Vol. 124, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 668-678 Copyright C) 1975 American Society for Microbiology
Peptidoglycan Synthesis in L-Phase Variants of Bacillus licheniformis and Bacillus subtilis J. B. WARD Division of Microbiology, National Institute for Medical Research, London NW7 1AA, England Received for publication 11 June 1975
Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-Lala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-,3-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glumA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-/8-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine. et al. [24] is used) of Streptococcus pyogenes (5) and S. aureus (6, 8), although devoid of an organized cell wall, have been shown to accumulate the nucleotide precursors of peptidoglycan. A second, independently isolated strain of S. aureus H, while not accumulating such precursors, was able to synthesize peptidoglycan when membrane preparations were supplied with the appropriate precursors (3). These observations suggest that stable L-phase variants of the same organism may not have the same lesion in peptidoglycan synthesis. The present study was undertaken to determine at which stage peptidoglycan synthesis was blocked in a number of stable L-phase variants of Bacillus licheniformis and Bacillus subtilis.
The structural integrity of the bacterial cell is maintained by the presence of peptidoglycan in the cell walls. Consequently, the removal of this polymer either by inhibition of the biosynthetic process with one of several substances, including f3-lactam antibiotics, D-cycloserine, glycine, and several D-amino acids, or by hydrolysis of peptidoglycan with suitable lytic enzymes results in the bacterium becoming osmotically fragile (4, 16, 28). Such processes have been used to isolate unstable L-forms from a number of bacteria, including Staphylococcus aureus (5) and various bacilli (7, 40), by growing the organisms on a suitable osmotically protective medium. The conversion of these unstable L-forms to stable L-forms has been found to occur after a variable number of transfers in the presence of the inhibitor. After stabilization has occurred, the L-forms may be grown in the absence of the inhibitor without their reversion to the bacterial form. This method of isolation and the osmotic fragility of these L-forms suggests that they are lacking in some enzyme(s) involved in the biosynthesis of peptidoglycan. L-phase variants (the terminology of McGee
MATERIALS AND METHODS The bacterial strains used were B. subtilis 168 trp C2, B. subtilis W23, B. licheniformis 6346 His-, and B. licheniformis 94 (36), a f3-lactamase-deficient mutant derived from B. licheniformis 6346 MH-3 (9). Certain of the L-phase variants used, including those derived from B. subtilis 168 trp C2 (these will be referred to in the present work by the designation
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PEPTIDOGLYCAN SYNTHESIS IN L-PHASE VARIANTS
Slf and this organism as Slf-1 [39]), B. subtilis 168 trp C2 rod B (Slf-rod B), B. licheniformis 6346 His(Slf-His-) (40), and B. licheniformis 6346 (Slf-wt) (7) have been described previously. Others, including those derived from B. subtilis W23 (Slf-W23) and B. licheniformis 94 (Slf-94), have been isolated during the course of this investigation using methods similar to those described previously (40). In addition, experiments have been carried out using one of the L-phase variants of B. subtilis AC2 trp C2 strr (SlfAC2) obtained by transformation using deoxyribonucleic acid isolated from Slf-1 (39). Media and cultural conditions. The L-phase variants were grown at 35 C with shaking in a modification of DP medium (21) containing 0.5 M sodium succinate, pH 7.3; 0.02 M MgCl2; acid-hydrolyzed casein, 0.5%; K2HPO4, 0.35%; KH2PO4, 0.15%; glucose, 0.5%; and tryptophan, 0.01%. When required, agar (Difco) was added to a final concentration of 0.8%. The L-phase variants were maintained by weekly subculture in DP medium. In addition, stock cultures were also kept in DP medium containing 10% dimethyl sulfoxide held at -150 C in liquid N2. The bacilli, maintained as spores in H20 at 4 C, were grown either on DP medium under identical conditions to the L-phase variants or in tryptoneyeast extract medium containing 1% tryptone, 1% yeast extract (both from Difco), and 0.5% KH2PO4. Experiments to establish auxotrophic requirements of the L-phase variants were carried out using a minimal medium containing 0.5 M sodium succinate, pH 7.3; 0.05 M potassium phosphate buffer, pH 7.3; 0.02 M MgCl2; glucose, 0.5%; arginine, 0.25%; ferrous sulfate-7H2O, 1.0 ,ug/ml; and manganese sulfate, 0.1 gg/ml. Auxotrophic requirements were routinely added to a final concentration of 50 ,ug/ml. Isolation and characterization of nucleotidebound peptidoglycan precursors. Cultures (500 ml) of L-phase variants were harvested by centrifuging at 18,000 x g for 20 min. The cell pellets were then extracted with cold 10% trichloroacetic acid (10 ml) at 0 C for 20 min. Cell debris was removed by centrifuging (10,000 x g for 5 min), and the pellet was re-extracted with trichloroacetic acid (2.5 ml). The supernatants were combined and extracted four times with an equal volume of ether to remove most of the acid. The concentration of "bound" N-acetylhexosamine in the aqueous phase was determined, and the nucleotides were then separated on a column of Dowex-1 (1.5 by 25 cm) equilibrated with 0.01 N HCI. Nucleotides were eluted with a linear gradient of NaCl (to 0.5 M final concentration) in 0.01 N HCI (600 ml). The absorbance of the eluate at 254 nm was followed, and fractions (5 ml) were assayed for bound N-acetylhexosamine. Appropriate fractions were pooled, and the nucleotide precursors were purified by absorption and elution from charcoal (25 mg of charcoal per ,umol of nucleotide). Their purity was further checked by chromatography in solvents A and B. Samples were hydrolyzed and analyzed for amino sugars and amino acids as described below. Paper chromatography and electrophoresis. Whatman 3MM paper was used unless otherwise
669
