Membrane Lipid Composition of Obligately Facultatively … · Vol. 168, No. 1 MembraneLipid Composition ofObligately and Facultatively Alkalophilic Strains ofBacillus spp. SANDACLEJAN,1*

Vol. 168, No. 1

Membrane Lipid Composition of Obligately and FacultativelyAlkalophilic Strains of Bacillus spp.

SANDA CLEJAN,1* TERRY A. KRULWICH,2 KATHERINE R. MONDRUS,2 AND DONNA SETO-YOUNG2

Department of Pathology, City Hospital Center at Elmhurst, and Mount Sinai School of Medicine, City University ofNew York,' and Department of Biochemistry, Mount Sinai School of Medicine, City University of New York,2

New York, New York 10029

Received 22 May 1986/Accepted 28 July 1986

The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were

characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were madefrom pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultativestrains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membraneprotein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probablynot prerequisites for growth at very high pH since one or another of the facultative strains failed to show theseproperties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids.Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, includingphosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the othermajor phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of thealkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of thetotal phospholipid and individual fractions revealed two features that distinguished between the obligate andfacultative strains. Membranes from the obligately alkalophilic species contained a high concentration ofbranched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively highcontent of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost nounsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligatealkalophiles or B. subtilis.

Bacteria and other microorganisms have been attractiveexperimental vehicles for studies of the nature and possibleroles of individual membrane lipids. Among the bacteria thatgrow at extremes of pH, the membrane lipids of extremeacidophiles have been studied far more extensively (21) thanthose of alkalophiles. In a study of the total cellular lipids ofalkalophilic Bacillus sp. A-007, Koga et al. (16) identified themajor neutral lipids as diacylglycerols, squalene anddehydrosqualene, and the major polar lipids as phosphatidyl-glycerol, phosphatidylethanolamine, and cardiolipin. Theseinvestigators also identified bis(monoacylglycero)phosphatein Bacillus sp. A-007 (26) and two other alkalophilic bacillibut failed to find this compound in a fourth alkalophilicBacillus species (16).

Currently, the basis for obligate alkalophily is not under-stood; some data suggest that the membranes of obligatealkalophiles lose integrity at near-neutral pH values (17). If,as this indicates, the membrane of these strains retains fullbarrier function only at alkaline pH values, the membranelipids might reflect relevant properties. A comparative studyof obligate and facultative strains should clarify this possi-bility. In addition, a comparative study of several obligateand facultative alkalophiles might provide useful indicationsof properties of the membrane that relate to its capacity tofunction under conditions in which the external leaflet existsat pH values as high as 10.5 to 11 while the internalmembrane leaflet is exposed to a cytoplasm that is main-tained near pH 8.5 during active growth (17). And finally, themembrane lipids may be an important component of thebioenergetic machinery of the cell. We undertook a compre-

* Corresponding author.

hensive characterization of the membrane lipid compositionsof two obligately alkalophilic species, B. alcalophilus and B.firmus RAB, two recently isolated facultatively alkalophilicbacilli, strains OFI and OF4 (9), and B. subtilis.

MATERIALS AND METHODSStrains and growth conditions. B. subtilis BD99 was ob-

tained from Anthony Garro. It was grown in Spizizen saltsmedium (31), pH 7.0, supplemented with 0.1% yeast extract,50 ,ug of L-histidine per ml, 50 ,ug of L-threonine per ml, and50 p.g of L-tryptophan per ml. D,L-Malate (50 mM) was usedas the carbon source. B. firmus RAB and the two facultativestrains OF1 and OF4 were isolated in this laboratory (7, 9).B. alcalophilus was obtained from the American TypeCulture Collection (ATCC 27647). The alkalophilic strainswere grown in pH 10.5 carbonate-buffered medium supple-mented with 50 mM D,L-malate and 0.1% yeast extract (10).The same medium was used for growth of OF1 and OF4 atpH 7.5 except that the carbonate buffer was replaced by 25mM sodium phosphate buffer. All of the organisms were

grown at 30°C in 20-liter carboys with forced aeration.Unless otherwise noted, the cells were grown to stationaryphase and harvested by a Millipore ultrafiltration unit(Pellicon cassette system). For the experiment on the effectsof the growth stage on lipid composition, growth of strainOF4 was monitored with a Klett-Summerson colorimeter.One-third of the carboy was harvested in the early log phase,another one-third of the culture was harvested in the mid-logphase, and the remainder of the culture was harvested in theearly stationary phase.

