337

R~och~mica et ~iopkysicu Ada, 424 (1976) 337-350 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BRA 56734

LIPIDS AND FA’ITY ACIDS OF A MODERATELY HALOPHILIC BACTERIUM, NO. 101

YOSHIMI OHNO, IKUYA YANO, TAKASHI HIRAMATSU and MASAMIKI MASUI department of ~cteriology, Osaka (Japan)

Osaka City

Unive~i~y~e~~ca~School,

Asahimacki,

Abeno-ku,

(Received August 2&b, 1975)

Summary The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaCl, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospho~ycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and enzymatic treatment with phospholipase Aa, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The extractable lipid composition varied greatly with the concentrations of NaCl in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were Clb: ,, , C16: 1, Cl8 :-, , Cl8 :, and cyclopropanoic C 1, and Cl9 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid. Introduction Halophilic microorganisms, requiring high concentration of salts for growth, have been shown to be useful for investigating membrane structures and func-

338

tions [l--5]. cutirubrum,

Among various halophilic or halotolerant bacteria, Halobacterium an extremely halophilic bacterium requiring 4 M NaCl for optimal growth, has been shown to have a very unusual cell wall structure lacking a peptidoglycan layer [6,7], and the hydrophobic residues in the membrane lipids substituted entirely of long-chain branched alkyl ethers instead of fatty acid esters [S-l 51. In contrast, moderately halophilic bacteria grown at wide ranges of NaCl concentrations between 0.5 and 4 M (2 M for optimum), having cell walls consisting of three layers including the diaminopimelic acid type peptidoglycan layer, seems to be more suitable for examining various membrane functions, comparing with other gram-negative bacilli. Several reports concerning with the lipids of moderately halophilic bacteria have been already published and the major lipids of these cases were reported to be diester mixtures of phospholipids and glycolipids [ 16-191. However, to our knowledge, the isolation and characterization of membrane fragments from moderately halophilic bacteria have not yet been described. Recently, we have isolated the cell envelopes [20-221 and the outer membrane fragments [ 21,231 from moderately halophilic bacterium No. 101-W3 and examined the lipid composition of fractionated cellular preparations. The present paper describes the analysis of extractable and tightly bound lipids of whole cells, cell envelopes and isolated outer membranes from No. 101-W3 cells grown in a medium containing 2 M NaCl. Materials and Methods Growth of organism

A wild-type strain of an unidentified moderately halophilic gram-negative rod, No. lOl-W3 [ 241, donated by Dr. A. Yamada, Research Laboratory of Nippon Suisan Co., Ltd., Tokyo, was grown in a medium containing 2 M NaCl, 0.4% MgS04 * 7Hz0, 1% polypeptone (Daigo-Eiyo Chem. Co., Osaka), 0.1% yeast extract and 0.2% NaN03, pH 7.2. After the cells were incubated for 42 h at 30°C in a shaker, they were harvested by centrifugation at 13 500 X g for 15 min. Preparation of cell envelopes No. lOl-W3

and outer

membranes

from

halophilic

bacterium

The crude cell envelopes were prepared by the method of Ohtani and Masui [20]. The outer membranes were released from the cells grown in a medium containing 2 M NaCl by mild desalting in a 20% sucrose solution containing 50 mM Tris . HCl buffer, pH 7.8 and then, purified by CsCl buoyant density centrifugation. The detailed procedure will be reported in a separate paper [ 231. The lipopolysaccharides were prepared by the method of Westphal et al. [ 251. Extraction

and fractionation

of free lipids

Lipids were extracted with 10 vol. of chloroform/methanol (2 : 1, by vol.). After washing by the method of Folch et al. [ 261, the combined extracts were condensed to dryness, dissolved again and separated by thin-layer chromatography on Silica Gel G (Merck, Darmstadt, G.F.R.). The plates were developed with the following solvent systems: chloroform/methanol/water (65 : 25 : 4,

339

by vol.) (A), chloroform/meth~ol/a~eti~ acid/water (100 : 20 : 12 : 5, by vol.) (B), n-hexane/diethyl ether (80 : 20, by vol.) (C) or chloroform/methanol/ ammonia/water (70 : 30 : 4 : 2, by vol.) (D). Total lipids were demonstrated by charring the plates after spraying with 9 M HzS04. For the de~ction of phosphorus, amino groups and reducing sugars were used Dittmer’s, ninhydrin and anthrone reagents, respectively. For preparative purpose, lipids were detected with 2’,7’-dichlorofluorescein, scraped off and then extracted from the gels with chlorofo~~meth~ol (1 : ‘2, then 1 : 4, by vol.). Hydrolysis of phospholipids