stated and was washed extensively with 2 M acetic acid and water before use. Descending paper chromatography was carried out in the following solvent systems: A, isobutyric acid-0.5 M NH3 (5:3, vol/vol); B, ethanol-i M ammonium acetate, pH 7.5 (5:2, vol/vol); C, butan-l-ol-acetic acid-water (3:1:1, volV vol/vol). Electrophoresis was carried out at 70 V/cm on Whatman 3MM paper in the following buffer systems: D, pyridine-acetic acid-water (1:10:989, volV vol/val), pH 3.5; E, pyridine-acetic acid-water (50: 2:948, vol/vol/vol), pH 6.5. Nucleotides were located by absorption of ultraviolet light, and amino acids and amino sugars were located with ninhydrin. Analytical methods. Protein was determined by the method of Lowry et al. (22) with bovine serum albumin as the standard. Amino acids and amino sugars were determined with a Beckman-Spinco automatic amino acid analyzer after hydrolysis of the samples in 4 M HCI for 4 h at 100 C for amino sugars and 6 M HCI for 16 h at 100 C for amino acids. The boundN-acetylhexosamine content of nucleotide precursors was determined after hydrolysis of the sample in 0.02 M HCI for 10 min at 100 C (30), using uridine 5'-diphosphate (UDP)-N-acetylglucosamine as the standard. Radioactivity on paper was located by radioautography and counted directly in a Packard Tri-Carb liquid scintillation spectrometer (model 3375), using a toluene-based scintillation fluid [toluene containing 0.4% 2,5-diphenyloxazole and 0.01% 1,4-bis-(4-methyl-5-phenyloxazole-2yl)benzene]. The counting efficiency for 14C was 72%. Preparation of enzymes. (i) Membranes. Membrane preparations for the assay of peptidoglycan synthesis and D-alanine carboxypeptidase activity were prepared as follows. L-phase variants in the late-exponential phase of growth were harvested and osmotically lysed in 50 mM-Tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.5, containing 60 mM MgCl2 and 1 mM dithiothreitol (Tris-Mg-dithiothreitol). After treatment with deoxyribonuclease I (Sigma, grade CL), the membranes were recovered by centrifuging at 38,000 x g for 20 min, washed twice with Tris-Mg-dithiothreitol, and finally resuspended in the same buffer to give a final protein concentration of 10 to 15 mg of protein per ml. Membrane preparations from the parent bacilli were prepared as described previously (36). Membrane preparations to be utilized in the determination of phospho-N-acetylmuramyl pentapeptide translocase were first treated to remove endogenous phospho-N-acetylmuramyl pentapeptide by prior incubation with uridine 5'-monophosphate, as described by Hammes and Neuhaus (17). (ii) Cytoplasmic enzymes. Cell-free extracts to be used for the assay of the various enzymes involved in the synthesis of diaminopimelic acid and the nucleotide precursors of peptidoglycan were made by disruption of the cells by passage of the cell suspension through a French pressure cell. Extracts for the assay of the ligases involved in the synthesis of the nucleotide precursors were prepared in 20 mM potassium phosphate buffer, pH 7.5. Cell debris was removed by centrifuging at 38,000 x g for 20 min, and
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3 volumes of saturated ammonium sulfate (pH 7.5; containing 0.1 mM ethylenediaminetetraacetic acid) were added to the supernatant solutions. After 30 min at 0 C the precipitate was recovered by centrifuging, re-dissolved in 20 mM potassium phosphate buffer, pH 7.5, to give a concentration of approximately 10 mg of protein per ml, and stored at -20 C. Aspartokinase and aspartate semialdehyde dehydrogenase were assayed in extracts prepared as described above to the stage of precipitation with ammonium sulfate. The precipitates were then dissolved in 50 mM diethanolamine-hydrochloride buffer, pH 8.5, containing 1 mM ,8-mercaptoethanol and 1 mM ethylenediaminetetraacetic acid and dialyzed against 100 volumes of the same buffer for 4 h at 2 C before use. Extracts for the assay of dihydrodipicolinate synthase and dihydrodipicolinate reductase were prepared as described for aspartokinase extracts, except that the buffer used was 50 mM Tris-hydrochloride, pH 8.0, containing 10 mM MgCl2 and 1 mM ethylenediaminetetraacetic acid. Finally, transaminase and deacylase (see below) were assayed in extracts prepared in 20 mM potassium phosphate buffer containing 1 mM f-mercaptoethanol and 0.5 mM pyridoxal-5-phosphate and dialyzed against 100 volumes of the same buffer for 4 h at 2 C before use. Enzyme assays. (i) Aspartokinase (adenosine 5' - triphosphate:L - aspartate - 4 - phosphotransfer ase). Aspartokinase (EC 2.7.2.4) was assayed as described by Paulus and Gray (29), and the specific activity of the enzyme preparation was calculated as nanomoles of ,8-aspartohydroxamate formed per milligram of protein per minute using an E520 for /3aspartohydroxamate of 400 (29). (ii) Aspartate semialdehyde dehydrogenase. Aspartate semialdehyde dehydrogenase (EC 1.2.1.11) was assayed essentially as described by Hegeman et al. (18). The specific activity of the enzyme preparation was calculated as nanomoles of nicotinamide adenine dinucleotide phosphate reduced per milligram of protein per minute. (iii) Dihydrodipicolinate synthase. Dihydrodipicolinate synthase (EC 4.2.1.52) was assayed by the O-aminobenzaldehyde method described by Shedlarski (31). One unit of activity was defined as giving an increase of 0.001/min at 540 nm. The specific activity of the enzyme preparations was calculated as units per milligram of protein. (iv) Dihydrodipicolinate reductase. Dihydrodipicolinate reductase was measured as described by Tamir (35). Specific activity of the enzyme preparations was calculated as nanomoles of reduced nicotinamide adenine dinucleotide phosphate oxidized per milligram of protein per minute. All values were corrected for the oxidation of reduced nicotinamide adenine dinucleotide phosphate occurring in the absence of added substrate. (v) N -acetylamino-keto-pimelate:L-glutamate transaminase. Attempts to measure this activity by reversal of the biosynthetic reaction as described by Sundharadas and Gilvarg (34) were unsuccessful. Therefore, the enzyme activity was measured in the forward direction as conversion of N-acetylaminoketo-pimelate, synthesized as described below, to
