Binding of Radioactive Benzylpenicillin to Sporulating Bacillus Cultures: Chemistry and Fluctuations in Specific Binding Capacity

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1 JOURNAL OF BACTERIOLOGY, Nov. 1971, p Copyright American Society for Microbiology Vol. 108, No. 2 Printed in U.S.A. Binding of Radioactive Benzylpenicillin to Sporulating Bacillus Cultures: Chemistry and Fluctuations in Specific Binding Capacity PAUL J. LAWRENCE, MARVIN ROGOLSKY, AND VO THI HANH Laboratory for the Study of Hereditary and Metabolic Disorders, Departments of Biological Chemistry and Medicine, and Department of Microbiology, University of Utah Medical School, Salt Lake City, Utah Received for publication 30 July 1971 The chemistry of the binding of 14C-benzylpenicillin to sporulating cultures of Bacillus megaterium and B. subtilis is similar to that in a 4-hr vegetative culture of Staphylococcus aureus. Unlabeled penicillins prevent the binding of 14C-benzylpenicillin, but benzylpenicilloic acid and benzylpenilloic acid do not. Bound antibiotic can be removed from cells with neutral hydroxylamine at 25 C. Sporulating cultures display two intervals of enhanced binding, whereas binding to stationaryphase S. aureus cells remains constant. The first period of increased binding activity occurs during formation of the spore septum or cell wall primordium development, and the second coincides with cortex biosynthesis. Penicillins kill growing cultures of bacteria by inhibiting the terminal steps in the biosynthesis of functional cell wall peptidoglycan. Penicillins irreversibly bind to a component of the cytoplasmic membrane (3), subsequently inhibiting the glycopeptide transpeptidase activity of intact cells (25, 26, 31; Fed. Proc. 25:344, 1966) or of membrane particles (1, 2, 13, 14). Specific inactivation of this enzyme is dependent upon the irreversible binding of the antibiotic (J. T. Park and E. M. Wise, Int. Congr. Biochem. 7th, Abstr. D-44, 1969), and penicillin is believed to bind directly to the glycopeptide transpeptidase. In vitro demonstration of a peptidoglycan transpeptidase and its irreversible inhibition by bound penicillins has been accomplished only with a particulate enzyme preparation from Escherichia coli Y-10 (1, 2, 13, 14). Radioactive penicillin also binds covalently to particulate enzyme preparations from Bacillus subtilis and E. coli capable of synthesizing peptidoglycan in vitro (16). Bound antibiotic irreversibly inhibits D-alanine carboxypeptidase activity in the former system. Neutral hydroxylamine removes the bound antibiotic, and enzyme activity is restored at a rate identical to the rate of removal of bound penicillin (16, 17). The D-alanine carboxypeptidase activity observed in the B. sublilis system may reflect an uncoupled transpeptidase which can react with water but not with its normal amino acid acceptor (13, 17). The irreversible inhibition of both the transpeptidase in E. coli and the D-alanine carboxypeptidase in B. subtilis by bound penicillins is compatible with the hypothesis that penicillin is a substrate analogue of the D-alanyl-D-alanine residue at the carboxyl end of a pentapeptide chain in the peptidoglycan polymer before transpeptidation or removal of a D-alanine residue (25). In either case, the enzyme is acylated by penicillin, and bound radioactive penicillin reflects the quantity of enzymes which participate in the terminal steps of peptidoglycan biosynthesis. Although bacterial sporulation has been described as a modified or atypical cell division (11), the role of peptidoglycan biosynthesis in this process remains obscure. Bacterial sporulation involves a sequence of seven clearly defined morphological stages (11). Stage II in this sequence is characterized by the development of a forespore septum at the apical end of the sporulating cell. The fourth stage in sporulation includes the biosynthesis of a mucopeptide polymer called the spore cortex. Antibiotics (penicillin G, D-cycloserine, bacitracin, etc.) which inhibit cell division by interfering with peptidoglycan synthesis can prevent spore septum formation (10) and cortex biosynthesis (8, 19). The precise mechanisms of inhibition of spore septum formation are not known, since the spore septum itself apparently lacks cell wall material (11). Moreover, the structure of the B. subtilis spore cortex peptidoglycan differs substantially from that of the peptidoglycan from the vegetative cell wall. 662