Acid hydrolysis was performed with 2 M HCl for 48 h at 125°C in an oil bath. Mild alkaline hydrolysis was carried out essentially by the method of Dawson [27], The resultant water-soluble products were separated by paper chromatography with a solvent of phenol/water/acetic acid/ethanol (80 : 20 : 10 : 12, by vol.). The deacylated phosphate esters were detected under ultraviolet light after molybdenic acid reagent spray, and by ninhydrin and alkaline AgN03 reagent [281. Hydrolysis of bbund lipids or lipopolysaccharides

After the exhaustive extraction of the cellular lipids with the neutral solvents, the residues were hydrolyzed with alkaline hydroxylamine in ethanol for 3 min at 63”C, according to the method of Snyder [ 291. The esterified fatty acids liberated were extracted with n-hexane after acidifying with acetic acid, additional hydrolysis of the residues was carried out with 3 M HCl for 2 h at 100°C and then the amide-linked fatty acids were extracted. The fatty acids of lipopoly~cch~des were separated similarly into esterified and amidelinked groups, and then analyzed. Infrared spectrometric analyst

Infrared spectrometic analysis of phospholipids were performed with a Hitachi Grating Infrared Spectrophotometer Model EPI-G3 (Hitachi Co., Tokyo) as KBr discs. Enzymatic hydrolysis of phosph~lipids

Hydrolysis of isolated phospholipids

by phospholipase AZ (EC 3.1.1.4) from

Vipera ruse& (Sigma) was carried out essentially according to the method of

Bonsen et al. [ 301. The positional dis~bution of fatty acids on glycerol moiety of phospholipid was determined by the gas chromatographic analysis of both methyl esters derived from free acids and lysophospholipids separated on thinlayer plates using the solvent system A. Hydrolysis of phospholipids by phospholip~ C (EC 3.1.4.3) from ~ci~~us cereus (Boehringer Mannheim, GmbH, G.F.R) was carried out by a modified method of Haverkate et al. [31]. The reaction mixtures contained: 2.5 mg phospholipids, 1.8 ml 0.1 M Tris * HCl buffer pH 7.4, 1.0 ml 0.02 M Ca&, 2.0 ml diethyl ether and 0.3 ml enzyme solution (2 mg pro~in/ml~ in a stoppered tube. The mixtures were incubated with shaking for 64 h at 37°C. Diethyl ether was added at 22 to 24 h intervals. After the reaction was stopped

340

by adding chloroform/methanol (2 : 1, by vol.) solution, reaction products (and any substrate) were extracted and separated on thin-layer chromatography. The plate was developed about 11 cm with solvent A first, dried and then developed again with solvent C. Hydrolysis of phospholipids by phospholipase D (EC 3.1.4.4) from carrot extracts was carried out by the method of Kates [32]. For the convenience, 14C-labelled phospholipids isolated from the cells grown in a medium containing [2-14C] glycerol were used. The reaction mixtures contained: 1.0 mg (about lo4 dpm) “C-labelled phospholipids, 2.0 ml 0.2 M acetate buffer, pH 5.52, 2.0 ml diethyl ether and 1.0 ml enzyme solution (carrot extracts). After the incubation for 4 h at 37”C, 1.0 ml of 1 M HC104 was added and centrifuged. The precipitate was extracted with chloroform/methanol (1 : 1, by vol.), neutralized, and redissolved with chloroform/methanol (2 : 1, by vol.). Thin-layer chromato~aphic separation was performed with solvent system B or D for basic plate prepared with 0.05 M Na2C03 coated silica. The radioactivity was monitored by Nihon Musen Radio Chromatographic Scanner. Acetolysis and separation of phospholipid molecular species Acetolysis of phospholipids was carried out according to the method of Renkonen [33]. The resultant diacylacetylglycerols were analyzed by gas chromatography-mass spectrometry, as reported earlier [ 341. Chemical analysis Lipid phosphorus was estimated by King’s method [ 351, hexose by anthrone method [ 36 J, glycerol by Hanahan et al. f37] after hydrolysis with 2 M HCl for 45 h at 125°C and uranic acid by carbazole method /38). The densi~me~c analysis of phospholipids separated on thin-layer plates was carried out using Shimadzu Dual Wave Length Chromatoscanner (CS 900 type) with a linearizer. The relative quantities of individual phospholipids were calculated from the peak areas for the total. Fatty acids were determined by the weight and their molar concentrations were calculated as the arbitrary weight, 300. Identification of hexose in phosphoglycolipid The unidentified phosphoglycolipid isolated on thin-layer plates was methanolyzed with 2 M HCl in anhydrous methanol for 3 h at 100°C. After the fatty acid methyl esters were extracted with n-hexane, the residues were evaporated off and trimethylsilyla~d with S&prep reagent (Applied Science Lab., Pa., U.S.A.). The identification of Me~Si-methylglycosides and -glycerol were carried out by gas-liquid chromatography and gas chromatography-mass spectrometry [ 393. Gas-liquid ch~mato~aphic and muss spectrometric analysis Fatty acids of each lipid were tr~sme~y~a~d with benzene/meth~ol/ HzS04 (10 : 20 : 1, by vol.) under reflux for 1 h and the resultant fatty acid methyl esters were then extracted with n-hexane. Gas-liquid chromatography of fatty acid methyl esters was carried out on a Shimadzu GC-3BF apparatus equipped with a hydrogen flame ionization detector. The glass coiled column used was 2.5 m X 3 mm packed with 15% ethylene-