J. BACTERIOL.
diaminopimelate utilizing the N-acetyl-L-diaminopimelate deacylase also present in the crude enzyme preparations. The reaction mixture contained 0.1 M potassium phosphate buffer, pH 8.3, 1.4 mM N-acetylamino-keto-pimelate, 15 mM Na-L-glutamate, pH 7.0, 0.4 mM CoCl2, and 10 ug of pyridoxal-5-phosphate and enzyme in a volume of 65 ul. After incubation at 37 C, 35 ,ul of concentrated HCl was added, and the diaminopimelic acid formed was determined by reaction with ninhydrin (13), utilizing a heating period of 5 min at 100 C. All values obtained were corrected for zero-time controls, and a requirement for the presence of L-glutamate could be demonstrated. In addition, the crude enzyme preparations used also contained diaminopimelate epimerase and diaminopimelate decarboxylase activity, converting small amounts of the L,L-diaminopimelate formed to lysine. For this reason the specific activity of the transaminase, given as nanomoles of diaminopimelate synthesized per milligram of protein per hour, is underestimated by this amount. (vi) N-acetyl-L-diaminopimelate deacylase. Nacetyl-L-diaminopimelate deacylase was assayed by the method of Sundharadas and Gilvarg (34). Specific activities are given as nanomoles of per hour. (vii) Enzymes involved in the synthesis of UDPN-MurAc peptides. The L-alanine-adding enzyme UDP-MurAc:L-alanine ligase was assayed by the method of Mizuno et al. (26). The remaining adding enzymes, UDP-MurAc-L-ala-D-glu:A2pm ligase, UDP-MurAc-L-ala-D-glu-A2pm:D-ala-D-ala ligase, Dalanyl-D-alanine ligase, and alanine racemase, were assayed essentially as described previously (23). The reaction products were separated by electrophoresis at pH 3.5 and located by radioautography. The specific activities of the enzyme preparations were calculated as nanomoles of nucleotide precursor synthesized per milligram of protein per minute. (viii) Phospho-N-acetylmuramyl pentapeptide translocase. The exchange assay of Hammes and Neuhaus (17) was modified as follows. After incubation at 25 C for 14 min, carrier UDP-MurAc pentapeptide (0.04 ,umol) was added and the complete reaction mixture was chromatographed in solvent A. Spots were located by radioautography and the absorbance of ultraviolet light, appropriate areas were cut out, and the radioactivity was determined. Exchange activity was calculated from the firstorder rate equation for the reaction (33). The rate of exchange is given as moles exchanged per liter per minute. (ix) D-Alanine carboxypeptidase. The previously described (10) assay was modified in that after termination of the reaction the products were separated by electrophoresis at pH 3.5. The specific activity of the enzyme preparations is given as nanomoles of Dalanine released per milligram of protein per hour. (x) Peptidoglycan synthesis. The determination of peptidoglycan synthesis was carried out as described previously for membrane preparations of B. licheniformis (36). The specific activity of the membrane preparations is given as nanomoles of diaminopimelic acid incorporated per milligram of protein per hour. Preparation of substrates. UDP-MurAc, iso-
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PEPTIDOGLYCAN SYNTHESIS IN L-PHASE VARIANTS
lated from a temperature-sensitive mutant of S. aureus TOF 33 (15), was a gift of Anne Wyke of this department. UDP-MurAc-L-alanyl-D-glutamic acid was isolated from lysine-deprived S. aureus H (32).
UDP-MurAc-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine, referred to as UDPMurAc pentapeptide, which was labeled with either [3H]diaminopimelic acid or [1,7-'4C]diaminopimelic acid, was prepared enzymically as described previously (37). Similarly, UDP-MurAc pentapeptide labeled in either both or only the terminal r-alanine residue of the D-alanyl-i-alanine dipeptide was synthesized by the enzymic addition of the radioactive dipeptide to UDP-MurAc-L-alanyl-D-glutamyl-mesodiaminopimelic acid obtained from cultures of B. licheniformis inhibited with D-cycloserine and chloramphenicol (36). Nonradioactive UDP-MurAc-pentapeptide was obtained from cultures of B. licheniformis incubated in the absence of Mg2+ (11). D, L-Aspartate-13-semialdehyde was prepared by ozmolysis of D,L-allyl-glycine as described by Black and Wright (1). The concentration of L-aspartic acid semialdehyde present was determined using partially purified homoserine dehydrogenase from bakers' yeast (2). Dihydrodipicolinate synthase for the generation of dihydrodipicolinate in assays of dihydrodipicolinate reductase was purified from Escherichia coli B to step (iv) (31). The final preparation had an activity of 15.26 U/mg of protein using the imidazole assay. N-acetyl-L-diaminopimelic acid was prepared by acetylation of L,L-diaminopimelic acid as follows. L,L-Diaminopimelic acid (107,umol) (purified by P. J. White; a gift from H. R. Perkins, Department of Microbiology, University of Liverpool) was dissolved in 1.5 ml of water and adjusted to pH 8 with 1 M NaOH. Acetic anhydride (106 ,umol) was added, and after readjusting to pH 8.0 the mixture was kept at 22 C overnight. The mixture was chromatographed on a column (1 by 10 cm) of Dowex 50 (H+) equilibrated with 0.01 M HCl and eluted with a linear gradient (400 ml) of 0.01 to 2.0 M HCl. Fractions were assayed for diaminopimelic acid by the acid ninhydrin method (13), using a heating period of 20 min at 100 C. Appropriate samples were pooled and concentrated, and HCl was removed in vacuo. The pooled material gave a single spot on electrophoresis at both pH 3.5 and 6.5 and on chromatography on Whatman no. 1 paper in solvent C. The yield of Nacetyl-L-diaminopimelate was 50.7 Zimol (47%). N-acetylamino-keto-pimelate was synthesized from N-acetyl-L-diaminopimelate by heating with pyridoxal in the presence of a metal (14). The reaction mixture contained 150 .tmol ofN-acetyl-L-diaminopimelate (diammonium salt), 150 ,umol of aluminium ammonium sulfate, 300 ,mol of pyridoxal hydrochloride, and 3.75 mmol of potassium acetate buffer, pH 4.5, in a total volume of 14 ml. The mixture was heated at 100 C for 20 min, and the slight precipitate which formed was removed by centrifuging. The supernatant solution was passed through a column (0.8 by 12 cm) of Dowex 50 (H+). Fractions (2 ml) were collected and assayed for the presence of an aketo acid (14). Appropriate fractions were pooled, concentrated, and neutralized with KHCO3 to pH
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6.0. N-acetylamino-keto-pimelate was further purified by chromatography on a column (0.8 by 12 cm) of Dowex 1 (Cl-) eluted with a linear gradient (400 ml) of 0.01 to 0.5 M HCl. Fractions (5 ml) were collected and assayed for keto acid as described above. Appropriate fractions were pooled, and HCl was removed in vacuo by repeated evaporation of water. The final product gave a single spot on electrophoresis in buffer E with an R glutamate of 1.51, which was ninhydrin negative and gave a positive reaction for an a-keto acid when sprayed with 0.1% 2,4-dinitrophenyl hydrazine in 2 M HCl followed by alkali. The yield of N-acetylamino-keto-pimelate, assayed as an a-keto acid with pyruvate as the standard, was 28.2 ,umol (18.8%).
