JOURNAL OF BACTERIOLOGY, Dec. 1974, p. 999-1003Copyright ( 1974 American Society for Microbiology

Vol. 120, No. 3Printed in U.S.A.

Effect of pH on the Proportions of Polar Lipids, in ChemostatCultures of Bacillus subtilis

D. E. MINNIKIN AND H. ABDOLRAHIMZADEH

Department of Organic Chemistry, The University, Newcastle upon Tyne NE] 7RU, Great Britain

Received for publication 6 September 1974

Significant changes in the relative proportions of the individual polar lipids oftwo strains of Bacillus subtilis were observed when the pH of their chemostatcultures was varied. In phosphate- and magnesium-limited cultures of B. subtilisvar. niger NCIB 8058, lysylphosphatidylglycerol was present in higher propor-

tions at low pH (5.1) than at neutral pH. With magnesium-limited cultures ofthis strain harvested at pH 8.0, lysylphosphatidylglycerol and phosphatidyleth-anolamine were not detected. Phosphate-limited cultures of B. subtilis NCIB3610 contained no phosphatidylethanolamine or lysylphosphatidylglycerol atneutral pH, but at low pH (5.1) both these lipids were present in substantialproportions. The proportions of phosphatidylglycerol in actively dividing cells ofchemostat cultures of bacilli were always greater than those of lysylphosphatidyl-glycerol. The reverse is commonly found in batch cultures of bacilli andstaphylococci harvested at low pH. Changes in the proportions of the other polarlipids present in these bacilli (diphosphatidylglycerol and diglucosyl diacyl-glycerol) with pH were also noted. Certain cultures of both strains of B. subtiliscontained small proportions of a peptidolipid.

Certain strains of Bacillus subtilis, Staphylo-coccus aureus, and other gram-positive bacte-ria, when cultivated in or exposed to media oflow pH, produce substantially increased propor-tions of lysylphosphatidylglycerol (lysyl-PG) atthe expense of phosphatidylglycerol (PG) (12,13, 22). The exact physiological role of aminoacid esters of PG in bacteria has not beenconclusively determined, but a body of evidenceexists which suggests that the production ofpositively charged phospholipids such as lysyl-PG may be linked to the control of the cationpermeability at the gram-positive bacterialmembrane (9, 11). In studying the effect of aparticular environmental parameter on mem-brane lipid composition, it is extremely impor-tant to ensure as far as possible that only asingle parameter is varied at one time. Anapproach to such ideal conditions is difficultwhen batch cultures are employed, but the useof continuous-flow chemostat cultures allows fora more exact control of the growth environment.Previous studies on the effect of the growthmedium pH on the proportion of lysyl-PG in themembranes of gram-positive bacteria have in-volved the use of batch cultures prepared on avariety of different media. The use of chemo-stats should certainly be a means wherebyinfluences of pH on membrane lipid composi-tion might be clarified.

MATERIALS AND METHODS

Growth of bacteria. Phosphate- and magnesium-limited cultures of B. subtilis var. niger NCIB 8058and phosphate-limited continuous cultures of B.subtilis NCIB 3610 were prepared in a 600-ml chemo-stat at a temperature of 37 C and a dilution rate of 0.2per h (20). Media containing phosphate or magnesiumions as growth-limiting nutrients were prepared asdescribed previously (6). The pH of the culture fluidwas maintained at close to pH 5, 7, or 8 by theautomatic addition of 2 N ammonia solution. Contin-uous cultures were kept for at least 48 h in a steadystate before samples (300 ml) were removed, and thecells were harvested by centrifugation. Under eachparticular nutrient limitation and pH condition atleast two samples were taken. Following a shift in pHfrom 7 to 5 or 8, the culture was returned to pH 7 sothat the reversibility of any changes in lipid composi-tion might be assessed. A batch culture (1 liter) of B.subtilis var. niger was prepared (16 h) at 37 C on agyratory shaker in a medium shown previously tosupport the production of lysyl-PG at low pH (13, 22).Cells from one-half of this batch culture (pH 5.4) wereharvested immediately, but the pH of the other halfwas raised quickly to 8.0 by the addition of 2 Nammonia before the cells were harvested.

Lipid analysis. Lipids were extracted from cellpellets by a modified Bligh and Dyer procedure (27).Polar lipids were analyzed by two-dimensional thin-layer chromatography on silica gel (Merck PF254 )impregnated with sodium acetate, as described previ-ously (17). Individual lipids were identified by co-

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MINNIKIN AND ABDOLRAHIMZADEH

chromatography with authentic standards and bytheir reactions with specific spray reagents (18). Alllipids were revealed by spraying the chromatogramswith sulfuric acid diluted to 70% (wt/vol) with asaturated aqueous solution of potassium dichromatefollowed by charring at 200 C for 45 min (21). Therelative proportions of individual polar lipid compo-nents were estimated by densitometry (Joyce-LoeblChromoscan) of the charred plates. This procedurehas been shown to give reproducible quantitativeanalyses of phospholipids provided that, as in thepresent case, only minor proportions of unsaturatedfatty acids are present (21). The densitometric re-sponse of glycosyl diacylglycerols and peptidolipidshas not been systematically investigated, and thequantitative data obtained for these lipids do not,therefore, bear exact comparison with that for thephospholipids. This inexactitude does not, however,detract from the utility of the densitometric methodof demonstrating large changes in lipid composition.Each lipid sample was analyzed in triplicate usingslightly different sample loadings on each chromato-gram until three consistent results were obtained. Theprecision of the quantitative analysis by densitometryranged between 0 and ±5%.

