Bacillus subtilis Mutant with Temperature-Sensitive Net Synthesis of ...

JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 473-484 Copyright c 1977 American Society for Microbiology

Vol. 132, No. 2 Printed in U.S.A.

Bacillus subtilis Mutant with Temperature-Sensitive Net Synthesis of Phosphatidylethanolamine VIVEKA LINDGREN,* ERIK HOLMGREN, AND LARS RUTBERG Department of Bacteriology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Received for publication 9 June 1977

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45°C, whereas the mutant did only at 32°C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45°C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation. The cytoplasmic membrane is the major membrane structure in most true bacteria. It is also the dominant lipid-containing structure in these cells. The lipids serve to give the membrane its characteristic hydrophobic and fluid properties, and different classes of lipids are required for the proper functioning of various membrane-bound enzymes (e.g., 26, 31, 33, 37, 38, 47, 48). The lipid classes found in various bacteria are of taxonomic significance (19, 25), but the relative amounts of different lipids in a bacterial species vary with the growth conditions (7, 11, 12, 46, 49). Various types of temperature-sensitive (Ts) mutants affected in lipid metabolism have been isolated from Escherichia coli, e.g., mutants with temperature-sensitive acylation of snglycerol 3-phosphate (G3P) (15, 16) and mutants with a temperature-sensitive phosphatidylserine (PS) synthetase (L-serine:CDP-diglyceride transferase, EC 2.7.8.8) (40-42) or phosphatidylserine decarboxylase (21, 22). The membrane structure of E. coli is comparatively complex. Besides the cytoplasmic membrane, these bacteria also contain an outer lipoprotein membrane. This complicates studies of the role of various lipid classes in cellular functions. Patterson and Lennarz have studied phospholipid metabolism in a Bacillus species (39). The pathways for synthesis of phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) are similar to those for E. coli (27; Fig. 1),

whereas the mechanism for cardiolipin (CL) synthesis has not been established in bacilli. In E. coli (23), Staphylococcus aureus (50), and Lactobacillus plantarum (10), CL is formed by fusion of two PG molecules, with release of one glycerol moiety. The cytoplasmic membrane is essentially the only membrane structure in vegetative cells of B. subtilis, and it contains most (if not all) of the cell phospholipids. PG, CL, PE, and lysylPG are the dominant phospholipid classes in B. subtilis (8, 43). B. subtilis is a spore-forming organism. The effects of biochemically defined alterations in phospholipid metabolism on sporulation and spore germination should be of interest since these processes are connected with major alterations of the cell surfaces. MATERIALS AND METHODS Bacterial strains. The bacterial strains are listed in Table 1. They are all derivatives of B. subtilis 168. Strain 3G18 is characterized by a higher transformability than that of B. subtilis wild type (9). Strain KA2 is an Ade+ recombinant obtained by transforming 3G18 with the deoxyribonucleic acid from strain 168. Strain KA3 was constructed by congression; i.e., 3G18 was transformed with a saturating concentration of deoxyribonucleic acid from the temperature-sensitive mutant KA1, and Ade+ transformants were selected and scored for the unselected temperature-sensitive character. The asaA mutation in strain QUA73 is a point mutation and makes the bacteria sensitive to 10-3 473

474

LINDGREN, HOLMGREN, AND RUTBERG

TABLE 1. Bacterial strains'