RESULTS Cell wall analysis of L-phase variants. Lphase variants (stable L-forms) have been described (20, 25) that continue to synthesize wall material, whereas such material is not detectable in L-phase variants from other organisms (3). To determine into which of these two classes the L-phase variants of B. licheniformis and B. subtilis would fall, cells from overnight cultures were analyzed for the presence of muramic acid and diaminopimelic acid as specific components of the peptidoglycan. In addition, membranes, prepared from similar cultures, were also analyzed. Hydrolysates of both Slf-1 and Slf-wt contained small quantities of muramic acid equivalent to 1.3 and 2.9 ,umol/100 mg (dry weight), respectively. In addition, Slf-wt contained an equivalent amount of diaminopimelic acid, whereas this was not detected in Slf1. Neither muramic acid nor diaminopimelic acid was detected in hydrolysates of the membrane preparations. Accumulation of cell wall precusors by Lphase variants. One possible explanation for the above results was the accumulation by the L-phase variants of UDP-MurAc peptides which, together with UDP-N-acetyl-glucosamine, are the cytoplasmic precursors of peptidoglycan. To investigate this possibility, cultures of the various L-phase variants, grown for 10 and 18 h in DP medium, were analyzed for bound N-acetyl-hexosamine as a measure of accumulated precursor. This was found in all the L-phase variants examined (Table 1), although there were considerable differences in the total amounts detected. Under identical conditions, the parent bacilli accumulated only negligible amounts ofprecursor. In general, continued incubation of the L-phase variants into stationary phase (i.e., 18 h) resulted in lower levels of bound N-acetylhexosamine being present in the cells. This may be due to the release of accumulated material as a consequence of cell lysis. Small quantities of bound N-acetyl-
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TABLE 1. Accumulation of peptidoglycan precursors by L-phase variants of bacilli Bound Organism
L-phase variants Slf-1 Slf-rod B Slf-AC2 Sif-W23 Slf-wt Slf-HisSlf-94 Bacilli B. subtilis 168 B. licheniformis 6346
Precursor accumulated
N-acetylhexosamine (jimol/100 mg
[dry wtl)
Expt Ta
Expt II
UDP-MurAc-L-ala-D-glu UDP-MurAc-Lrala-D-glu UDP-MurAc UDP-MurAc-L-ala-D-glu UDP-MurAc UDP-MurAc-L-ala-D-glu-A2pm-D-ala-D-ala UDP-MurAc-L-ala-D-glu UDP-MurAc-L-ala-D-glu
0.95 0.87 2.92 0.95 1.25 3.68 2.09 1.16
2.17 1.73 5.74
Not determined Not determined
0.13 0.07
1.71 4.08 2.71 1.29
a The organisms were grown in DP medium for 18 h in experiment I and for 10 h in experiment II.
hexosamine were detected in the medium after absorption and elution of the nucleotides from charcoal, particularly with the longer incubation period. However, at no time did the amounts recovered represent more than 10 to 15% of the precursor accumulated by the cells. Accumulation of precursor is not a phenomenon associated with the stationary phase of growth, since bound N-acetylhexosamine was detectable throughout the growth cycle of both Slf-1 and Slf-wt (Fig. 1). In addition, replacement of sodium succinate, the osmotic stabilizing agent in DP medium, with either 0.5 M sucrose or 0.5 M sorbitol did not affect the amount of precursor found. This observation eliminates the high ionic strength of the growth medium as the cause of accumulation. Identification of the accumulated nucleotide precursors. The purified nucleotide precursors, isolated as described in Materials and Methods, were identified by amino sugar and amino acid analysis. Five of the L-phase variants accumulated UDP-MurAc-L-alanyl-D-glutamic acid (UDP-MurAc-dipeptide) (Table 1), although in the case of Slf-AC2 this was the minor product in organisms grown for 18 h. In a second experiment, where cells of Slf-AC2 were harvested after 10 h of incubation, only UDPMurAc was found. UDP-MurAc was also accumulated by Slf-W23, whereas the complete precursor UDP-MurAc-pentapeptide was isolated from Slf-wt. On one occasion, this organism accumulated UDP-MurAc-L-alanyl-D-isoglutamyl-A2pm rather than the complete precursor. Since the L-phase variants were not cloned on solid media prior to these experiments, the pos-
sibility exists that the cultures were mixtures of two L-phase variants. However, it should be noted that the lesions in peptidoglycan synthesis found in these organisms (described in later sections) still provide a rational explanation for the nature of the precursor accumulated. The nature of the accumulated precursors was not altered when several of the L-phase variants were grown in medium containing sucrose or sorbital or in minimal medium. However, in the latter case a two- to threefold reduction occurred in the amount of precursor accumulated. The addition of either cloxacillin (20 ,ug/ml) or D-cycloserine (200 ug/ml) to DP medium had no effect on either the amount or the nature of the accumulated precursor. The parent bacilli incubated under these conditions accumulated the expected UDP-MurAc-pentapeptide and UDP-MurAc-tripeptide. In vitro activities of enzymes involved in the synthesis of UDP-MurAc-pentapeptide. The accumulation of incomplete nucleotide precursors such as observed in six of the seven Lphase variants examined may result from a defect in one of the synthetic enzymes. Such lesions have been described in mutants of E. coli (23) and S. aureus (15). To test this possibility these enzymes were assayed using crude cell-free extracts from the L-phase variants, with the results given in Table 2. The activities of the various enzymes in these extracts were compared with those obtained using similar preparations obtained from the parent bacilli grown under identical conditions. Clearly, the accumulation of UDP-MurAc-dipeptide by the L-phase variants did not result from a defi-
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PEPTIDOGLYCAN SYNTHESIS IN L-PHASE VARIANTS
VOL. 124, 1975 2 SIf-l
SIf - wt
Ii
E
8 E
0
0 0
Q. 0 .
S
0 U
01
2
0
4
r24 0 Time (h.)
6
I
1
I
2
4
6
III
-8V4
FIG. 1. Accumulation of bound N-acetylhexosamine by cultures of Slf-1 and Slf-wt. Cultures of the two Lphase variants were grown in DP medium and the optic density at 600 nm (-) was followed. At the times shown, samples (100 ml) were harvested, and the accumulated bound N-acetylhexosamine (0) was measured as described in Materials and Methods.