341

glycolsuccinate or 1% OV-1 on Chromosorb W, silanized, SO/l00 mesh. The column temperature was 155°C for Me&-methylglycosides or 180°C for fatty acid methyl esters. Gas chromatographic and mass spectrometric analysis were carried out on a Shimadzu-LKB 9000. The mass spectra were recorded at an electron energy of 22.5 eV, a trap current of 60 PA, an accelerating voltage 3.5 kV and ion source temperature of 250°C. Helium was used as carrier gas at a flow rate of 30 ml/ min. Chemicals All the chemicals used were of the highest purity commercially available. Organic solvent were redistilled before use. The lipids and fatty acids as reference standards were purchased from Applied Science Laboratories, State College, Pa., U.S.A. Results Lipid con tent and general analysis The amounts of extractable lipids obtained from whole dry cells, the cell envelopes and the outer membranes were 7.9, 20.1 and 21.6%, respectively. In addition, the amount of bound lipids (mainly extracted as fatty acids) from those preparations were 1.4, 35.4 and 41.6%, respectively. The extractable lipids from whole cells contained about 3.1% of lipid phosphorus, 1.0% of hexose (as glucose) and 0.4% of uranic acid, this indicating the major components of the cellular lipids were phospholipids and a smaller amount of glycolipids. When the total lipids of the bacterium were chromatographed on a thin-layer plate, at least 8 different components were observed. The individual lipids were tentatively identified on the basis of the specific color reactions and the comparison of RF values with the authentic standards. The major phospholipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol which consist of about 80% of the total phospholipids. Besides these main components, it was noted that an unidentified phosphoglycolipid with a lower RF value than phosphatidylglycerol, showing positive reaction for both anthrone and Dittmer’s reagents, occurred. As indicated in Table I, this lipid increased significantly in the cells grown in the presence of 1% glucose in the medium comprising about 25% or more of the total cellular phospholipids and seemed to be mainly concentrated (23%) in the outer membrane preparations, although the distribution of other phospholipids among each cellular preparations was similar. On the other hand, the relative amount of individual phospholipid was shown to vary dramatically under various culture conditions. Fig. 2a shows the changes in lipid composition with the stages of bacterial growth. The younger cells had a high proportion of phosphatidylglycerol and phosphatidylethanolamine, while the older cells had a high proportion of diphosphatidylglycerol. The concentration of phosphoglycolipid was shown to be between 10 to 15% of the total, consistently during the stages of growth. Since the moderately halophihc bacterium No. lOl-W3 can grow over a wide range of NaCl concen-

342

Fig, 1. Thin-layer chromatograms of phospholipids from moderately halophilic bacterium No. 101 grown in 2 M N&l medium, The plate was developed with solvent (B). (1) and (5). whole cells grown without glucose; (2), whole cells grown with 1% glucose; (3). outer membrane without ghmose: (4), cell envelope without glucose; NL. neutral lipids; CL, diphosphatidylglycerol; PE, phosphatidylethanolamine: PG. phosphatidylglycerol; PGL, unidentified phosphoglycolipid; X1-X3. unidentified minor components; 0, origin.