Preparation of total membrane lipids. Lipids were pre-pared from right-side-out membrane vesicles that were made

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LIPID COMPOSITION OF ALKALOPHILIC BACILLI 335

from whole cells by the lysozyme method of Kaback (11) asdescribed previously (23). The vesicles thus prepared hadundetectable levels of contaminating muramic acid (8). Theconcentration of membrane protein in each vesicle prepara-tion was determined by the method of Lowry et al. (22) withbovine serum albumin as the standard. Total lipids wereextracted from the right-side-out membrane vesicles by themethod of Bligh and Dyer (4). The extracted lipids werestored at - 80°C under N2.

Purification and separation of neutral and polar lipid frac-tions. The total membrane lipids were fractionated on asilicic acid column with stepwise elution with chloroform,acetone, and then methanol. Individual lipid fractions thateluted from the column were pooled and purified to singlelipid species with silica gel thin-layer chromatography. Pu-rified lipids were obtained by extracting the scraped gel withchloroform and methanol (2:1, vol/vol). In other experi-ments, phospholipids and glycolipids were separated fromneutral lipids by acetone precipitation (19).

Fractionation, identification, and quantitation of neutrallipids. Neutral lipids were developed on a thin-layer chro-matography plate with petroleum ether-diethyl ether-aceticacid (80:20:3, vollvol/vol) and visualized by exposure toiodine vapor. Five fractions were quantitated by densi-tometry (Beckman CDS 200). Fraction I was cochro-matographed with free fatty acids. Fractions II and III werecochromatographed with authentic 1,2-diacylglycerol and1,3-diacylglycerol, respectively. Fractions IV and V con-tained squalene, C40 isoprenoid acid, and C50 isoprenoids.Fraction IV was scraped from the plate, extracted, andrechromatographed with ethyl ether-hexane (0.25:99.75,vol/vol). The following fractions were identified with theauthentic standards tetrahydrosqualene (Rf, 0.55), squalene(Rf, 0.41), dehydrosqualene (Rf, 0.26), phytoene (Rf, 0.19),and 1-carotene (Rf, 0).The mass spectra recorded with an MS-9 mass spectrom-

eter (70 eV) gave the expected molecular ion peaks: dehy-drosqualene mle, 408; squalene mle, 410; tetrahydro-squalene mle, 414; phytoene mle, 544; and 1-carotene mle,536. The fragmentation patterns were similar to those ofauthentic 13-carotene and phytoene. Gas-liquid chromatogra-phy, which was performed on a 3.5-mm by 60-cm stainlesssteel column (SE30 on Chromosorb G) at 295°C and 1.0kg/cm2, produced the expected retention times: squalene-tetrahydrosqualene, 1.59 min; phytoene, 9.27 min; and 1-carotene, 14.61 min.The infrared spectra for squalene (Beckman IR 20

spectrophotomer) showed absorption maxima at 2,960 to2,850 cm-1 (C-H stretching), 3,050 to 3,020 cm-'(C=C-H stretching), 1,665 cm- (C=C stretching), 1,465cm 1 (C-H methylene), 1,450 cm- 1 (C-CH3 asymmetric),1,383 cm-' (C-CH3) symmetric), and 840 cm-' (C-Hdeformation modes) for squalene.The nuclear magnetic resonance spectra (Varian T-60