2 VOL. 108, 1971 BINDING BENZYLPENICILLIN TO SPORULATING BACILLUS The former contains a lactam of muramic acid not found in the latter and is substantially less cross-linked than the peptidoglycan from the vegetative cell wall (29). The binding of radioactive penicillins to vegetative cells has been extensively studied (3, 4, 6, 7, 18, 20, 23), and good correlation was found between the binding of penicillins to bacterial cells and the resulting inhibition of growth (7). Despite the obvious importance of the penicillin target in the sporulation process, the binding of penicillins to sporulating cells has not been examined. The binding of 14C-benzylpenicillin to sporulating cells was studied, therefore, to determine whether the chemistry of binding is similar to that reported for vegetative cells and to examine the role of peptidoglycan biosynthesis in bacterial sporulation. MATERIALS AND METHODS Organisms. The bacterial strains used in these studies were B. megaterium ATCC 19213, B. subtilis Porton, B. cereus NRRL 569, and Staphylococcus aureus Copenhagen. All strains were grown in 15 liters of medium under forced aeration in a 20-liter carboy. B. megaterium was grown in the synthetic sucrose-salts medium of Kolodziej and Slepecky (15). B. subtilis was grown in the sporulation medium described by Donnellan et al. (5), modified to contain a final concentration of 0.05% glucose and supplemented with 0.2% casein hydrolysate, 20 gg of tryptophan (filter sterilized) per ml, 0.4 mm K2HPO4, and 0.18% lactic acid. The final ph of this medium was adjusted to 7.2. B. cereus 569 and S. aureus Copenhagen were grown in the media of Pollock (20) and Wiley (30), respectively. Growth was measured turbidimetrically with a Klett- Summerson photoelectric colorimeter with a no. 66 filter. Samples (500 ml) were taken at various intervals, harvested by centrifugation, and washed three times with a buffer containing tris(hydroxymethyl) aminomethane (0.01 M), succinate (0.04 M), and magnesium acetate (0.01 M), adjusted to ph 7.2. The cells were then suspended in a volume of buffer (in milliliters) equal to their wet weight (in grams) and frozen at -20 C. Sporulation septa in B. megaterium were counted by the staining technique of Gordon and Murrell (9). Sporulation septa in B. subtilis could be identified neither with this staining technique nor with Normarski optics. Refractile sporangia were distinguished by dark-contrast phase microscopy. Penicillins and derivatives. '4C-benzylpenicillin (penicillin G) and 6-aminopenicillanic acid (6-APA) were obtained from Amersham Searle and Sigma, respectively. Ampicillin, cloxacillin, dicloxacillin, methicillin, and oxacillin were gifts from Bristol Laboratories. Benzylpenicilloic acid and benzylpenilloic acid were prepared by the procedure of Pruess and Johnson (22). Binding assay. A 75-Mliter quantity of suspended cells was incubated with 25 uliters of appropriately diluted '4C-benzylpenicillin for 20 min at 0 C (B. cereus for 8 min). The cells were washed with 2 ml of buffer, collected on a membrane filter, and washed four times with 5 ml of 95% ethanol. The samples were dried and counted to 3% accuracy in a scintillation counter (Nuclear-Chicago Corp.). Control incubations were done in a similar manner, but cells were incubated for 20 min with unlabeled benzylpenicillin (25 mg/ml) before exposure to radioactive penicillin. Both the controls and the experimental incubations were performed in triplicate. For each determination, the radioactivity bound nonspecifically to the control sets was subtracted from that bound in the experimental incubation. Direct competition experiments. Cells were incubated with '4C-benzylpenicillin in the presence of semisynthetic penicillins or derivatives of benzylpenicillin as outlined above. Reversal of binding with hydroxylamine. Cells were incubated with 14C-benzylpenicillin as described above, washed with 2 ml of buffer, resuspended, and incubated with neutral hydroxylamine or tris(hydroxymethyl) aminomethane buffer for 20 min at 25 C. The cells