanoate (Hawke, Hansen, and Shorland,1959). An infrared spectrum of this fraction in-dicated the presence of both an iso- and anteiso-configuration (Kaneda, 1963)."D" corresponded to 15-methylhexadecanoate

and neoheptadecanoate (Ackman, Burgher, andJangaard, 1963). An infrared spectrum indicatedthe presence of both the iso- and tertiary-con-figurations in this fraction."E" corresponded to a mixture of heptadeceno-

ate, 16-methylheptadecanoate, and 9,10-methyl-enehexadecanoate (Gray, 1962). An infraredspectrum of "E" indicated the presence of a cis-

configuration but no cyclopropane absorption(Kaneshiro and Marr, 1961)."F" was another triplet containing 9-methyl-

octadecanoate (Cornelius and Shone, 1963), cis-9-octadecenoate (oleate), and cis-11-octadecenoate(cis-vaccenate). Had lactobacillate (cis-11,12-methylene octadecanoate) been present, it wouldhave emerged with the branched member of thistriplet. However, no cyclopropane absorptioncould be observed in the infrared spectrum ofthis fraction.

Evidence exists for the inclusion of two ho-mologous series of branched-acids besides theiso-series, although the exact nature of thesetrace components has not been established.Moreover, the ApL column indicated that, inopposition to what has been found for otherbacteria, n-hexadecanoic acid constitutes only asmall percentage of the fatty acid content of S.lutea. The bulk of the material designated 16:0in Table 1 eluted slightly before authentic hexa-decanoate.

DISCUSSION

In S. lutea, the largest class of lipids was foundto be the simple lipids, consisting of free fattyacids and glycerides. The cell content of freefatty acids is usually higher in bacteria than inmost other life forms. A number of bacteria havebeen reported to contain free fatty acids com-prising more than 20% of the fatty acid content(Asselineau and Lederer, 1960). Although mono-,di-, and triglycerides have been reported in manybacteria (Porter, 1946; Pennell, 1950), theirconcentrations are usually lower than in plantand animal cells. In fact, many bacteria report-edly lack glycerides (Asselineau and Lederer,1960). S. lutea, on the other hand, was found tocontain lipids with only 2.1% free fatty acidsand 51.0% glycerides. Recent studies (Hanahan,1960) suggest that reports of high free fatty acidcontents in lipid material may be due to lipolysisduring extraction and chromatography on silicicacid. As ether is known to activate certain lipo-lytic enzymes (Hanahan, 1952), the use of thissolvent for extraction or column chromatographywas avoided in this study. Moreover, ketones areknown to inhibit the action of certain lipases(Weinstein and Wynne, 1935). Thus, the use ofacetone as the initial extracting solvent mayhave prevented the lipolysis of S. lutea lipids.The complex lipids were not characterized for

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this study except for a gross determination oftheir fatty acid content. The fact that severalninhydrin-positive and -negative componentswere detected by TLC indicates that this fractionis justifiably termed "complex."An interesting point is the relatively high per-

centage of hydrocarbon material (20.5%) ex-tracted with the lipids. Since S. lutea has a yellowpigment, it is not unreasonable to expect thathydrocarbons might be present in the extract.,y-Carotene was reported in several bacterialspecies (Chargaff, 1934; Chargaff and Lederer,1935; Takeda and Ohta, 1940), although fewquantitative data are available. Further com-ment will have to await a more complete char-acterization of this material.The major group of fatty acids found in S.

lutea was the branched-acids, comprising over40% (ca.) of the C8 to C22 acids. With the excep-tion of two, all of the branched-acids present inamounts greater than 0.1% belonged to a ho-mologous series of iso-acids. The complete seriesof iso-acids from C12 to C01 was observed. Thetwo exceptions were 12-methyltetradecanoate and9-methyloctadecanoate. Akashi and Saito (1960)reported a branched-acid (C15) in the lipids ofSarcina, which they named sarcinic acid. Saito(1960a, b), in subsequent studies, reported theisolation and identification of 13-methyltetra-decanoic acid and 15-methylhexadecanoic acidfrom Bacillus subtilis, but did not report whetherthe C15 acid from B. subtilis was identical withsarcinic acid. Asselineau (1961) also reportedfinding two branched-acids in B. subtilis, oneC,5 and the other C16. In the same paper thereis mention of a C19 branched-acid that was alsofound in Pasteurella pestis. Kates, Kushner, andJames (1962) reported that B. cereus contained ahomologous series of branched-acids from C12 toC18, but did not report the position of the branch.They reported also that the major fatty acid ofthis organism was a branched C15 acid. Agre andCason (1959) reported a series of branched-acidsfrom C,5 to Cig in tubercle bacillus.The relatively low unsaturated fatty acid con-

tent was concentrated mainly in the C16 and C18portions of the chromatograms. The hexadeceno-ate corresponds to cis-9-hexadecenoate. Both thisacid and cis-11-hexadecenoate were found inbacteria (Hofmann and Tausig, 1955a; Law,1961). Oleic acid was reported in streptococci(Hofmann and Tausig, 1955a) and the tubercle

bacillus (Hofmann and Tausig, 1955b), whereascis-11-octadecenoate was reported in severalmicroorganisms (Hofmann and Tausig, 1955a;Law, 1961; Cason and Tavs, 1959; Hofmannand Sax, 1953). Since cis-11-octadecenoate is aprecursor for lactobacillic acid (Hofmann andLiu, 1960), the low concentration of unsaturatesmay have resulted in too little lactobacillatebeing produced by the organism for infraredanalysis.A more detailed quantitative study of the dis-

tribution of the fatty acids of S. lutea in relationto culture conditions is required before any sig-nificant biochemical conclusions can be drawn.

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