spectrometer) for squalene showed signals at 1.60, 1.68, 2.0,2.04, and 5.09. Both infrared and nuclear magnetic reso-nance spectra were identical with those of authentic samplesfor all squalenes, 13-carotene, and phytoene, and the assign-ments of all protons were as expected for their respectivestructures. Fraction V was tentatively identified as a polyenehydrocarbon with no functional groups because no absorp-tion bands characteristic of tertiary hydroxyl groups wereobserved. The infrared spectra showed absorption bands at3,025, 2,960, 2,930, 2,860, 1,450, 1,365, and 960 cm- 1. Whenthe compound was dehydrated in dry pyridine with phos-phorus oxychloride at 50°C (15), both infrared and proton

nuclear magnetic resonance spectra were the same as thoseof the nonhydrated product. These data tend to confirm thestructure as a tetraanhydrobacterioruberin.

Preparation of 32P-labeled phospholipids. B. firmus RABwas grown in flasks containing 1 liter of the carbonate-buffered medium described above, to which 32p, as inorganicphosphate, was added to 500 ,uCi. Twenty percent of the 32pwas incorporated into the cells, and 10% of the incorporated32p was recovered in the membrane fraction. Lipids wereprepared from those membranes as described above.

Fractionation, quantitation, and identification of polarlipids. Separation of polar lipids was achieved by two-dimensional thin-layer chromatography. The plates weredeveloped by using chloroform-methanol-water (65:25:4)and dried in a desiccator for 45 min under N2. Plates werethen turned 90° and developed in a second solvent system,which consisted of chloroform-acetone-methanol-glacialacetic acid-water (6:8:2:2:1). The plates were allowed to dryovernight at room temperature. The following reagents wereused for detection: molybdenum blue (30), periodate-Schiff(13), a-naphthol (13), Dragendorff (13) iodine vapor, andninhydrin. Identification was based on the relative mobilitiesof the various components compared with those of authenticstandards, as well as by reaction with specific detectionreagents. Identification was further confirmed by mild alka-line deacylation followed by paper chromatography as de-scribed by Kates (13). The relative amounts of the phos-pholipids were determined by phosphorus analysis by amodification of the method described by Bartlett (2). Thelimit of phosphorus detection was about 20 nmol. The resultsby this method agreed well (±+ 3%) with those of radioactivecounting of 32P-labeled phospholipids. The ester bond (28),sugar content (29), and glycero moiety (5) were also deter-mined.

Fatty acid analysis. Fatty acid methylesters were preparedby the method of Morrison and Smith (25) by using borontrichloride-methanol instead of boron trifluoride-methanolreagent. The methylesters were extracted with pentane andexamined by gas-liquid chromatography (Varian 4600 gaschromatograph with data station) and mass spectrometry(Hewlett-Packard 5985GC/MS). Fatty acid methylester sep-aration was performed by using a two-column system asdescribed by Miller (24). For gas-liquid chromatographylinked to mass spectroscopy, a glass capillary column wasused (30 m) which was heated from 125 to 300°C at 10°C/min.

Materials. Authentic lipids were purchased from Serdary,Sigma Chemical Co., or Alltech Associates, Inc., AppliedScience Div. Fatty acid methylesters were from Supelco andAiltech. Organic solvents were obtained from Aldrich Chem-ical Co. and were spectrophotometric grades. 32P-phosphatewas obtained from Amersham Corp. (8 mCi/ml, 296mEq/ml). Bis(monoacylglyceryo)phosphate was a generousgift from Y. Koga of the Department of Chemistry, Univer-sity of Occupational and Environmental Health, Kita-kyushu, Japan.