TABLE 2. Activity of the enzymes involved in the biosynthesis of UDP-MurAc pentapeptide Enzyme (Sp aCta)
L-Alanine-
Organism
adding enzyme
mesoDiaminopimelic acid-
adding
D-Alanyl-D-
D-Alanyl-D-
alanineadding enzyme
alanine synthetase
934 1,032 720 1,145 1,038 1,219 567 545 437 587
109 115 66 145 83 113 94 171 130 83
Alanine racemase
enzyme
B. subtilis 168 Slf-1 Slf-rod B Slf-AC2 B. subtilis W23 Slf-W23 B. licheniformis 6346 Slf-wt Slf-HisSlf-94
9.7 NDb ND
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 668-678 Copyright C) 1975 American Society for Microbiology
Peptidoglycan Synthesis in L-Phase Variants of Bacillus licheniformis and Bacillus subtilis J. B. WARD Division of Microbiology, National Institute for Medical Research, London NW7 1AA, England Received for publication 11 June 1975
Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-Lala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-,3-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glumA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-/8-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine. et al. [24] is used) of Streptococcus pyogenes (5) and S. aureus (6, 8), although devoid of an organized cell wall, have been shown to accumulate the nucleotide precursors of peptidoglycan. A second, independently isolated strain of S. aureus H, while not accumulating such precursors, was able to synthesize peptidoglycan when membrane preparations were supplied with the appropriate precursors (3). These observations suggest that stable L-phase variants of the same organism may not have the same lesion in peptidoglycan synthesis. The present study was undertaken to determine at which stage peptidoglycan synthesis was blocked in a number of stable L-phase variants of Bacillus licheniformis and Bacillus subtilis.
The structural integrity of the bacterial cell is maintained by the presence of peptidoglycan in the cell walls. Consequently, the removal of this polymer either by inhibition of the biosynthetic process with one of several substances, including f3-lactam antibiotics, D-cycloserine, glycine, and several D-amino acids, or by hydrolysis of peptidoglycan with suitable lytic enzymes results in the bacterium becoming osmotically fragile (4, 16, 28). Such processes have been used to isolate unstable L-forms from a number of bacteria, including Staphylococcus aureus (5) and various bacilli (7, 40), by growing the organisms on a suitable osmotically protective medium. The conversion of these unstable L-forms to stable L-forms has been found to occur after a variable number of transfers in the presence of the inhibitor. After stabilization has occurred, the L-forms may be grown in the absence of the inhibitor without their reversion to the bacterial form. This method of isolation and the osmotic fragility of these L-forms suggests that they are lacking in some enzyme(s) involved in the biosynthesis of peptidoglycan. L-phase variants (the terminology of McGee
MATERIALS AND METHODS The bacterial strains used were B. subtilis 168 trp C2, B. subtilis W23, B. licheniformis 6346 His-, and B. licheniformis 94 (36), a f3-lactamase-deficient mutant derived from B. licheniformis 6346 MH-3 (9). Certain of the L-phase variants used, including those derived from B. subtilis 168 trp C2 (these will be referred to in the present work by the designation
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Slf and this organism as Slf-1 [39]), B. subtilis 168 trp C2 rod B (Slf-rod B), B. licheniformis 6346 His(Slf-His-) (40), and B. licheniformis 6346 (Slf-wt) (7) have been described previously. Others, including those derived from B. subtilis W23 (Slf-W23) and B. licheniformis 94 (Slf-94), have been isolated during the course of this investigation using methods similar to those described previously (40). In addition, experiments have been carried out using one of the L-phase variants of B. subtilis AC2 trp C2 strr (SlfAC2) obtained by transformation using deoxyribonucleic acid isolated from Slf-1 (39). Media and cultural conditions. The L-phase variants were grown at 35 C with shaking in a modification of DP medium (21) containing 0.5 M sodium succinate, pH 7.3; 0.02 M MgCl2; acid-hydrolyzed casein, 0.5%; K2HPO4, 0.35%; KH2PO4, 0.15%; glucose, 0.5%; and tryptophan, 0.01%. When required, agar (Difco) was added to a final concentration of 0.8%. The L-phase variants were maintained by weekly subculture in DP medium. In addition, stock cultures were also kept in DP medium containing 10% dimethyl sulfoxide held at -150 C in liquid N2. The bacilli, maintained as spores in H20 at 4 C, were grown either on DP medium under identical conditions to the L-phase variants or in tryptoneyeast extract medium containing 1% tryptone, 1% yeast extract (both from Difco), and 0.5% KH2PO4. Experiments to establish auxotrophic requirements of the L-phase variants were carried out using a minimal medium containing 0.5 M sodium succinate, pH 7.3; 0.05 M potassium phosphate buffer, pH 7.3; 0.02 M MgCl2; glucose, 0.5%; arginine, 0.25%; ferrous sulfate-7H2O, 1.0 ,ug/ml; and manganese sulfate, 0.1 gg/ml. Auxotrophic requirements were routinely added to a final concentration of 50 ,ug/ml. Isolation and characterization of nucleotidebound peptidoglycan precursors. Cultures (500 ml) of L-phase variants were harvested by centrifuging at 18,000 x g for 20 min. The cell pellets were then extracted with cold 10% trichloroacetic acid (10 ml) at 0 C for 20 min. Cell debris was removed by centrifuging (10,000 x g for 5 min), and the pellet was re-extracted with trichloroacetic acid (2.5 ml). The supernatants were combined and extracted four times with an equal volume of ether to remove most of the acid. The concentration of "bound" N-acetylhexosamine in the aqueous phase was determined, and the nucleotides were then separated on a column of Dowex-1 (1.5 by 25 cm) equilibrated with 0.01 N HCI. Nucleotides were eluted with a linear gradient of NaCl (to 0.5 M final concentration) in 0.01 N HCI (600 ml). The absorbance of the eluate at 254 nm was followed, and fractions (5 ml) were assayed for bound N-acetylhexosamine. Appropriate fractions were pooled, and the nucleotide precursors were purified by absorption and elution from charcoal (25 mg of charcoal per ,umol of nucleotide). Their purity was further checked by chromatography in solvents A and B. Samples were hydrolyzed and analyzed for amino sugars and amino acids as described below. Paper chromatography and electrophoresis. Whatman 3MM paper was used unless otherwise