PA CTP I PPi gLcr -

CDP-diglyceride

L-serine

G3P CMP

PE

( PGP) Pi

Strain BR102 KA1 3G18 KA2 KA3

QUA73 QB935

CMP

PS 3:C 2

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5

PG

FIG. 1. Pathways for PE and PG syntheses; adopted from reference 39. Phosphatidylglycerol phosphate (PGP) has not been detected in Bacillus membranes (39). PPi, Inorganic pyrophosphate; Pi, inorganic phosphate. The reactions are catalyzed by the following enzymes: (1) CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41); (2) L-serine:CDP-diglyceride transferase (EC 2.7.8.8); (3) PS decarboxylase; (4) G3P:CDP-diglyceride transferase (EC 2.7.8.5); and (5) PGP phosphohydrolase (EC 3.1 .3.2 7 M arsenate (1). The QB935 strain belongs to a kit of standard reference strains constructed for mapping new mutations on the B. subtilis chromosome (R. A. Dedonder, J.-A. Lepesant, J. Lepesant-Kejzlarova, A. Billault, and F. Kunst, Abstr. 3rd Eur. Meet. Bacterial Transformation Transfection, 1976, p. 30). Media. PB is Penassay broth (antibiotic medium no. 3) from Difco Laboratories, Detroit, Mich. TBAB plates were made from Difco tryptose blood agar base. Min-CH medium consists of the minimal salt solution described by Anagnostopoulos and Spizizen (4) supplemented with glucose (5 g/liter), Difco casein hydrolysate (0.5 g/liter), 10-5 M MnCl2, and L-tryptophan (20 mg/liter). Minimal agar contains the components of Min-CH but with casein hydrolysate omitted and is supplemented with 20 mg of required amino acids per liter. The medium is solidified with 18 g of Difco Noble agar per liter. Asa+ and Asa phenotypes were scored on arsenatesupplemented nutrient agar (1). Mutant isolation. Strain BR102 was treated with 100 ,g of N-methyl-N'-nitro-N-nitrosoguanidine (K & K Laboratories, Inc., Hollywood, Calif.) per ml for 20 min by the method of Adelberg et al. (2). After nitrosoguanidine treatment, the bacteria were washed once with 0.1 M phosphate buffer (pH 7.4), inoculated in PB, and grown overnight at 32'C in an incubator shaker (gyratory model G25, New Brunswick Scientific Co., Inc., New Brunswick, N.J.) to allow phenotypic expression of mutations. The mutagenized cultures were then appropriately diluted and spread on TBAB plates, which were incubated at 32'C until the next day. Temperaturesensitive mutants growing on TBAB at 32'C but not at 45°C were subsequently isolated by replica plating. After purification of the temperature-

Genotype trpC2 hisB trpC2 hisB Ts39-2 trpC2 met ade trpC2 met trpC2 met Ts39-2

Origin or derivation J. Spizizen BR102, NTG G. Venema 3G18, 168-DNA 3G18, KA1-DNA nic-l asaA4 aroD4 A. Adams-Lindahl trpC2 lys-1 aroD120 R. Dedonder

a Strain 3G18 is characterized by a higher transformability than that of wild-type B. subtilis (9). Strain KA2 is an Ade+ recombinant obtained by transforming strain 3G18 with strain 168 deoxyribonucleic acid (DNA). Strain KA3 was constructed by congression; i.e., 3G18 was transformed with a saturating concentration of DNA from the temperature-sensitive mutant KA1, Ade' transformants were selected and scored for the unselected temperature-sensitive character. NTG, Nitrosoguanidine.

sensitive clones, it was determined that they retained the nutritional requirements of the parental strain, BR102. Other genetic techniques. Deoxyribonucleic acid was extracted essentially by the method of Marmur

(36). The growth regimen described by Bron and Venema was followed to obtain 3G18 bacteria competent for transformation (9K. Strain QUA73 was grown to competence by the method of Anagnosto-

poulos and Spizizen (4). Transducing PBS1 lysates were prepared as described by Young et al. (53). PBS1-mediated transduction crosses were performed by the protocol of Hoch (24). Growth of bacteria and lipid extraction for screening of phospholipid composition. The bacteria were inoculated into baffled Erlenmeyer flasks containing 0.10 the flask volume of PB and 10 ,zCi of [2--3H]glycerol (specific activity, 202 mCi/mmol) per mol or 5 1Ci of VH]acetate (specific activity, 686 mCi/mmol) per ml. The starting cell density was 1 x 10' to 3 x 107 bacteria per ml, corresponding to an absorbance at 650 nm of 0.2 to 0.3. The flasks were incubated at 32tC with shaking (200 rpm) in a G25 incubator until an absorbance value at 640 nm of about 1 was reached. The cultures were then centrifuged. ln the experiments quoted in Tables 3 and 4, half of the bacteria were subjected to lipid extraction. Those remaining were diluted 2.5-fold into fresh PB containing unlabeled glycerol or acetate at a 100-fold-larger concentration than that of the labeled compound in the preceding culture. After 20 min of growth at 32"C, the temperature was raised to 45-C, and 20 min later 1 j.4Ci of 12-'4C]acetate (specific activity, 58 mCi/mmol) per ml or 0.5 A.LCi of L1,3-'4C]glycerol (specific activity, 56 mCi/mmol) per ml was added. The bacteria were then grown for another 90 min at 45-C. The amount of isotope added was in excess. Before the bacteria were harvested, after the 90-min growth period in the presence of the '4C-labeled compounds, samples were taken as controls for the temperature-sensitive character of the bacteria. The bacteria were then centrifuged and washed once with an equal volume of 0.99k NaCl. Lipids were then extracted from whole cells as described by Ames (3). A 100-ml PB