RESULTS

Total membrane lipids. The two obligately alkalophilicspecies in the study exhibited the highest ratios of total lipidto protein in the membrane (Table 1). Facultative strain OF4grown at either pH 7.5 or 10.5 and OF1 grown at pH 7.5showed similar lipid contents relative to the protein in themembrane; at pH 10.5, OF1 exhibited somewhat lowermembrane lipid/protein ratios, approximating the ratio ob-served in membranes from B. subtilis. The two obligate

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TABLE 1. Membrane lipids of alkalophilic bacilli and B. subtilis"

Growth Mean (± SD) total Neutral/polarBacterial strain pH membrane lipid (mg/mg lipid ratiob

of membrane protein) (%/%)

B. subtilis 7.0 0.72 ± 0.12 (n = 9) 30/70B.firmus RAB 10.5 1.11 ± 0.25 (n = 12) 45/55B. alcalophilus 10.5 1.08 ± 0.16 (n = 12) 45/55OF1 10.5 0.76 + 0.16 (n = 9) 25/75OF1 7.5 0.92 ± 0.20 (n = 9) 35/65OF4 10.5 0.94 ± 0.18 (n = 9) 25/75OF4 7.5 1.06 ± 0.21 (n = 8) 35/65

a Lipids were extracted from isolated membranes from the strains shown;the growth values are indicated. Total membrane lipid and neutral and polarlipids were measured as described in Materials and Methods. The number ofdeterminations is shown next to each value for total membrane lipid. At leastthree independent preparations of each strain were examined.

b The values were calculated from 8 to 10 determinations on at least threeindependent samples with an error of ±5%.

alkalophiles also exhibited high ratios of neutral/polar lipids,with 45% of the total membrane lipid represented by neutrallipid. By contrast, B. subtilis membranes contained 30% ofits lipid in the neutral lipid fraction. The two facultativealkalophiles had 25% of their lipid in the neutral fractionwhen grown at pH 10.5 and 35% in that fraction when grownat pH 7.5.

Neutral lipids. B. subtilis membranes had a very simpleneutral lipid composition, which consisted of 1,2-diacylglycerol (94%) and free fatty acids (6%) (Table 2). Theneutral lipid compositions of the alkalophilic strains weremuch more complex. In addition to 1,2-diacylglycerol, theyalso contained 1,3-diacylglycerol, squalene, dehydro-squalene, and tetrahydrosqualene, as well as C40 and C50isoprenoids. The principal C40 isoprenoids were r-caroteneand phytoene. The obligate alkalophiles and pH 10.5-grownOF4 contained very similar levels of C40 isoprenoids. OF1had generally lower levels of these lipids but shared with theother facultative strain the property of having higher con-centrations of C40 isoprenoids during growth at pH 7.5 thanat pH 10.5. C50 isoprenoids were found in small amounts inthree of four of the alkalophiles, being undetectable in OFI.

Polar lipids. Six or seven different polar lipids weredetected in preparations from all of the alkalophile mem-brane lipid extracts. All of these polar lipids werephospholipids rather than glycolipids or phosphoglycolipids.Each lipid fraction contained 6 to 12 ,ug of phosphate, but nosample contained detectable sugar, and none of them reacted

with at-naphthol reagent. All of the alkalophilic bacteriacontained phosphatidylglycerol as their major membranephospholipid and appreciable amounts of phosphatidyl-ethanolamine (Table 3). The alkalophilic bacteria alsoshowed high concentrations of cardiolipin (13 to 25%) exceptthat OF1 grown at pH 10.5 had somewhat lower cardiolipinand correspondingly higher phosphatidic acid contents. Thepolar lipids of the alkalophiles were thus largely negativelycharged.

Small amounts of bis(monoacylglycero)phosphate wereidentified in the alkalophilic bacilli, except for OF4, in whichthis compound was not detected. One spot which waspositive to molybdate spray but negative to ninhydrin,periodate-Schiff, and u-naphthol could not be identified.The chloroform-ethanol (3:1)-soluble products of mild

alkaline methanolysis of each phospholipid fraction wereanalyzed by thin-layer chromatography in petroleum ether-diethyl ether-acetic acid (80:20:3). All contained only fattyacid methylesters. Thus, the fatty acids in the membranes ofalkalophilic bacteria were bound by ester linkages and not byether linkages.