TABLE I PHOSPHOLIPID COMPOSITION OF CELLULAR XI,X2.X3:

FRACTION

unidentified minor components. .._~___

-_I__

Whole cell * glucose(+) glucose(-) -I~____ Diphosphatidy~ycerol Phosphatldyl~thano~mine Phosphatidylglycerol Xl + X2 Phosphoglycolipid X3

25.3% 28.5 30.3 6.4 8.7 0.8

23.1% 41.3 9.1 tr 26.5 tr

* Cells were incubated in a medium containing 2 M NaCl.

Cell envelope * glucose(-)

Outer membrane * glucose(-)

____27.9% 23.3 33.1 2.9 8.1 4.7

9.3% 33.5 26.0 5.0 23.5 2.3

343

(b)

growth

0’

I

3

20

40 60 Time of growth

60 (h)

0.5

1.0

1.5 2.0 2.5 3.0 NaCl (M) Fig. 2. Changes in the relative proportions of the major phospholipids by the growth stages and the NaCl concentrations. The lipids were separated on a thin-layer plate with solvent (B). After spraying with 9 M H+OJ and charring, each band scanned with Shimadzu Dual Wave Length Chromatoscanner (CS 900 type) with linearizer. In (a), cells were grown in 2 M NaCl. In (b), cells were hnwested at 40 h after inoculation at 3O’C. Dotted line shows growth of the cells (A56m). For abbreviations see the legend to Fig. 1.

trations, the changes in lipid composition due to the NaCl concentrations of the culture medium have been examined. Fig. 2b shows that the most abundant phospholipid of the cells grown in lower NaCl medium (0.5 to 1.0 M NaCl) was phosphatidylethanolamine, and phosphatidylglycerol comprised only l/2 of the former, in contrast, the amounts of both phospholipids were almost equal in high NaCl medium (3 M or more NaCl). The relative amounts of diphosphatidylglycerol and phosphoglycolipid were consistently maintained at 10 to 15%. Characterization of phosphoglycolipid The individual lipids which separated on thin-layer plates were recovered from the gel, confirmed for purity, and then lipid phosphorus, glycerol, fatty acid and hexose were estimated. The molar ratios of Pi, glycerol and fatty acid of phosphatidylethanolamine and phosphatidylglycerol almost coincided with their calculated values. The acid hydrolysis of phosphoglycolipid formed Pi (2.3%), glycerol (14.2%) and hexose (14.8%) as water soluble components, while long chain fatty acids were detected as sole hexane soluble extracts. The molar ratios of Pi, glycerol, fatty acid and hexose of phosphoglycolipid were 1.0 : 2.15 : 1.86 : 1.05, respectively. Since similar phosphoglycolipids were reported to occur in gram-positive cocci [ 40-431 or gram-negative bacilli [ 18, 191, we have analyzed to characterize the chemical structure in more detail. The gas chromatogram of the Me&-derivatives of water soluble components from phosphoglycolipid after methanolysis showed peaks corresponding to Me@-methylglucoside. No other peaks with the retention times of Me&methylgalactoside or -methylmannoside were detected. The mass spectra of the Me&-methylhexoside from phosphoglycolipid coincided with that of authentic Me&-methylglucoside [39], which also sup-

344

ported the hexose moiety of phosphoglycolipid was exclusively glucose. The infrared spectra of phosphoglycolipid showed a resemblance to that of phosphatidylglycerol, in absorption at 1740 cm-’ due to ester bond, 2900 cm-’ due to a methylene derivative and 1020-1050 cm-’ due to CO stretching. The only differences were deep and broad absorption at the 3500 cm-’ region due to hydroxyl function and slight but distinctive absorption at 845 cm-’ due to a-glycosidic linkage in phosphoglycolipid, this indicating that the phosphoglycolipid possessed more hydroxyl groups than phosphatidylglycerol and the glucose appeared to be attached glycosidically to the hydroxyl group of glycerol or phosphate. On a paper chromatography, the water soluble compound obtained by mild alkaline hydrolysis of phosphoglycolipid gave a phosphorusand alkaline AgN03 positive spot with a lower RF value (RF = 0.13) than that of phosphatidylglycerol (RF = 0.41 1441, most likely glyce~lphospho~lglycerol).