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TEMPERATURE-SENSITIVE SYNTHESIS OF PE

culture gave 0.4 ml of lipid extract. Analysis of lipid extracts by TLC. Precoated silica gel 60 thin-layer chromatography (TLC) plates (Merck) were activated for 1 h at 120°C. A 25-A.l portion of the lipid extract was applied to each of two TLC plates. In all routine assays the lipid extracts were chromatographed in chloroform-methanol-acetic acid (65:25:8 [vol/vol], solvent system A). The other solvent systems used were: (B) chloroform-methanol-acetone-acetic acid-water (50:10:20:10:5, vol/vol); (C) chloroform-methanol-water (65:25:4, vol/vol); and (D) chloroform-methanol-7 M ammonia (60:35:5, vol/vol). One of the TLC plates was then sprayed with 0.5% ninhydrin in butanol for localization of amino groups and with the phosphate ester reagent described by Dittmer and Lester (17). The other TLC plate was used for radioautography. The film (No-Screen film from Agfa-Gevaert) was exposed to the plate for about 70 h. The spots on the TLC plate were then localized by comparison with the film and also by exposing the plate to iodine vapor (51). The spots were scraped off the TLC plates directly into scintillation vials containing 10 ml of Aquasol (New England Nuclear Chemicals GmbH, Dreieichenhain, West Germany). Radioactivity was counted in a Nuclear-Chicago Mark I liquid scintillator computer, model 60. Spillover of '4C activity into the 3H channel was 25 to 30% for samples from spots with an Rf value less than 0.7 and 10 to 15% for samples from spots with a higher Rf value. All 3H activities given are corrected for spillover. Recovery of radioactivity from unfractionated lipid extracts on silica gel in Aquasol was about 50% for both 3H and 14C (data not shown). The fact that different phospholipid classes may be eluted differently by Aquasol (32) does not influence the interpretation of the results, since all experiments were performed as comparisons between lipids from wild-type and mutant bacteria. Elution of phospholipids from TLC silica gel. The phospholipid fractions were scraped off the TLC plate after localization by radioautography and were then eluted from the silica gel with 6 ml of chloroform-methanol (1:1, vol/vol) followed by 6 ml of methanol (3). Except for the phospholipid designated PX NH2 (fraction I of BR102 phospholipids), less than 10% of the radioactivity remained with the silica gel after elution (data not shown). Growth of bacteria and preparation of membranes for enzyme assays. The bacteria were grown in 8 liters of PB in a stirred fermentor at 32°C. Aeration of the cultures was 6 liters/min. Before cell harvest in late exponential growth phase, the temperature-sensitive character and nutritional requirements of the bacteria were controlled by plating on appropriate agar media. After the cells were washed with 1.5 liters of 0.1 M potassium phosphate buffer (pH 7.4), they were kept at -78°C overnight. The next day the cells were thawed and suspended in 500 ml of 0.05 M potassium phosphate buffer (pH 8.0) with 300 ,ig of lysozyme per ml. The suspension was incubated at 32°C with stirring for 30 min. Deoxyribonuclease I (from bovine pancreas; 10 gg/ ml) and 10 mM MgSO4 were then added, and incubation continued for 20 min at 32°C. Membranes