Fatty acid composition. Seventeen fatty acid methylestercomponents were detected by gas-liquid chromatography(Table 4). The methylesters were primary anteiso- andiso-branched-chain fatty acids. The major branched fattyacid components of both obligately alkalophilic species andB. subtilis were anteiso-C150, iso-C15:0, and iso-C17:0, al-though the proportion of anteiso-C15:0 and iso-C17:0 washigher in B. subtilis. The obligate alkalophiles had moremonounsaturated species than B. subtilis, the major onebeing n-C16:1.The fatty acid composition of the facultative alkalophiles

was strikingly different from that of the obligate alkalophileswith respect to unsaturated fatty acids. The facultativealkalophiles were almost completely devoid of unsaturatedfatty acids when they grew at pH 7.5, and only 2 to 3% of thefatty acids were unsaturated (n-C16:1) in membranes from pH10.5-grown cells. The same trends of fatty acid distributionwere seen in the phosphatidylglycerol (Table 5), phospha-tidylethanolamine (Table 6), and cardiolipin (Table 7) frac-tions.The second striking difference between the fatty acids in

membrane lipids from the obligate and facultative strainswas in the levels of branched-chain fatty acids (Table 4).Membrane phospholipids from the two obligate alkalophileshad levels of branched-chain fatty acids that were compara-ble to those found in the membrane phospholipids from B.

TABLE 2. Neutral lipid composition of membranes from alkalophilic bacilli and B. subtilisa

% of total lipid found in cells of:Lipid(s) B. subtilis B. firmus RAB B. alcalophilus OFI OF1 OF4 OF4

(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

Free fatty acids 6 5 4 4 5 3 21, 3-Diacylglycerol 0 20 21 26 23 22 201, 2-Diacylglycerol 94 32 30 39 38 30 28Squalene 0 10 11 12 14 10 10Dehydro- or 0 12 14 12 10 12 6

tetrahydrosqualeneC40 isoprenoids 0 18 16 7 10 19 30

(n-carotene, phytoene)C50 isoprenoids (tetra- 0 3 4 0 0 4 4

hydrobacterioruberin)a These results represent averages of at least 10 experiments with at least two independent preparations of the lipid extracts from cells grown at the indicated

pH value. The standard deviation of the values was 1 to 5%.

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LIPID COMPOSITION OF ALKALOPHILIC BACILLI 337

TABLE 3. Membrane polar lipid composition of alkalophilic bacilli and B. subtilisa

% of total polar lipids found in membranes of:Polar lipid B. subtilis B. firmus RAB B. alcalophilus OF1 OF1 OF4 OF4

(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

Phosphatidylglycerol 70 56 57 63 58 50 56Phosphatidylethanolamine 12 20 19 15 20 18 20Cardiolipin 4 13 15 8 13 25 20

Bis(monoacylglycero)phos- 0 5 5 3 5 0 0phate

Phosphatidic acid 0 2 1 7 2 1 1Phosphoglycolipid 5 0 0 0 0 0 0Monoglycosyl di- 2 0 0 0 0 0 0

acylglycerolDiglycosyldiacylglycerol 4 0 0 0 0 0 0Aminoacyl phos- 2 0 1 1 2 2 3

phatidylglycerolUnidentified 1 4 2 3 0 4 0

a These results represent the averages of at least 12 experiments with at least three independent lipid preparations from cells grown at the indicated pH. Thestandard deviations were + 1 to 7%.

subtilis, whereas much lower levels of branched-chain fattyacids were present in the membrane phospholipids from thefacultative strains. The low level of branched-chain fattyacids was particularly pronounced in the cardiolipin fraction(Table 7).