Fig. 3. Thin-layer chromatograms of the reaction products obtabted after phospholipase C treatment of phosphoglycolipid (PGL). The plate was first developed with solvent (A) about 11 cm, dried and then developed again with solvent (C). Spots were visualized by charring after spraying with 9 M H2SO4. (1). pho~hatidyleth~ol~~e; (Z), the products after phospholipase C hydrolysis of phosphatidy~e~anolamine; (3). authentic 1.2diPalmitoylglycerol: (4), the products after phospholipasc C hydrolysis of phosphoglycolipid; (5), phosphoglycolipid: (6), authentic tripahnitoylglycerol (TG) and 1,3_dipalmitoyklycerol (1.3-DG).

345

The phosphoglycolipid was hydrolyzed by phospholipase AZ, spiitting free fatty acids and lysophosphoglycolipid, whereas phosphoglycolipid was rather resistant to hydrolysis by phospholipase C. Complete hydrolysis by phospholipase C was achieved after 64 h incubation, and thin-layer chromato~phy of the reaction products extracted with chloroform/methanol (2 : 1, by vol.) showed that the only products was l,Zdiacylglycerol with the concomitant loss of phosphoglycolipid (Fig. 3). On the other hand, phospholipase D digestion of 14C-labelled phosphoglycolipid using carrot enzyme formed two radioactive peaks, one large peak corresponding to phosphatidic acid and the other to a neutral lipid, besides the original phosphoglycolipid peak. This indicates the phosphoglycolipid was hydrolyzed to phosphatidic acid and water soluble compounds, and then the resultant phosphatidic acid may be further degraded to diglycerides and phosphate. Furthermore, the thin-layer chromatogram of acetolysis products of phosphoglycolipid shows the formation of 1,2-diacyl-3-acetylglycerol predominantly, as in the cases of other phospholipids [ 343. From the results obtained above, the phosphoglycolipid seemed not to have the glucosyldiacylglycerol structure but have a phosphodi~ylglycerol structure. Therefore, this lipid appeared most likely to be a glucosyl derivative of phosphatidy~lycerol. Fatty acid composition of extractable and bound lipids

Table II shows the fatty acid composition of the individual phospholipids from this bacterium at 42 h-growth. The major fatty acids were palmitic (CM:o), Cl8 monoenoic, Cl9 and C1, cyclopropanoic acids, while Cl8 saturated and C& monoenoic acids were’ present only in a small amount. It is characTABLE

II

FATTY

ACID COMPOSITfON

tr, Trace; Fatty

composition

acid

14: 0 16 : 0 16 : 1 17 : 0 Cyclopropanoic 11 : 0 18 : 0 18 : 1 Cyclopropanoic 19 : 0 Unknown ** Total cvcloptapanoie

Total lipids

OF THE EXTRACTABLE

expressed

LIPIDS

*

as percentage.

Neutral lipids

Phosphatidyl-

Dipho~hat~dylglycerol position

Phosphatidylethanolamine position

position

1

2

1

2

1

2

1

2

&TerIJl

Phosphogfycolipid position

0.3 42.8 1.0 tr

0.9 44.0 tr 0.8

1.5 59.2 tr tr

tr 16.5 1.3 tr

1.1 64.0 0.8 tr

0.5 36.1 0.2 tr

tr 43.0 1.1 tr

1.0 12.8 0.9 tr

tr 58.2 1.1 tr

tr 17.8 2.4 tr

6.5 2.1 3.1

2.3 2.8 5.7

2.6 4.2 2.6

12.1 3.5 5.4

6.1 4.3 2.4

11.9 2.9 1.9

2.0 8.6 2.6

11.6 2.6 3.4

1.9 7.8 4.8

9.3 3.5 5.7

39.2 5.2

24.3 18.5

30.7 -

61.3 -

20.2 1.1

46.3 0.3

42.6 -

67.8 -

25.3 -

61.4 -

45.7

26.6

33.3

73.4

26.3

58.2

44.6

79.4

27.2

70.7

* Cells were incubated in a medium containing 2 M NaCl. ** Contained one to several peaks detected by gas chromatography.