669
stated and was washed extensively with 2 M acetic acid and water before use. Descending paper chromatography was carried out in the following solvent systems: A, isobutyric acid-0.5 M NH3 (5:3, vol/vol); B, ethanol-i M ammonium acetate, pH 7.5 (5:2, vol/vol); C, butan-l-ol-acetic acid-water (3:1:1, volV vol/vol). Electrophoresis was carried out at 70 V/cm on Whatman 3MM paper in the following buffer systems: D, pyridine-acetic acid-water (1:10:989, volV vol/val), pH 3.5; E, pyridine-acetic acid-water (50: 2:948, vol/vol/vol), pH 6.5. Nucleotides were located by absorption of ultraviolet light, and amino acids and amino sugars were located with ninhydrin. Analytical methods. Protein was determined by the method of Lowry et al. (22) with bovine serum albumin as the standard. Amino acids and amino sugars were determined with a Beckman-Spinco automatic amino acid analyzer after hydrolysis of the samples in 4 M HCI for 4 h at 100 C for amino sugars and 6 M HCI for 16 h at 100 C for amino acids. The boundN-acetylhexosamine content of nucleotide precursors was determined after hydrolysis of the sample in 0.02 M HCI for 10 min at 100 C (30), using uridine 5'-diphosphate (UDP)-N-acetylglucosamine as the standard. Radioactivity on paper was located by radioautography and counted directly in a Packard Tri-Carb liquid scintillation spectrometer (model 3375), using a toluene-based scintillation fluid [toluene containing 0.4% 2,5-diphenyloxazole and 0.01% 1,4-bis-(4-methyl-5-phenyloxazole-2yl)benzene]. The counting efficiency for 14C was 72%. Preparation of enzymes. (i) Membranes. Membrane preparations for the assay of peptidoglycan synthesis and D-alanine carboxypeptidase activity were prepared as follows. L-phase variants in the late-exponential phase of growth were harvested and osmotically lysed in 50 mM-Tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.5, containing 60 mM MgCl2 and 1 mM dithiothreitol (Tris-Mg-dithiothreitol). After treatment with deoxyribonuclease I (Sigma, grade CL), the membranes were recovered by centrifuging at 38,000 x g for 20 min, washed twice with Tris-Mg-dithiothreitol, and finally resuspended in the same buffer to give a final protein concentration of 10 to 15 mg of protein per ml. Membrane preparations from the parent bacilli were prepared as described previously (36). Membrane preparations to be utilized in the determination of phospho-N-acetylmuramyl pentapeptide translocase were first treated to remove endogenous phospho-N-acetylmuramyl pentapeptide by prior incubation with uridine 5'-monophosphate, as described by Hammes and Neuhaus (17). (ii) Cytoplasmic enzymes. Cell-free extracts to be used for the assay of the various enzymes involved in the synthesis of diaminopimelic acid and the nucleotide precursors of peptidoglycan were made by disruption of the cells by passage of the cell suspension through a French pressure cell. Extracts for the assay of the ligases involved in the synthesis of the nucleotide precursors were prepared in 20 mM potassium phosphate buffer, pH 7.5. Cell debris was removed by centrifuging at 38,000 x g for 20 min, and
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3 volumes of saturated ammonium sulfate (pH 7.5; containing 0.1 mM ethylenediaminetetraacetic acid) were added to the supernatant solutions. After 30 min at 0 C the precipitate was recovered by centrifuging, re-dissolved in 20 mM potassium phosphate buffer, pH 7.5, to give a concentration of approximately 10 mg of protein per ml, and stored at -20 C. Aspartokinase and aspartate semialdehyde dehydrogenase were assayed in extracts prepared as described above to the stage of precipitation with ammonium sulfate. The precipitates were then dissolved in 50 mM diethanolamine-hydrochloride buffer, pH 8.5, containing 1 mM ,8-mercaptoethanol and 1 mM ethylenediaminetetraacetic acid and dialyzed against 100 volumes of the same buffer for 4 h at 2 C before use. Extracts for the assay of dihydrodipicolinate synthase and dihydrodipicolinate reductase were prepared as described for aspartokinase extracts, except that the buffer used was 50 mM Tris-hydrochloride, pH 8.0, containing 10 mM MgCl2 and 1 mM ethylenediaminetetraacetic acid. Finally, transaminase and deacylase (see below) were assayed in extracts prepared in 20 mM potassium phosphate buffer containing 1 mM f-mercaptoethanol and 0.5 mM pyridoxal-5-phosphate and dialyzed against 100 volumes of the same buffer for 4 h at 2 C before use. Enzyme assays. (i) Aspartokinase (adenosine 5' - triphosphate:L - aspartate - 4 - phosphotransfer ase). Aspartokinase (EC 2.7.2.4) was assayed as described by Paulus and Gray (29), and the specific activity of the enzyme preparation was calculated as nanomoles of ,8-aspartohydroxamate formed per milligram of protein per minute using an E520 for /3aspartohydroxamate of 400 (29). (ii) Aspartate semialdehyde dehydrogenase. Aspartate semialdehyde dehydrogenase (EC 1.2.1.11) was assayed essentially as described by Hegeman et al. (18). The specific activity of the enzyme preparation was calculated as nanomoles of nicotinamide adenine dinucleotide phosphate reduced per milligram of protein per minute. (iii) Dihydrodipicolinate synthase. Dihydrodipicolinate synthase (EC 4.2.1.52) was assayed by the O-aminobenzaldehyde method described by Shedlarski (31). One unit of activity was defined as giving an increase of 0.001/min at 540 nm. The specific activity of the enzyme preparations was calculated as units per milligram of protein. (iv) Dihydrodipicolinate reductase. Dihydrodipicolinate reductase was measured as described by Tamir (35). Specific activity of the enzyme preparations was calculated as nanomoles of reduced nicotinamide adenine dinucleotide phosphate oxidized per milligram of protein per minute. All values were corrected for the oxidation of reduced nicotinamide adenine dinucleotide phosphate occurring in the absence of added substrate. (v) N -acetylamino-keto-pimelate:L-glutamate transaminase. Attempts to measure this activity by reversal of the biosynthetic reaction as described by Sundharadas and Gilvarg (34) were unsuccessful. Therefore, the enzyme activity was measured in the forward direction as conversion of N-acetylaminoketo-pimelate, synthesized as described below, to