Effects of growth stage on the membrane lipid compositionof OF4. The levels of total lipids and diacylglycerides in themembranes of OF4 were essentially unchanged as a functionof growth stage (Fig. 1A). However, the relative amounts ofcarotenoids (Fig. 1A), cardiolipin (Fig. 1B), and iso-C15:0fatty acid (Fig. 1C) increased as the growth of the cultureprogressed. Phosphatidylethanolamine and phosphatidyl-glycerol decreased when the cells entered the stationaryphase (Fig. 1B). The calculated total branched-chain fattyacids decreased at the late-stationary phase, and the anteiso-

C15:0 fatty acid also decreased, but more gradually, towardthe latter part of the growth curve (Fig. 1C),

DISCUSSION

The goal of this investigation was to gather data on themembrane lipids of obligate and facultative alkalophiles toprovide the basis for designing critical experiments whichmay (i) indicate features of the membrane lipids that arerequired for growth at extremely alkaline pH; (ii) identifyproperties of the membrane that preclude growth of theobligate strains at near-neutral pH values, whereas thefacultative strains can grow over a broader range; and (iii)clarify possible roles of membrane lipids in the bioenergeticfunctions of the cells. B. subtilis was included for compara-

TABLE 4. Fatty acid composition of total membrane lipid extracts from alkalophilic Bacillus species and B. subtilis

% of fatty acid(s) in total membrane lipid extracts of:

Fatty acid(s)a B. subtilis B. firmus RAB B. alcalophilus OF1 OFI OF4 OF4(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

Iso-C12:0 1 2 2 3 3 3 5n-C13:0 NDb ND ND ND 4 ND 5Iso-C14:0 1 1 1 8 8 10 10n-C140 1 1 1 8 7 9 9Iso-C15:0 15 23 21 20 24 18 18Anteiso-C15:0 40 28 29 34 30 33 26Iso-C16:0 5 5 6 2 2 2 1n-C16:0 5 7 5 8 5 8 6n-C16:1 7 12 13 3 ND 2 NDIso-C17:0 20 10 12 4 3 4 6Anteiso-C17:0 1 1 1 ND ND ND NDn-C17:0 ND ND ND 10 14 11 14Iso-C17:1 1 2 2 ND ND ND NDAnteiso-C17:1 1 2 2 ND ND ND NDn-C18:0 1 2 2 ND ND ND NDn-C8:1 0.5 2 1 ND ND ND NDn-C18:2 0.5 2 2 ND ND ND NDUnsaturated fatty acids 10.0 20 20 3 0 2 0Branched-chain fatty acids 93 90 92 74 70 72 66

a Fatty acids are abbreviated such that the number of carbon atoms precedes the colon and the number of double bonds follows the colon. The prefixes anteisoand iso represent types of branched-chain structure.

b ND, None detected. These results represent averages of at least five experiments with at least two independent total lipid preparations from cells grown atthe indicated pH.

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TABLE 5. Fatty acid distribution of the phosphatidylglycerol fraction of phospholipids from alkalophilic bacilli and B. subtilis

% of fatty acid(s) in phospatidylglycerol fraction of phospholipids from:Fatty acid(s)" B. subtilis B. firmus RAB B. alcalophilus OF1 OF1 OF4 OF4

(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

Iso-C13:0 1 2 2 3 4 3 2n-C13:0 1 NDb ND 2 5 5 6Iso-C14:0 3 3 3 2 2 3 1n-C14:0 3 2 3 2 5 3 4Iso-C15:0 17 15 17 13 12 21 22Anteiso-C15.0 32 33 30 27 27 20 20n-C15:0 ND ND ND 14 7 6 9Iso-C16:0 9 11 12 7 5 7 4n-C16:0 10 6 5 14 19 18 21n-C16:1 4 7 8 ND ND ND NDIso-C17:0 15 17 15 14 12 12 10Anteiso-C17:1 3 2 3 2 2 2 1n-C18:0 2 2 2 ND ND ND NDUnsaturated fatty acids 4 7 8 0 0 0 0Branched-chain fatty acids 84 90 90 68 64 68 60

a Fatty acids are abbreviated such that the number of carbon atoms precedes the colon and the number of double bonds follows the colon. The prefixes anteisoand iso represent types of branched-chain structure.