were collected on membrane filters, and the bound radioactivity was determined. RESULTS Penicillin-binding characteristics of sporulating B. subtilis and B. megaterium cells. Sporulating B. subtilis and B. megaterium cells exhibit penicillin-binding properties similar to those of previously studied vegetative cultures of S. aureus and Micrococcus pyogenes (4, 7, 18, 23) and membrane fragments of B. subtilis (16). The saturation phenomenon seen with a 4-hr vegetative culture of S. aureus is also apparent with 14- hr cultures of B. subtilis and B. megaterium (Fig. 1), but the concentration of antibiotic required to saturate the available binding sites is higher with the sporulating organisms. The antibiotic concentration required for half maximal binding to sporulating B. subtilis cells (0.15,gg/ml) is similar to that required for membrane a z 3 C.) 14C PENICILLIN G (gg/mi) 663 FIG. 1. Effect of 14C-penicillin G concentration on the extent of binding to Bacillus subtilis, B. megaterium (14-hr cultures), and Staphylococcus aureus (4- hr culture). Symbols: 0, S. aureus; 0, B. subtilis; A, B. megaterium.

3 664 LAWRENCE, ROGOLSKY, AND HANH J. BACTERIOL. fragments obtained from vegetative cells of the same organism (16). In all subsequent binding experiments, the radioactive penicillin concentration was the minimal required for maximal binding by the given organism. The antibiotic concentration used in experiments with B. cereus cultures was that which was previously reported (6). Unlabeled semisynthetic penicillins compete with radioactive benzylpenicillin for the available binding sites on sporulating B. subtilis and B. megaterium cells and those of vegetative S. aureus cells (Table 1). Unlabeled benzylpenicillin prevents the binding of 14C-benzylpenicillin to all of the organisms tested. Methicillin, oxacillin, cloxacillin, and dicloxacillin are less effective competitors in S. aureus cells, as was shown by Edwards and Park (7), although they compete favorably in sporulating cultures of B. subtilis and B. megaterium. Even at a concentration of 100 ug/lml, 6-APA competes poorly for the available binding sites of B. megaterium cells, although it does prevent the binding of radioactive 14C-benzylpenicillin to S. aureus and B. subtilis cells. Although unlabeled benzylpenicillin prevents the binding of 14C-benzylpenicillin to vegetative cells of S. aureus and to sporulating B. subtilis or B. megaterium cells (Table 1), benzylpenicilloic acid and benzylpenilloic acid exert no such effect (Table 2). Neutral hydroxylamine, which reverses the binding of benzylpenicillin to particulate enzyme preparations from vegetative B. subtilis cells (16), also reverses the binding to intact S. aureus, B. subtilis, and B. megaterium cells (Table 3). The conditions for reversal (20 min at 25 C, ph 7.0) are identical to those required for TABLE 1. Competition between unlabeled penicillins and 14C-benzylpenicillin for the penicillin binding sites of S. aureus (4-hr culture), B. subtilis, and B. megaterium (14-hr cultures)a.concn Aio(1lg/ml ) "4C-penicillin G bound (nmoles/g of dry wt) aureus tilis terium Penicillin G APA Methicillin 0... ṭ Oxacillin Cloxacillin Ampicillin Dicloxacillin a Unlabeled, semisynthetic penicillins were added to the cells simultaneously with the appropriate concentration of "4C-benzylpenicillin. TABLE 2. Competition between unlabeled benzylpenicilloic acid and unlabeled benzylpenilloic acid for the penicillin binding sites ofs. aureus (4-hr culture), B. subtilis, and B. megaterium (14-hr culture)a '4C-penicillin G bound Addition (nmoles/g of dry wt) (10 g/mil) B. sub- B. mega- S. aureus tilis terium Penicilloic acid Penilloic acid a Vegetative S. aureus cells or sporulating Bacillus cells were incubated with "4C-benzylpenicillin alone or with '4C-benzylpenicillin plus the unlabeled derivatives indicated. The amount of radioactive penicillin bound was determined as described in Materials and Methods. TABLE 3. Reversal of the binding of 14Cbenzylpenicillin to S. aureus (4-hr culture), B. subtilis, and B. megaterium (14-hr cultures) with hydroxylaminea Treatment 4C-penicillin G bound (nmoles/g of dry wt) S. aureus B. subtilis B. megaterium (%)b (%)b (%)b I M Tris 1.59 (100) 0.75 (100) 1.50 (100) I M NH2OH 