475

and remaining whole cells were collected by centrifugation at 45,000 x g for 30 min and then suspended in two 10-ml portions of 0.05 M potassium phosphate buffer (pH 7.4) containing 0.5 mM dithiothreitol (buffer A). Each 10-ml sample was sonically oscillated, while chilling in an ice bath, for two 30-s bursts with a 1-min interval in a 100-W ultrasonic disintegrator (Measuring & Scientific Equipment Ltd., London) operating at maximum output. The sonically treated samples were pooled and centrifuged at 3,000 x g for 30 min to remove whole cells. Membranes were collected from the supernatant by centrifugation at 100,000 x g for 30 min and then washed twice with 5 ml of buffer A. Finally, the membranes were suspended in buffer A at a protein concentration of about 15 mg/ml, and the suspension was divided into 0.5-ml samples, which were stored at -78°C. The protein concentration of the membrane preparations was determined by the method of Lowry et al. (34). The number of viable cells in the membrane preparations did not exceed 1,000/ml. Enzyme assays. In vitro net synthesis of PS, PE, and PG was assayed by the incorporation of 14Clabeled water-soluble precursor (i.e., uniformly labeled L-serine or G3P) as described by Patterson and Lennarz (experiment II in Table V'I, reference 39). For assay of PS and PE net synthesis, 0.5 mM L-[U-_4C]serine (5 mCi/mmol) was added at zero time to a mixture containing 25 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 8.0), 250 mM KCl, 4 mM MgCl2, 0.125 mM cytidine 5'triphosphate (CTP), and 1.0 mM phosphatidic acid in a sonically oscillated water dispersion (or 0.135 mM sonically oscillated cytidine 5'-diphosphate [CDP]-diglyceride instead of CTP and phosphatidic acid) plus membranes (about 1 mg of membrane protein) in a total volume of 0.4 ml. When CDP-diglyceride was used as a substrate, the complete assay system contained 0.25% Triton X-100. In the assay with phosphatidic acid and CTP as the substrates, the detergent was omitted. For the assay of PG synthesis, 0.5 mM [U-14C]G3P was substituted for [14C]serine. A control containing membranes preincubated at 100°C for 20 min was always included. The assay was performed at 32°C and stopped by adding 6 ml of chloroform-methanol (2:1, vol/vol containing unlabeled carrier lipids from B. subtilis BR102. Lipid extraction was performed as described previously (39). After the chloroform phase was washed with 4.5 ml of methanol-0.9% NaCl (pH 7.0; 1:1.25, vol/vol), 3 ml of the chloroform phase was taken for evaporation. The residual material was dissolved in 0.1 ml of chloroform, and the whole sample was fractionated by TLC in solvent system A. The spots were localized by radioautography and by exposure to iodine vapor and were scraped directly into scintillation vials with 10 ml of Aquasol; radioactivity was then counted. In the assay of PS and PE net synthesis, only two radioactive fractions were found in TLC; both fractions gave positive reactions with ninhydrin and the phosphate ester reagents and had Rf values corresponding to PS and PE. TLC fractionation of samples from the assay of PG and CL net synthesis gave three radioactive

476

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LINDGREN, HOLMGREN, AND RUTBERG

spots. Two reacted with the phosphate ester reagent (but not with ninhydrin) and had Rf values corresponding to PG and CL. The third radioactive spot did not react with the phosphate ester reagent or

with ninhydrin and had an Rf value higher than those of the phospholipids. Enzyme activities are given as counts per hour per milligram of protein, since exact quenching values are not known. Chemicals. Phosphatidic acid was purchased from Supelco, Inc., Bellefonte, Pa.; CTP, CDP, diglyceride, Triton X-100, dithiothreitol, and deoxyribonuclease were from Sigma Chemical Co., St. Louis, Mo.; [3H]acetate, [1,3-'4C]glycerol, [U14C]glycerol 3-phosphate, and L-[U-_4C]serine were from New England Nuclear Chemicals GmbH; [23H]glycerol and [2-'4C]acetate were from The Radiochemical Centre, Amersham, England.

RESULTS

values for fraction I were obtained by applying the unfractionated BR102 lipid extract on TLC plates, which were then developed in one dimension in the four solvent systems. Since only fraction I and fraction II were ninhydrin positive, fraction I could be identified in the different solvent systems. On the basis of the chromatographic properties of fractions II, III, and IV and their reactions with ninhydrin and the phosphate ester reagent, fractions II, III, and IV were identified as PE, PG, and CL, respectively. These three phospholipids have previously been identified as major phospholipids in B. subtilis (5, 8, 43). The structure of an amino group-containing phospholipid other than PE has previously been shown to be lysyl-PG (43) and is probably represented by fraction I of the BR102 lipid