346

teristic

that this bacterium contains a large amount of cyclopropanoic acids Cl,), 45% or more for total extractable lipids, 50% or more for phos(Cl9 pholipids and 26.6% for neutral lipids. The positional distribution of the component fatty acids is also demonstrated. The fatty acids for position 1 of the glycerol moiety are from the lysophospholipids and those of position 2 are from free fatty acids liberated after phospholipase AZ treatment. The l-position of phosphatidyl moiety of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphoglycolipid mainly associated with Cl6 and Cl8 saturated acids (50-70%) with a smaller amount of C16 and C18 monoenoic and C1, and C19 cyclopropanoic acids (30-45%), while the 2position associated with a large amount of C 17 and C19 ~ycloprop~oi~ acids (60-75%). This fact coincided with several reports which showed that saturated fatty acids such as palmitic and stearic acids presented in l-position of phosphatidyl moiety while cyclopropanoic and monoenoic acids presented in 2-position in gram-negative bacteria [ 191, in contrast to gram-positive bacteria 142,431. The fatty acid composition varied with the growth stages dramatically. The younger cells contained more C16 and Cl8 monoenoic acids, while the cells of late log stages contained C17 and C19 cyclopropanoic acids up to 10 and 50%, respectively, as demonstrated in Fig. 4. The fatty acid composition of bound lipids differed distinctively from those of extractable lipids and the differenti~ analysis of both ester- and amidelinked fatty acids revealed that a large amount of hydroxy (C,,) and shorter chain (CIO-C14) fatty acids occurred. The mass spectra of the most abundant hydroxy fatty acid methyl ester, postulated tentatively as 3-hydroxydodecanoic acid from the retention times on gas-liquid chromatography, is demon+

20 Time

40 of growth

! h)

Fig. 4. Changes in the fatty acid composition of extractable lipids during the growth of moderately baloNumbers in figures indicate philic bacterium No. 101. Dotted line shows growth of the cells (A550nm). the fatty acid carbon and double bond numbers. CPA. cyclopropanoic fatty acids.

347

Fig. 5. Mass spectra of major hydroxy fatty acid methyl esters obtained from bound Iipids of moderately halophi& bacterium No. 101. upper: methyl 3-hydroxydodecanoate. lower; trimethylsiIy1 derivative of the upper ester. The conditions for mass spectrometry are described in the text.

TABLE III FATTY ACID COMPOSITION OF THE BOUND LIPIDS * tr. Trace; composition expressed as percentage. Fatty acid

10 : 0 3-OH-10 : 0 12 : 0 12 : 1 3-OH-12 : 0 14 : 0 14 : 1 3-OH-14 : 0 16 : 0 16 : 1 Cyclopropanoic 17 : 0 18 : 1 Cyclopropanoic 19 : 0 Unknown **

Whole ceII total bound lipids

-

CeII envelope

Outer membrane

Lipopolysaccharide

Ester

Amide

Ester

Amide

Ester

Amide

tr 5.7 tr

tr -

tr 21.7 tr

tr -

tr 30.7 tr

tr 92.7

5.0

8.7 1.9 32.2 1.4 3.6 tr 19.2 1.3

16.8 0.7 2.3

92.8 -

39.0 4.4 5.9

86.7 -

61.8 2.4 0.7

tr 31.2 2.9

tr

tr -

tr 2.4 0.5

tr

-

tr 20.9 2.6

2.0 2.1

17.7

-

-

-

-

-

-

-

-

22,7

-

22.6 -

3.2

2.5

5.5

* Cells were incubated in.a medium containing 2 M NaCl. ** Contained one to several peaks detected on gas chromatogram.