0.63 (38.5) 0.17 (23.0) 0.58 (38.7) acells were exposed to "4C-benzylpenicillin for 20 min at 0 C, and unbound antibiotic was removed by washing and centrifugation. The cells were resuspended and treated either with tris(hydroxymethyl)aminomethane (Tris) buffer or with neutral hydroxylamine. Bound radioactive antibiotic was determined as described in Materials and Methods. bvalue in parentheses indicates percentage of 14Cbenzylpenicillin bound to the cells after treatment with hydroxylamine as compared to that bound to cells treated with I M Tris buffer, ph 7.0. the reversal of binding to a particulate enzyme preparation from B. subtilis (16). Binding of 14C-benzylpenicillin to stationaryphase cells of S. aureus, and to sporulating cells of B. megaterium, B. subtilis, and B. cereus. The specific binding activity of S. aureus cells in the stationary phase of growth is essentially the same as that of vegetative cells, and the binding activity remains constant over the 9-hr period of stationary growth examined (Fig. 2). The specific binding activity of sporulating cultures of B. megaterium, B. subtilis, and B. cereus decreases substantially during the presporulation and early sporulation events (Fig. 3, 4, and 5), but the decrease is not continuous. In B. megaterium and B. subtilis cultures, the initial decrease is fol-

4 VOL. 108, 1971 BINDING BENZYLPENICILLIN TO SPORULATING BACILLUS lowed by two periods of increased binding activity. The first increase occurs before the appearance of refractile forms, and a second increase is coincident with the formation of refractile endospores. In sporulating B. megaterium cultures, the initial increase in specific binding activity is concurrent with sporulation septation (stage II) and rises proportionately with the number of septated cells (Fig. 3). The time of septation indicated in Fig. 3 is consistent with the observation of Hitchins and Slepecky (10), who noted that spore septum formation in B. megaterium began 2.5 hr after the end of logarithmic growth and reached a maximum between 3.5 and 4 hr into the stationary phase of growth. They concluded that septum formation, or its initiation, requires peptidoglycan synthesis, because penicillin and cycloserine added 2.0 or 2.5 hr into the stationary phase of growth prevented septum formation (10). Although septa could not be de- tected in sporulating cells of B. subtilis with Normarski optics or with the staining technique of Gordon and Murrell (9), spore septum formation is believed to coincide with the first peak of specific binding activity, which preceded refractility by approximately 2 hr (Fig. 4). The penicillin binding pattern of a sporulating B. cereus (Fig. 5) culture inducible for penicillinase is similar to that of B. megaterium (Fig. 3) and B. subtilis (Fig. 4). The binding of penicillin to vegetative B. cereus cells is required for penicillininduced penicillinase formation (6, 20, 21), but it is not known if the binding site for induction of this enzyme is distinct from that responsible for the bactericidal activity of the antibiotic. In the sporulating cultures examined, the initial increase in binding began approximately 2.5 hr after the end of logarithmic growth and prior to the accumulation of refractile forms. The second peak occurred during the formation of refractile endospores (Fig. 5). The fluctuations in specific binding activity described for the sporulating organisms were reproducible under the condi o z150 ' Downloaded from FIG. 2. Binding of "4C-penicillin G to Staphylococcus aureus cells during stationary phase of growth. Symbols: 0, growth; A, penicillin binding.?50 01 I E z M m D INCUBATION TIME (HOURS) FIG. 4. Binding of "4C-penicillin G to sporulating cells of Bacillus subtilis. Sporulation septa could not be observed in B. subtilis. Symbols: *, growth; A, refractile forms; A, penicillin binding. on December 30, 2018 by guest -i INCUBATION TIME (HOURS) FIG. 3. Binding of "4C-penicillin G to sporulating cells of Bacillus megaterium. Symbols: 0, growth; 0, cells with septa; A, refractile forms; A, penicillin binding. INCUBATION TIME (HOURS) FIG. 5. Binding of 1"C-penicillin G to sporulating cells of Bacillus cereus. Symbols: 0, growth; A, refractile forms; A, penicillin binding.