RESULTSThe polar lipid patterns obtained from che-

mostat-grown cells of B. subtilis var. nigerprepared at different pH values are shown inFig. 1. The chromatograms presented in Fig. 1were chosen as representing a good mean of allthe results obtained for different lipid samples.The previous history of the culture had nosignificant effect on its lipid composition. Forexample, cells harvested at pH 7.0 before andafter the pH was changed to another value hadpractically the same polar lipid pattern. Theoverall lipid composition was in general agree-ment with published data for strains of B.subtilis (7) and the phospholipids were appar-ently identical to those of another strain of B.subtilis var. niger (2). Phosphate-limited cul-tures contained a small amount of a componentcorresponding to the peptidolipid encounteredin similar cultures of B. subtilis NCIB 3610 (20)(Fig. 2) and traces of a more polar glycolipid (?)were also noted. The polar lipid composition ofthe two halves of the batch culture of B. subtilisvar. niger were identical (phosphatidyleth-anolamine [PE], 33%; PG, 11%; diphospha-tidylglycerol [DPG], 5%; lysyl-PG, 28%;diglucosyl diacylglycerol [DGDG], 23%), thusindicating that differences in pH during lipidextraction have no significant effect on the pro-portions of lysyl-PG.The polar lipid composition of phosphate-

limited B. subtilis NCIB 3610 at 7.3 (Fig. 2b)was similar to that recorded previously (20) at aslightly lower pH. The most notable feature of

the lipids of such cultures was the absence ofPE. Magnesium-limited cultures of B. subtilisNCIB 3610 also contained no PE at pH 7 (20). Areduction in the pH of the phosphate-limitedculture to 5.1 produced a dramatic change inlipid composition (Fig. 2a),- PE and lysyl-PGbecoming significant components. The polarlipid composition of these two strains of B.subtilis was different when cultivated underclosely comparable chemostat conditions. PEand lysyl-PG occurred in all chemostat culturesof B. subtilis var. niger, except the magnesium-limited cells harvested at pH 8.0 (Fig. le).These two polar lipids were observed to beinsignificant components of the lipids derivedfrom chemostat cultures of B. subtilis NCIB3610 at neutral pH (Fig. 2b) (20). Circum-stances did not allow the preparation of mag-nesium-limited cultures of B. subtilis NCIB3610 at low pH for further comparative studies.

DISCUSSIONMagnesium-limited cultures of B. subtilis

var. niger contained significantly higher propor-tions of PG in comparison with phosphate-limited cultures. This finding is consistent withthe previous suggestion (20) that this lipid mayhave an important role in the binding andassimilation of metal ions in gram-positivebacteria. Under conditions of both phosphateand magnesium limitation, lysyl-PG increasedas a proportion of the total polar lipids as thepH was reduced. This result supports the origi-nal suggestion (12) that production of thispositively charged lipid is a physiological re-sponse to increased proton concentrations in theexternal environment. Artificial membranescomposed of lysyl-PG were found (11) to be veryimpermeable to protons. Similar membranesprepared from the acidic phospholipids PG andDPG were cation selective (11). Liposomescomposed of lysyl-PG were shown to be moreloosely packed than those prepared from PGand were more permeable to nonelectrolytes,such as erythritol, but less permeable to 86Rb+(9). For intact cells of S. aureus, erythritolpermeability increased with increasing lysyl-PG-to-PG ratio, but valinomycin-mediatedtransport of 86Rb+ decreased when this ratiowas increased. It was concluded that cationpermeability of both model systems and intactcells is dependent on the polar lipid head-groups (9).The proportions of PG in cells from continu-

ous cultures of B. subtilis var. niger and B.subtilis NCIB 3610 were always greater (Fig. 1and 2) than those of lysyl-PG. In the batch

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POLAR LIPIDS IN CULTURES OF B. SUBTILIS STRAINS

PL(7 /0)

DPG; :I

(12 l)PE4(15 04J

PG23 * DGOG(230Jo3 (27%)

0- LysyfI PG(16 OQ

?2

pH51

PE *(1304)

2t 1

a

PL -(i8%)

DPG(25 04)

P043limited

2t:~-dl 1

* DPG(11'/)

Mg2+limfted

P..G* * DGDG(:M'/O ..(32 1)

* LySYI PG7 (14%)

pH 51C-

PE(14 'V.)