-

-

4.2

1.4

-

2.0

348

&rated in Fig. 5a and 5b. This shows base peak at m/e 103 due to [ CH -CHI

-

OH COZ-CHj], fragment peak at m/e 157 EM-73 3, m/e 180 EM-503 and molecular ion peak at m/e 230, indicating methyl-3-hydroxydodecanoate. On the other hand, the mass spectra of the Me,Si-derivative of hydroxydodecanoic acid methyl ester was also demonstrated. The molecular weight is indicated at m/e 302 [M] + and more intense peak at m/e 287 due to loss of methyl. The base peak, m/e 175, appeared to be due to C3-C4 cleavage of 3-0-Me,Si methyl ester, this supporting the 3-hydroxy fatty acid structure. The results obtained were summarized in Table III. It was noted that 3-hydroxydodecanoic acid appeared to be associated specifically with amide linkages to the lipopolysaccharide molecule. The detailed structural analysis of lipopolysaccharide obtained from No. 101”W3 will be reported elsewhere. Discussion A moderately halophilic gram-negative rod, No. 101-W3 was originally isolated from rock salts in 1949 f 241. As reported previously, the most characteristic point of this bacterium is that the outer membrane fragment can be liberated easily when the cells were in a sucrose solution which protected their lysis in a demineralized water [20,21]. On the other hand, the isolated cell envelopes have been shown to possess a strikingly high Na’ content [ 221. Since the membrane phospholipids have a variety of roles such as barriers or cofactors for membrane bound enzymes, we have tried to find out specific components. The acid hydrolysis of the outer membrane preparation yielded a high concentration of acidic amino acids [21] and the extractable lipids contained abundant amounts of acidic phospholipids. Since Hopper et al. [45] and Papahadjopoulos et al. [46] found that the negatively charged phospholipid bilayers were more permeable for cations than positively charged ones, the occurrence of anionic phospholipids in high concentration may contribute to the regulatory mechanism of the permeation barrier in cationic environments. The changes of cellular phospholipid composition, increase in phosphatidylglycerol and decrease in phosphatidyleth~olamine in higher NaCl medium, may also support this hypothesis. The phospholipid composition of No. 101W3 roughly resembled the other gram-negative slightly halophilic bacilli such as Vibrio [47] and marine Pseudomonads (481, but differed in having an unidentified phosphoglycolipid. Peleg and Tietz have already reported the occurrence of glucosylphosphatidylglycerol and glucuronosyldiacylglycerol as major lipid components of a moderately halophilic bacterium [16-191. From the lipid composition, our strain closely resembled this bacterium, however, we could not detect such a glucuronosyldiacylglycerol in any appreciable amount and instead the amount of phosphoglycoplipid increased significantly when glucose was added to the medium. Prom the results obtained with chemical and enzymatic hydrolysis, the phosphogly~olipid which we have isolated appeared most likely to be a glucosyl derivative of phosphatidylglycerol such as that reported by Peleg and Tietz [16-191. They described the phosphoglycolipid as being resistant to phos-

349

pholipase C (from Bacillus cereus) digestion and only less than 40% was hydrolyzed after the treatment overnight. In our case, however, the complete hydrolysis of phosphoglycolipid was achieved and the resultant lipid products were identified as 1,2-diacylglycerol, exclusively. From the substrate specificity of enzymes, this result gave more evidence for supporting the structure of phosphoglycolipid having a phosphatidyl moiety, this differing from phosphoglycolipid possessing glucosyldiacylglycerol structure obtained from various strains of Streptococci [40-431, The detailed structural analysis is now in progress. The fatty acid composition of extractable lipids of No. lOl-W3 is also characteristic in possessing a high concentration of Cl9 cyclopropanoic fatty acid. This also differs in that the extremely halophilic bacteria do not contain entirely fatty acids [8,13]. The physiological functions of cyclopropanoic fatty acids have not been well documented, however, and it is suggested that they may be related to the membrane fluidity and permeability, considered from their physical properties compared with those of unsaturated fatty acids. We have observed that the transient temperature of phosphatidylglycerol or phosphatidylethanolamine possessing mostly saturated and cyclopropanoic fatty acids was 3 to 4°C higher than those possessing saturated and monoenoic fatty acids (unpublished data). The fatty acid composition of lipopolysaccharide from a moderately halophilic bacterium, one of the most characteristic components of the outer membranes in gram-negative bacteria, was first described. The amide-linked fatty acids comprised mostly of 3-hydroxydodecanoic acid and this differs from those of gram-negative enteric bacilli possessing 3-hy~oxymy~stic acid as sole hydroxy fatty acid [48]. The lipid (extractable and bound) and fatty acid composition of moderately halophilic bacteria seemed to be considerably characteristic and these analytical data may present evidence for determining the phylogenetic situation of these bacteria in chemotaxonomy. Acknowledgments We would like to thank Professor K. Saito, Department of Medical ChemOsaka, for the performance of the early stages of this research and also to thank Professor A. Hayashi, Department of Chemistry, Faculty of Science and Technology, Kinki University, HigashiOsaka, Osaka, for his fruitful suggestions.

istry, Kansai Medical School, Mo~~chi,

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Lipids and fatty acids of a moderately halophilic bacterium, No. 101.

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were ...
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