5 666 LAWRENCE, ROGOLSKY, AND HANH J. BACTERIOL. tions used, as was the relatively constant binding pattern of stationary cultures of S. aureus. The minor weight fluctuations in the sporulating cultures examined were not sufficient to produce the described fluctuations in specific binding. DISCUSSION The periodic fluctuations in the specific binding activity of sporulating bacteria, in contrast to the constant binding activity of a nonsporulating organism in the stationary phase of growth, agrees with previous observations that functional cell wall synthesis is required during two distinct phases of sporulation (19, 27, 28). The first increase in binding activity corresponds to the period of spore septum formation or cell wall primordium biosynthesis; the second coincides with spore cortex formation. The incorporation of radioactive diaminopimelic acid into a hot trichloroacetic acid-insoluble fraction of sporulating B. cereus cells shows a similar pattern (27, 28). Vinter suggested that the first interval of enhanced incorporation reflects germ cell wall synthesis and the second represents cortical biosynthesis (27, 28). A cortexless mutant of B. cereus exhibits the first stage of enhanced diaminopimelic acid synthesis, but not the second (19). In the diaminopimelic acid incorporation experiments, however, the incorporation pattern of a non-spore-forming bacteria during its stationary phase of growth was not presented. Penicillins, and other antibiotics which interfere with the synthesis of vegetative cell wall peptidoglycan, prevent spore septum formation and can prevent functional cortex synthesis if added to a sporulating culture at the appropriate time intervals (8, 19). Warth and Strominger detected significant differences between the structures of cortical and vegetative cell wall peptidoglycans (29). These authors suggest that the structural differences may reflect the utilization of different sets of enzymes for the synthesis of each species. The existence of cortexless mutants with apparently normal vegetative cell wall peptidoglycan supports this hypothesis. Most of the products of genetically mapped spore genes in B. subtilis (12, 24) remain unidentified, but a number of these genes may function by regulating the production of enzymes required for the biosynthesis of functional peptidoglycan. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grants A (M.R.) and A (P.J.L.) from the National Institute of Allergy and Infectious Diseases. Competent technical assistance was provided by Faulalo Faamama. LITERATURE CITED 1. Araki, Y., A. Shimada, and N. Ishimoto Effect of penicillin on cell wall mucopeptide synthesis in an Escherichia coli particulate system. Biochem. Biophys. Res. Commun. 23: Araki, Y. A., R. Shimai, A. Shimada, and N. Ishimoto Enzymatic synthesis of cell wall mucopeptide in a particulate preparation of Escherichia coli. Biochem. Biophys. Res. Commun. 23: Cooper, P. D Site of action of radiopenicillin. Bacteriol. Rev. 20: Daniel, J. W., Jr., and M. J. Johnson Properties of the penicillin binding component of Micrococcus pyogenes. J. Bacteriol. 67: Donnellan, E. J., Jr., E. H. Nags, and H. S. Levinson Chemically defined, synthetic media for sporulation and for germination and growth of Bacillus subtilis. J. Bacteriol. 87: Duerksen, J. D Localization of the site of fixation of the inducer, penicillin, in Bacillus cereus. Biochim. Biophys. Acta 87: Edwards, J. R., and J. T. Park Correlation between growth inhibition and the binding of various penicillins and cephalosporins to Staphylococcus aureus. J. Bacteriol. 