PG * :* >DGD(16 'V.) (28 'V.)

S Lysyl PG(9 o%)

pH 70

DPG

PE * (12%)P(6%13)

PG _4~ * DGD.4..).2704)

* Lysyi PG(7/0%)

2*

It--O-1 pH 7-0

t'--A ~~~~~.1~

PG (61 %)

(39o/%)

pH 80

FIG. 1. Two-dimensional thin-layer chromatograms of the polar lipids obtained from chemostat cultures ofB. subtilis var niger. Chloroform-methanol-water (65:25:4, vol/vol) was used in the first direction andchloroform-acetic acid-methanol-water (80:18:12:5, vol/vol) in the second direction. (a) and (b) representphosphate-limited cultures prepared at pH 5.1 and 7.0, respectively. (c), (d), and (e) represent magnesium-limited cultures prepared at pH 5.1, 7.0 and 8.0, respectively. The parentheses show proportions of the lipids asestimated by densitometry after charring (19). Abbreviations: ?, unknown (staining properties of a glycolipid);PL, peptidolipid.

b

2

* * 1

i I

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MINNIKIN AND ABDOLRAHIMZADEH

2

i

2

t

FIG. 2. Two-diimgrams of the polarlimited chemostat cprepared at (a) p1

information refer to

cultures of B. suband in previous stNCIB 3610 (22)lysyl-PG than PCcells harvested atmulates in the n

the stationary pEpH, a high propctively reduce thevery low level. Acuous cultures, hability to accumtance between theproportions of thleast partially, c

membrane to suc:The proportions

tive to pH chant

tures of B. subtilis var. niger (Fig. la and b), butin magnesium-limited cells the proportions of

(17°L) PE decreases to zero as the pH is raised from 5.1to 8.0. A pH change from 5.1 to 7.3 in the

E: ". DPG>3 phosphate-limited cultures of B. subtilis 3610PE DP" ) did, however, lead to the elimination of PE as a(200/.) (18I.0) component of the polar lipids. An apparent

PG - DGDG interrelation between the proportions of PE and(25'IO) (1301.) DGDG in bacilli has been noted (18, 19, 20).

The present results suggest that, under condi-Lysyl PG tions of low pH, PE might possibly make an(7"1.) important contribution to the degree of ioniza-

tion of the membrane surface. An increaseda proportion of PE in batches of Bacillus

pH 5 1 stearothermophilus harvested at low pH hasbeen noted (14). An analogy between increasesin the proportion of PE and amino acid esters of

.. wr PG at low pH has been mentioned (12), and thePL . spossibility of PE giving additional membranetP8°h) stability under conditions unfavorable to

growth was raised (16).DPG PG appears to be closely interrelated with(380/, botb DPG and lysyl-PG. Indeed DPG and

PG43 O1:3GOG:3 lysyl-PG may be considered as modifications ofPGJ DGDG PG which enable the properties of the bacterial

°130I.) (41%) membrane to be adjusted rapidly in response toa change in external environment (23). It maybe significant that the biosynthesis of lysyl-PGinvolves the direct transfer of lysine from lysyltransfer ribonucleic acid to an intact molecule

H 73 b of PG in S. aureus (8), and DPG has been shownpH 7-3 to be formed by the condensation of two PG

tensional thin-layer chromato- molecules in several bacteria (4, 10, 24, 25, 26).lipids obtained from phosphate- The absence of DPG from certain magnesium-cultures of B. subtilis NCIB 3610 limited cultures of bacilli (20) (Fig. le) suggests( 5.1 and (b) pH 7.3. For other that this lipid is less essential than PG in theFig. 1. PL, Peptidolipid. membranes of bacilli. In cultures of B.

stearothermophilis incubated under growth-'tilis var. niger described here, inhibiting conditions, it was noted (3) that thetudies on batches of B. subtilis proportion of PG decreased and that of DPGand S. aureus (9, 13), more increased; a possible influence of DPG on thewas present in the lipids of heat stability of whole cells was mentioned.

low pH. Since lysyl-PG accu- It has been shown that cells from chemostationdividing conditions (15) of cultures of bacilli (5) and S. aureus (1) prepared.ase of batch cultures at low at low pH have higher proportions of ester-)rtion of this lipid may effec- bound alanine in their wall teichoic acids. Suchuptake of positive ions to a changes may well be related to the variations in

'tively dividing cells of contin- the proportions of lysyl-PG found in the presentiowever, must maintain the study. If the esterification of phosphate groupsalate essential cations. A bal- by amino acids is important in control of theamount of lysyl-PG and the metal ion binding capacity of the gram-positive

e other polar lipids may, at bacterial envelope, it appears that at low pH aontrol the selectivity of the partial neutralization of the negative charge ofh ions. the wall teichoic acid is sufficient, but a reversals of PE and DGDG are insensi- of the charge is preferable for at least some ofges in phosphate-limited cul- the phospholipids of the plasma membrane.

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LITERATURE CITED

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