I ND, None detected. These results represent averages of at least five experiments with at least two independent total lipid preparations from cells grown atthe indicated pH.

tive purposes. The findings for this species were similar tothose found by other investigators (e.g., reference 3).The two obligate alkalophiles had high membrane

lipid/protein ratios, as well as high proportions of neutralrelative to polar lipids in their membranes. Neither of thesefeatures seems crucial for life at very high pH, however,since strain OF1 did not exhibit especially high membranelipid/protein ratios when grown at pH 10.5 and neither of thefacultative alkalophiles exhibited very high neutral lipidcontents. On the other hand, all of the alkalophiles containedsqualene, squalene derivatives, and C40 isoprenoids amongtheir neutral lipids. The presence of squalene has beenreported in a number of other bacteria, including bacilli (1).Squalene, dehydrosqualene, and diacylglycerol have alsobeen reported in the total cellular lipids from alkalophilicBacillus sp. A-007 by Koga et al. (16). In all of the

alkalophilic strains studied here, the presence of dehydro-squalene and tetrahydrosqualene showed that the alkalo-philic bacilli were able both to desaturate squalene and tosaturate it.Of considerable interest was the finding of C50 isoprenoids

in most of the membranes from the alkalophiles and ofsubstantial amounts of C40 isoprenoids in all of thealkalophilic strains. It was notable that all of the alkalophilescontained 3-carotene and phytoene, two members of thePorter series (27). The presence of higher concentrations ofC40 isoprenoids in the facultative strains that were grown atnear-neutral pH indicates that the synthetic pathway func-tions better at somewhat lower cytoplasmic pH values thanat those obtained when cells grow at pH 10.5.Among the polar membrane lipids, there were compounds

that were notable for their absence or lack of universality in

TABLE 6. Fatty acid distribution of the phosphatidylethanolamine fraction of phospholipids from alkalophilic Bacillus species andB. subtilis

% of fatty acid(s) in phosphatidylethanolamine fraction of phospholipids from:Fatty acid(s)a B. subtilis B. firmus RAB B. alcalophilus OF1 OFI OF4 OF4

(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

ISO-C13:0 1 1 1 1 NDb 1 NDn-C13:0 ND ND ND 3 5 4 7ISOC14:0 3 1 3 2 ND 2 NDn-C14:0 3 2 1 1 1 1 1ISO-C15:0 28 33 32 30 28 28 29Anteiso-C15:0 23 30 30 24 24 25 21ISO_C16:0 10 7 8 9 8 7 7n-C16:0 16 12 13 22 26 26 31n-C16:1 4 3 4 ND ND ND NDn-C17:0 1 ND ND 3 4 2 3ISO-C17:0 10 8 7 5 4 4 2Anteiso-C17:0 1 1 1 ND ND ND NDUnsaturated fatty acids 4 3 4 0 0 0 0Branched-chain fatty acids 80 86 86 70 63 67 58

a Fatty acids are abbreviated such that the number of carbon atoms precedes the colon and the number of double bonds follows the colon. The prefixes anteisoand iso represent types of branched-chain structure.

b ND, None detected. These results represent averages of at least five experiments with at least two independent total lipid preparations from cells grown atthe indicated pH.

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LIPID COMPOSITION OF ALKALOPHILIC BACILLI 339

TABLE 7. Fatty acid distribution of the cardiolipin fraction of phospholipids from alkalophilic Bacillus species and B. subtilis

% of fatty acid(s) in cardiolipin fraction of phospholipids from:Fatty acid(s)a B. subtilis B. firmus RAB B. alcalophilus OFl OF1 OF4 OF4

(pH 7.0) (pH 10.5) (pH 10.5) (pH 10.5) (pH 7.5) (pH 10.5) (pH 7.5)