Characterization of phospholipids in B. extract. In addition to the phosphate-containing fracsubtilis BR102. TLC of lipid extracts from B. subtilis BR102 grown in PB in the presence of tions, there was one fraction, detected by ra['-'C]acetate revealed four phosphate-contain- dioautography, that migrated between PG and CL in solvent system A and four fractions ing fractions, two of which were also ninhydrin positive. BR102 lipids fractionated in solvent having Rf values of 0.8 to 1 in the same solvent system A (the solvent system routinely used in system. These five fractions, which did not further experiments) were scraped from the react with the phosphate ester reagent, were TLC plates, eluted, and rechromatographed in not studied further. Phospholipid composition of B. subtilis two-dimensional TLC with solvent system C in the first dimension and solvent system D in BR102 and temperature-sensitive mutants. the second dimension. Each of the eluted lipid The phospholipid composition of strain BR102 fractions still yielded one spot as revealed by grown at 32°C with [3H]glycerol, shifted to radioautography and by their reactions with 45°C, and then grown with [4C]acetate is ninhydrin and the phosphate ester reagent. shown in Table 3. Tritium activity in the phosFraction II co-chromatographed with standard pholipids from the 45°C cultures represents the PE, fraction III co-chromatographed with stan- sum of net incorporation at 32°C and turnover dard PG, and fraction IV co-chromatographed at 450C. PE, PG, and PX NH, were the predomapproximately with standard CL in four differ- inant phospholipids at both growth temperaent solvent systems (Table 2). The standard tures. In lipid extracts from membrane preparaCL, however, gave a longer trailing spot than fraction IV. Fraction I of BR102 phospholipids tions, the size of the CL fraction is increased eluted inefficiently from the silica gel. The Rf compared with that in extracts from whole TABLE 2. Rf values in TLC of BR102 phospholipids and standard phospholipids R, value in solvent system:

Reaction with:

Phospholipid

BR102 fraction I ........... BR102 fraction II ........... BR102 fraction III .......... BR102 fraction IV .......... Standard PS ............... Standard PE ............... Standard PG .............. Standard CL ............... Standard PA ...............

D-Lb

A

B

C

D

+

0.05

+

+

0.28

-

+

0.44 0.59 0.20 0.29 0.45 0.58-0.77 0.67

0.07 0.28 0.28 0.33 0.15 0.28 0.29 0.32-0.46 ND

0.07 0.33 0.21 0.17

0.17 0.38 0.45 0.50

0.11 0.33 0.23 0.16-0.27 0.25

0.13 0.36 0.44, 0.60 0.41-0 .58 ND

Ninhydrin +

+ + +

+

-

+

+ +

Standard PG gave two phosphate positive spots in solvent system D; the Rf value for the major one was 0.44. Standard CL gave long, trailing spots; the lower and upper boundaries for the spots are given. ND, Not done. bD-L, Phosphate ester reagent of Dittmer and Lester (17). PA, Phosphatidic acid. "


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TABLE 3. Phospholipid composition of strains BR102 and KA1 % 3H at 32°C

% 14C at 45°C

% 3H at 45°C

Phospholipid

BR102

PX NH2 ...................... 20 PS .1 PE .31 PG .30 CL .19

KA1

25 2 27 34 12

BR102

24 2 55 17 2

KA1

BR102

23 4

36 19 19

17 1 45 37 ,

5o 0 ,

3

Minutes

5.0

E

, c

C (D

2 E ° >> o 0

0

100

200 300 Mi n Ut e s

400

FIG. 2. Growth of BR102 (A) and KAI (B) in PB. After 185 min of growth at 32°C, the bactceria were centrifuged and diluted 1:3 into fresh PB Iprewarmed to either 32 or 45°C, and cultivation iwas continued at the respective temperature. Absorba nce

J. BACTERIOL.

5 to 10% for a mutation causing amino acid auxotrophy. This is probably due to counterselection of the temperature-sensitive recombinants, since KAI forms smaller colonies than wild-type bacteria at 32CC. The phospholipid patterns of the temperature-sensitive recombinant KA3 isolated by congression and the temperature-insensitive Ade+ 3G18 recombinant KA2 are shown in Table 4. The temperature-sensitive recombinant had a phospholipid pattern similar to that of the KAI mutant. Similar results, but with an increased CL fraction, were obtained when lipids were extracted from membranes instead of whole cells (data not shown). The results also indicate that PE is metabolically more stable at 450C than is PG, both in the tempera-