99: Fitz-James, P. C Spore formation in wild and mutant strains of B. cereus and some effects of inhibitors, p In M. J. C. Senez (ed.), Regulations chez les microorganismes. Centre National Recherche Science. Paris. 9. Gordon, R. A., and W. G. Murrell Simple method of detecting spore septum formation and synchrony of sporulation. J. Bacteriol. 93: Hitchins, A. D., and R. A. Slepecky Antibiotic inhibition of the septation stage in sporulation of Bacillus megaterium. J. Bacteriol. 97: Hitchins, A. H., and R. A. Slepecky Bacterial sporulation as a modified procaryotic cell division. Nature (London) 223: Hoch, J. A., and J. Spizizen Genetic control of some early events in sporulation of Bacillus subtilis, p In L. L. Campbell (ed.), Spores IV. American Society for Microbiology, Ann Arbor, Michigan. 13. Izaki, K., M. Matsuhashi, and J. L. Strominger Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Proc. Nat. Acad. Sci. U.S.A. 55: Izaki, K., M. Matsuhashi, and J. L. Strominger Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin sensitive enzymatic reactions in strains of Escherichia coli. J. Biol. Chem. 243: Kolodziej, B. J., and R. A. Slepecky Trace metal requirements for sporulation of Bacillus megaterium. J. Bacteriol. 88: Lawrence, P. J., and J. L. Strominger The binding of radioactive penicillin to the particulate enzyme preparation of Bacillus subtilis and its reversal with hydroxylamine or thiols. J. Biol. Chem. 245: Lawrence, P. J., and J. L. Strominger The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis. J. Biol. Chem. 245: Maass, E. A., and M. J. Johnson Penicillin uptake by bacterial cells. J. Bacteriol. 57: Pearce, S. M., and P. C. Fitz-James Sporulation of a cortexless mutant of a variant of Bacillus cereus. J. Bacteriol. 105: Pollock, M. R., and C. J. Perret The relation between fixation of penicillin sulfur and penicillinase adaptation in Bacillus cereus. Brit. J. Exp. Pathol. 32: Pollock, M. R A simple method for the production

6 VOL. 108, 1971 BINDING BENZYLPENICILLIN TO SPORULATING BACILLUS 667 of high titer penicillinase. J. Pharm. Pharmacol. 9: Pruess, D. L., and M. J. Johnson Enzymatic deacylation of S35-benzylpenicillin. J. Bacteriol. 90: Rogers, H. J The inhibition of mucopeptide synthesis in relation to the irreversible fixation of the antibiotic by staphylococci. Biochem. J. 103: Rogolsky, M Chromosomal regions which control sporulation in Bacillus subtilis. Can. J. Microbiol. 15: Tipper, D. J., and J. L. Strominger Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine. Proc. Nat. Acad. Sci. U.S.A. 54: Tipper, D. J., and Strominger, J. L Inhibition of cross-linking by penicillins and cephalosporins: studies in Staphylococcus aureus in vivo. J. Biol. Chem. 243: Vinter, V The fate of preexisting diaminopimelic acid-containing structures during germination and postgerminative development of bacterial spores. Folia Microbiol. (Prague) 10: Vinter, V Commencement of synthetic activities of germinating bacterial spores and changes in vulnerability of cells during outgrowth, p In L. L. Campbell and H. 0. Halvorson (ed.), Spores III. American Society for Microbiology, Anrl Arbor, Mich. 29. Warth, A. D., and J. L. Strominger Structure of the peptidoglycan of bacterial spores: occurrence of the lactam of muramic acid. Proc. Nat. Acad. Sci. U.S.A. 64: Wiley, B. B A new virulence test for Staphylococcus aureus and its application to encapsulated strains. Can. J. Microbiol. 7: Wise, E. M., and J. F. Park Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proc. Nat. Acad. Sci. U.S.A. 54: Downloaded from on December 30, 2018 by guest