ISO-C14:0 1 1 1 3 4 3 4n-C14:0 1 1 1 8 12 10 18ISO-C15:0 30 25 27 24 20 20 10Anteiso-C15:0 23 20 18 16 16 16 10ISO-C16:0 6 4 5 9 11 10 8n-C16:0 14 16 14 27 26 28 30n-C16:1 10 15 18 5 1 4 1ISO_C17:0 9 4 6 3 3 4 4n-C17:0 NDb ND ND 5 7 5 6ISO-C17:1 2 5 4 ND ND ND NDAnteiso-C17:1 1 3 4 ND ND ND NDn-C18:1 2 4 1 ND ND ND NDn-C18:2 1 2 1 ND ND ND NDUnsaturated fatty acids 16 29 28 5 1 4 1Branched-chain fatty acids 85 84 85 60 55 57 48

a Fatty acids are abbreviated such that the number of carbon atoms precedes the colon and the number of double bonds follows the colon. The prefixes anteisoand iso represent types of branched-chain structure.

b ND, None detected. These results represent averages of at least five experiments with at least two independent total lipid preparations from cells grown atthe indicated pH.

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FIG. 1. Effect of growth stage on the membrane lipids of pH10.5-grown OF4. Cells were grown to the indicated stages ofgrowth,and lipid fractions were prepared and analyzed as described inMaterials and Methods. (A) Total lipid (TL) is shown as milligramsper milligram of membrane protein; diacylglycerol (DG) and C40 +C50 isoprenoids (CAR) are shown as percentages of total neutrallipid. (B) Cardiolipin (CL), phosphatidylglycerol (PG), andphosphatidylethanolamine (PE) are shown as percentages of thetotal polar lipid fraction. (C) Total branched-chain, iSo-C15:0, andanteiso-C15:0 fatty acids are shown as percentages of total fatty acid.

the alkalophile membranes. Thus, glycolipids and phospho-glycolipids that are commonly found in gram-positive orga-nisms were absent. Similarly, ether lipids that have beenfound in several archaebacterial species that inhabit extremeenvironments of other kinds (14, 18, 20) were absent. More-over, bis(monoacylglycero)phosphate was found in the twoobligate alkalophiles and one of the facultative strains butwas absent from OF1 just as it has been absent in studies byKoga et al. (16) of alkalophilic Bacillus sp. A40-2. It isunlikely that this lipid is a marker for membranes fromalkalophiles, and its role is currently unknown.The polar lipid and its fatty acid composition did, how-

ever, point to a number of features that were common to thealkalophiles and two features that distinguished between theobligate and facultative strains. Except for phosphatidyl-ethanolamine, all of the phospholipid was anionic, thusconferring a highly negative charge upon the membrane. Thehigh concentration of cardiolipin in the membrane lipids ofalkalophilic bacilli is of special interest. In in vitro modelsystems, cardiolipin has been shown to form nonbilayerstructures (hexagonal II phase or lipidic particles) undercertain experimental conditions, e.g., in the presence ofCa2+ or cytochrome c (6, 32). Alkalophile membranes areknown to contain high concentrations of cytochrome c,especially pH 10.5-grown OF4 (9), which also had thehighest concentration of cardiolipin.The fatty acid compositions of the polar membrane lipids

from the obligate alkalophiles exhibited patterns of someintrinsic interest and also offered the two most clear-cutdifferences between these species and the facultativelyalkalophilic strains in this study. Branched-chain fatty acidsare proposed to increase the fluidity of the membrane (12).Since, in addition to a high proportion of branched-chainfatty acids, the obligate alkalophiles had a high concentra-tion of monounsaturated fatty acids, the membranes fromthese species seem to contain a very large proportion of fattyacids with low melting points. The possibility that thesefeatures pose a problem with respect to membrane integrity,at least at neutral pH, is put into focus by the finding that thefacultative alkalophiles have lower contents of branched-chain fatty acids and almost no unsaturated fatty acids.

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340 CLEJAN ET AL.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Serviceresearch grant GM28454 from the National Institutes of Health,grant DMB8504395 from the National Science Foundation, andcontract DEAC02 81ER10871 from the U.S. Department of Energy.

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