ture-sensitive mutant and the wild type. Finally, the lipid composition was analyzed in five temperature-sensitive AroD- recombinants from a PBS1-mediated transduction between an aroD recipient (QB935) and a Ts39-2 donor (KA3). The Ts39-2 mutation is closely linked to aroD (see below). All five temperature-sensitive recombinants showed the same lipid pattern as KA3 and KA1 bacteria, whereas a temperature-insensitive AroD+ recombinant had the wild-type lipid composition (data not shown). Other lipid alterations in KAI and KA3 bacteria. When total lipids were taken into account, it was found that incorporation of labeled precursors at 45CC into the lipid fraction moving with the front in the TLC system was severalfold increased in KA1 and KA3 bacteria compared with that in wild-type bacteria. The chemical composition of the front fraction has not yet been determined. Turnover of PE. To determine whether the low PE content in KA1 and KA3 bacteria grown at 45°C was due to an increased turnover rate of this phospholipid, the following experiments were performed. Strains KA2 and KA3 were grown in PB at 32°C with either L'4C]acetate, [H]acetate, or [14C]glycerol. In the late exponential phase, the cells were centrifuged and lipids were extracted from onehalf of each culture. The other half was inoculated in PB (at 45CC) containing a 1,000-fold excess of unlabeled acetate or a 100-fold excess of unlabeled glycerol. The bacteria were cultured at 45CC for 100 or 110 min, and total lipids were then extracted. at 650 nm (A650) in 32CC cultures (0) and in 45°C cultures (O). Colony-forming units per milliliter in 32°C cultures (A) and in 45CC cultures (A). Colonyforming ability was assayed on TBAB plates incubated at 32°C.

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The PE fraction, labeled at 32°C, was 20 to 40% less stable at 45°C in temperature-sensitive mutants than in temperature-insensitive bacteria (Table 5). The possible increase in PE turnover in the mutants cannot account alone, however, for the more than fivefold decrease in PE net synthesis at 45°C. Effect of hydroxylamine on in vivo phospholipid metabolism. Since an increased rate of PE turnover cannot explain the low content of this phospholipid in strain KA3 (and KAl), PE synthesis was examined further. The immediate precursor for PE in both a Bacillus species (39) and E. coli (27) is PS. Only traces of PS are normally found in lipid extracts from either of these species or from B. subtilis (19). KA1 and KA3 bacteria do not accumulate significant amounts of PS at either 32 or 45°C, indicating that defective decarboxylation of PS is not the (sole) reason for the PE low content. InE. coli, hydroxylamine inhibits PS decarboxylase activity, leading to PS accumulation (42, 45). To determine whether a block in PS decarboxylation could also be detected in B. subtilis, the following experiments were done. Strains KA2 and KA3 were grown at 32°C in PB with [3H]glycerol. The bacteria were then divided into four cultures, two at 45°C and two at 32°C. Twenty minutes later, hydroxylamine was added to one culture of each strain to a final concentration of 4 mM. After another 20 min, L-[U-'4Clserine was added to all cultures, and incubation continued for 80 min. The bacteria were then harvested, and their lipids

479

were extracted and analyzed (Table 6). In the presence of hydroxylamine, KA2 accumulated

'4C label in PS at both 32 and 45°C. In contrast, KA3 accumulated '4C label from serine in PS when grown in the presence of hydroxylamine only at 32°C. Incorporation of trichloroacetic acid-precipitable radioactivity from [U-'-'C]serine into whole cells of mutant KA3 is comparable to that into whole cells of KA2 during the first 40 min after the shift to 450C. These experiments thus show that the low PE content in the mutants is not due (only) to a deficient PS decarboxylase. The results do not rule out the possibility that the Ts39-2 mutation affects the activity of both PS synthetase and PS decarboxylase. In vitro synthesis of phospholipids. Since in vivo synthesis of PG and CL seems normal in KAI and KA3 bacteria grown at 450C, the decreased PE content cannot be caused by a mutation affecting a step in phospholipid synthesis before CDP-diglyceride. Therefore, in vitro net synthesis of PS and PE was assayed with membranes prepared from KA2 and KA3, respectively, grown in PB at 32°C. The general characteristics of the in vitro system are shown in Table 7. There is an absolute requirement for CTP- or CDP-diglyceride. The presence of Triton X-100 gave a relative decrease in PE formation. A similar effect of the detergent Cutscum has previously been reported for a Bacillus species (39). The presence of detergent is essential when CDP-diglyceride is used as a sub-

TABLE 4. Phospholipid composition of strains KA2 and KA30 % 3H at 32°C

% 3H at 45°C

% 14C at 45°C

Phospholipid KA2

KA3

KA2

KA3

KA2

KA3

12 6 32 31 19

20 0 40 39