J. BACTERIOL.
diaminopimelate utilizing the N-acetyl-L-diaminopimelate deacylase also present in the crude enzyme preparations. The reaction mixture contained 0.1 M potassium phosphate buffer, pH 8.3, 1.4 mM N-acetylamino-keto-pimelate, 15 mM Na-L-glutamate, pH 7.0, 0.4 mM CoCl2, and 10 ug of pyridoxal-5-phosphate and enzyme in a volume of 65 ul. After incubation at 37 C, 35 ,ul of concentrated HCl was added, and the diaminopimelic acid formed was determined by reaction with ninhydrin (13), utilizing a heating period of 5 min at 100 C. All values obtained were corrected for zero-time controls, and a requirement for the presence of L-glutamate could be demonstrated. In addition, the crude enzyme preparations used also contained diaminopimelate epimerase and diaminopimelate decarboxylase activity, converting small amounts of the L,L-diaminopimelate formed to lysine. For this reason the specific activity of the transaminase, given as nanomoles of diaminopimelate synthesized per milligram of protein per hour, is underestimated by this amount. (vi) N-acetyl-L-diaminopimelate deacylase. Nacetyl-L-diaminopimelate deacylase was assayed by the method of Sundharadas and Gilvarg (34). Specific activities are given as nanomoles of per hour. (vii) Enzymes involved in the synthesis of UDPN-MurAc peptides. The L-alanine-adding enzyme UDP-MurAc:L-alanine ligase was assayed by the method of Mizuno et al. (26). The remaining adding enzymes, UDP-MurAc-L-ala-D-glu:A2pm ligase, UDP-MurAc-L-ala-D-glu-A2pm:D-ala-D-ala ligase, Dalanyl-D-alanine ligase, and alanine racemase, were assayed essentially as described previously (23). The reaction products were separated by electrophoresis at pH 3.5 and located by radioautography. The specific activities of the enzyme preparations were calculated as nanomoles of nucleotide precursor synthesized per milligram of protein per minute. (viii) Phospho-N-acetylmuramyl pentapeptide translocase. The exchange assay of Hammes and Neuhaus (17) was modified as follows. After incubation at 25 C for 14 min, carrier UDP-MurAc pentapeptide (0.04 ,umol) was added and the complete reaction mixture was chromatographed in solvent A. Spots were located by radioautography and the absorbance of ultraviolet light, appropriate areas were cut out, and the radioactivity was determined. Exchange activity was calculated from the firstorder rate equation for the reaction (33). The rate of exchange is given as moles exchanged per liter per minute. (ix) D-Alanine carboxypeptidase. The previously described (10) assay was modified in that after termination of the reaction the products were separated by electrophoresis at pH 3.5. The specific activity of the enzyme preparations is given as nanomoles of Dalanine released per milligram of protein per hour. (x) Peptidoglycan synthesis. The determination of peptidoglycan synthesis was carried out as described previously for membrane preparations of B. licheniformis (36). The specific activity of the membrane preparations is given as nanomoles of diaminopimelic acid incorporated per milligram of protein per hour. Preparation of substrates. UDP-MurAc, iso-
VOL. 124, 1975
PEPTIDOGLYCAN SYNTHESIS IN L-PHASE VARIANTS
lated from a temperature-sensitive mutant of S. aureus TOF 33 (15), was a gift of Anne Wyke of this department. UDP-MurAc-L-alanyl-D-glutamic acid was isolated from lysine-deprived S. aureus H (32).
UDP-MurAc-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine, referred to as UDPMurAc pentapeptide, which was labeled with either [3H]diaminopimelic acid or [1,7-'4C]diaminopimelic acid, was prepared enzymically as described previously (37). Similarly, UDP-MurAc pentapeptide labeled in either both or only the terminal r-alanine residue of the D-alanyl-i-alanine dipeptide was synthesized by the enzymic addition of the radioactive dipeptide to UDP-MurAc-L-alanyl-D-glutamyl-mesodiaminopimelic acid obtained from cultures of B. licheniformis inhibited with D-cycloserine and chloramphenicol (36). Nonradioactive UDP-MurAc-pentapeptide was obtained from cultures of B. licheniformis incubated in the absence of Mg2+ (11). D, L-Aspartate-13-semialdehyde was prepared by ozmolysis of D,L-allyl-glycine as described by Black and Wright (1). The concentration of L-aspartic acid semialdehyde present was determined using partially purified homoserine dehydrogenase from bakers' yeast (2). Dihydrodipicolinate synthase for the generation of dihydrodipicolinate in assays of dihydrodipicolinate reductase was purified from Escherichia coli B to step (iv) (31). The final preparation had an activity of 15.26 U/mg of protein using the imidazole assay. N-acetyl-L-diaminopimelic acid was prepared by acetylation of L,L-diaminopimelic acid as follows. L,L-Diaminopimelic acid (107,umol) (purified by P. J. White; a gift from H. R. Perkins, Department of Microbiology, University of Liverpool) was dissolved in 1.5 ml of water and adjusted to pH 8 with 1 M NaOH. Acetic anhydride (106 ,umol) was added, and after readjusting to pH 8.0 the mixture was kept at 22 C overnight. The mixture was chromatographed on a column (1 by 10 cm) of Dowex 50 (H+) equilibrated with 0.01 M HCl and eluted with a linear gradient (400 ml) of 0.01 to 2.0 M HCl. Fractions were assayed for diaminopimelic acid by the acid ninhydrin method (13), using a heating period of 20 min at 100 C. Appropriate samples were pooled and concentrated, and HCl was removed in vacuo. The pooled material gave a single spot on electrophoresis at both pH 3.5 and 6.5 and on chromatography on Whatman no. 1 paper in solvent C. The yield of Nacetyl-L-diaminopimelate was 50.7 Zimol (47%). N-acetylamino-keto-pimelate was synthesized from N-acetyl-L-diaminopimelate by heating with pyridoxal in the presence of a metal (14). The reaction mixture contained 150 .tmol ofN-acetyl-L-diaminopimelate (diammonium salt), 150 ,umol of aluminium ammonium sulfate, 300 ,mol of pyridoxal hydrochloride, and 3.75 mmol of potassium acetate buffer, pH 4.5, in a total volume of 14 ml. The mixture was heated at 100 C for 20 min, and the slight precipitate which formed was removed by centrifuging. The supernatant solution was passed through a column (0.8 by 12 cm) of Dowex 50 (H+). Fractions (2 ml) were collected and assayed for the presence of an aketo acid (14). Appropriate fractions were pooled, concentrated, and neutralized with KHCO3 to pH
671
6.0. N-acetylamino-keto-pimelate was further purified by chromatography on a column (0.8 by 12 cm) of Dowex 1 (Cl-) eluted with a linear gradient (400 ml) of 0.01 to 0.5 M HCl. Fractions (5 ml) were collected and assayed for keto acid as described above. Appropriate fractions were pooled, and HCl was removed in vacuo by repeated evaporation of water. The final product gave a single spot on electrophoresis in buffer E with an R glutamate of 1.51, which was ninhydrin negative and gave a positive reaction for an a-keto acid when sprayed with 0.1% 2,4-dinitrophenyl hydrazine in 2 M HCl followed by alkali. The yield of N-acetylamino-keto-pimelate, assayed as an a-keto acid with pyruvate as the standard, was 28.2 ,umol (18.8%).
RESULTS Cell wall analysis of L-phase variants. Lphase variants (stable L-forms) have been described (20, 25) that continue to synthesize wall material, whereas such material is not detectable in L-phase variants from other organisms (3). To determine into which of these two classes the L-phase variants of B. licheniformis and B. subtilis would fall, cells from overnight cultures were analyzed for the presence of muramic acid and diaminopimelic acid as specific components of the peptidoglycan. In addition, membranes, prepared from similar cultures, were also analyzed. Hydrolysates of both Slf-1 and Slf-wt contained small quantities of muramic acid equivalent to 1.3 and 2.9 ,umol/100 mg (dry weight), respectively. In addition, Slf-wt contained an equivalent amount of diaminopimelic acid, whereas this was not detected in Slf1. Neither muramic acid nor diaminopimelic acid was detected in hydrolysates of the membrane preparations. Accumulation of cell wall precusors by Lphase variants. One possible explanation for the above results was the accumulation by the L-phase variants of UDP-MurAc peptides which, together with UDP-N-acetyl-glucosamine, are the cytoplasmic precursors of peptidoglycan. To investigate this possibility, cultures of the various L-phase variants, grown for 10 and 18 h in DP medium, were analyzed for bound N-acetyl-hexosamine as a measure of accumulated precursor. This was found in all the L-phase variants examined (Table 1), although there were considerable differences in the total amounts detected. Under identical conditions, the parent bacilli accumulated only negligible amounts ofprecursor. In general, continued incubation of the L-phase variants into stationary phase (i.e., 18 h) resulted in lower levels of bound N-acetylhexosamine being present in the cells. This may be due to the release of accumulated material as a consequence of cell lysis. Small quantities of bound N-acetyl-
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J. BACTERIOL.
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TABLE 1. Accumulation of peptidoglycan precursors by L-phase variants of bacilli Bound Organism
L-phase variants Slf-1 Slf-rod B Slf-AC2 Sif-W23 Slf-wt Slf-HisSlf-94 Bacilli B. subtilis 168 B. licheniformis 6346
Precursor accumulated
N-acetylhexosamine (jimol/100 mg
[dry wtl)
Expt Ta
Expt II
UDP-MurAc-L-ala-D-glu UDP-MurAc-Lrala-D-glu UDP-MurAc UDP-MurAc-L-ala-D-glu UDP-MurAc UDP-MurAc-L-ala-D-glu-A2pm-D-ala-D-ala UDP-MurAc-L-ala-D-glu UDP-MurAc-L-ala-D-glu
0.95 0.87 2.92 0.95 1.25 3.68 2.09 1.16
2.17 1.73 5.74
Not determined Not determined
0.13 0.07
1.71 4.08 2.71 1.29
a The organisms were grown in DP medium for 18 h in experiment I and for 10 h in experiment II.
hexosamine were detected in the medium after absorption and elution of the nucleotides from charcoal, particularly with the longer incubation period. However, at no time did the amounts recovered represent more than 10 to 15% of the precursor accumulated by the cells. Accumulation of precursor is not a phenomenon associated with the stationary phase of growth, since bound N-acetylhexosamine was detectable throughout the growth cycle of both Slf-1 and Slf-wt (Fig. 1). In addition, replacement of sodium succinate, the osmotic stabilizing agent in DP medium, with either 0.5 M sucrose or 0.5 M sorbitol did not affect the amount of precursor found. This observation eliminates the high ionic strength of the growth medium as the cause of accumulation. Identification of the accumulated nucleotide precursors. The purified nucleotide precursors, isolated as described in Materials and Methods, were identified by amino sugar and amino acid analysis. Five of the L-phase variants accumulated UDP-MurAc-L-alanyl-D-glutamic acid (UDP-MurAc-dipeptide) (Table 1), although in the case of Slf-AC2 this was the minor product in organisms grown for 18 h. In a second experiment, where cells of Slf-AC2 were harvested after 10 h of incubation, only UDPMurAc was found. UDP-MurAc was also accumulated by Slf-W23, whereas the complete precursor UDP-MurAc-pentapeptide was isolated from Slf-wt. On one occasion, this organism accumulated UDP-MurAc-L-alanyl-D-isoglutamyl-A2pm rather than the complete precursor. Since the L-phase variants were not cloned on solid media prior to these experiments, the pos-
sibility exists that the cultures were mixtures of two L-phase variants. However, it should be noted that the lesions in peptidoglycan synthesis found in these organisms (described in later sections) still provide a rational explanation for the nature of the precursor accumulated. The nature of the accumulated precursors was not altered when several of the L-phase variants were grown in medium containing sucrose or sorbital or in minimal medium. However, in the latter case a two- to threefold reduction occurred in the amount of precursor accumulated. The addition of either cloxacillin (20 ,ug/ml) or D-cycloserine (200 ug/ml) to DP medium had no effect on either the amount or the nature of the accumulated precursor. The parent bacilli incubated under these conditions accumulated the expected UDP-MurAc-pentapeptide and UDP-MurAc-tripeptide. In vitro activities of enzymes involved in the synthesis of UDP-MurAc-pentapeptide. The accumulation of incomplete nucleotide precursors such as observed in six of the seven Lphase variants examined may result from a defect in one of the synthetic enzymes. Such lesions have been described in mutants of E. coli (23) and S. aureus (15). To test this possibility these enzymes were assayed using crude cell-free extracts from the L-phase variants, with the results given in Table 2. The activities of the various enzymes in these extracts were compared with those obtained using similar preparations obtained from the parent bacilli grown under identical conditions. Clearly, the accumulation of UDP-MurAc-dipeptide by the L-phase variants did not result from a defi-
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PEPTIDOGLYCAN SYNTHESIS IN L-PHASE VARIANTS
VOL. 124, 1975 2 SIf-l
SIf - wt
Ii
E
8 E
0
0 0
Q. 0 .
S
0 U
01
2
0
4
r24 0 Time (h.)
6
I
1
I
2
4
6
III
-8V4
FIG. 1. Accumulation of bound N-acetylhexosamine by cultures of Slf-1 and Slf-wt. Cultures of the two Lphase variants were grown in DP medium and the optic density at 600 nm (-) was followed. At the times shown, samples (100 ml) were harvested, and the accumulated bound N-acetylhexosamine (0) was measured as described in Materials and Methods.
TABLE 2. Activity of the enzymes involved in the biosynthesis of UDP-MurAc pentapeptide Enzyme (Sp aCta)
L-Alanine-
Organism
adding enzyme
mesoDiaminopimelic acid-
adding
D-Alanyl-D-
D-Alanyl-D-
alanineadding enzyme
alanine synthetase
934 1,032 720 1,145 1,038 1,219 567 545 437 587
109 115 66 145 83 113 94 171 130 83
Alanine racemase
enzyme
B. subtilis 168 Slf-1 Slf-rod B Slf-AC2 B. subtilis W23 Slf-W23 B. licheniformis 6346 Slf-wt Slf-HisSlf-94
9.7 NDb ND
