Effects of Cell Wall Inhibitors on Cell Division in Streptococcus mutans

INFECTION AND IMMUNITY, July 1983, P. 28-32 19-9567/83/728-5$2./ Copyright 1983, American Society for Microbiology Vol. 41, No. 1 Effects of Cell Wall Inhibitors on Cell Division in Streptococcus mutans TIMOTHY A. KRALt AND LOLITA DANEO MOORE* Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 1914 Received 31 January 1983/Accepted 13 April 1983 The addition of inhibitors of cell wall biosynthesis to exponential-phase cultures of Streptococcus mutans may do one of three things, depending on the concentrations used: (i) prevent cell division at a time coincident with the onset of chromosome replication, (ii) prevent cell division later in the cell cycle coincident with or near completion of septation, or (iii) lead to limited cell lysis. The relative tolerance of S. mutans to inhibitors of cell wall biosynthesis may be due to the fact that S. mutans cultures treated with low levels of cell wall antibiotics seem to be blocked at a stage before initiation of autolytic activity, whereas cultures treated with high levels of these antibiotics seem to be blocked after termination of the autolytic phase. Thus, the cells escape the lytic death that is seen in other streptococci exposed to inhibitors of cell wall biosynthesis. In bacteria, tolerance to antibiotic inhibitors of cell wall biosynthesis leads to inhibition of growth without killing (9). Therefore, bacteria tolerant to the killing action of these antibiotics are useful tools for examining the effects of antibiotics on cell growth and division. In this study, we examined the effects of various inhibitors of cell wall biosynthesis on division of Streptococcus mutans GS-5, a bacterium previously found to be tolerant-(5). The method of analysis used in this study is based on the cell cycle age distribution of exponential-phase cells which is invariant (2). If such cells are blocked in division at a specific age by an antibiotic, then only the cells that are past the block will divide (1, 2). Thus, by determining cell numbers at time zero and at several hours after the block, one can obtain information on the position of the block in a cell division cycle. MATERIALS AND METHODS Organism and growth conditions. S. mutans strain GS-5 was obtained from A. S. Bleiweis, Department of Microbiology and Cell Science, University of Florida, Gainesville. Experiments were initiated by transferring 5 p.l of a frozen culture (a culture was grown to mid-exponential phase in Todd-Hewitt broth [Difco Laboratories, Detroit, Mich.] supplemented with 1% glucose, concentrated 1 times in Todd-Hewitt broth containing 3o glycerol, quick-frozen in dry ice-ethanol, and stored at -7 C) into 18-mm-diameter cuvettes containing a chemically defined medium (FMC [8]). From this culture, 11 and 1-2 dilutions were t Present address: Department of Botany and Microbiology, University of Arkansas, Fayetteville, AR 7271. made into the same medium. After inoculation of cultures (and all cultures in the subsequent experiments), 1,ul of a fresh sterile sodium bicarbonate solution (84 mg/ml) was added to 1 ml of each culture. The cultures were then incubated overnight at 37 C. Addition of antibiotics. From an overnight culture, fresh prewarmed defined medium was inoculated at a starting absorbance (1 absorbance unit is equivalent to approximately.39 p.g of cells [dry weight] per ml [8]) measured at an optical density of 675 nm (Spectronic 2; Bausch & Lomb, Rochester, N.Y.) of approximately 25. The total volume of medium used was 5 ml for each antibiotic plus 5 ml for a control. The culture was incubated at 37 C, and optical density measurements were recorded every 3 min. Additionally, 1- p.l samples were removed every 3 min and added to 4 p.l of 9.3% formaldehyde. The formaldehyde-fixed cells were covered with aluminum foil to prevent contamination with extraneous particulate matter and allowed to sit at room temperature for at least 3 min and no longer than 24 h before counting. When the culture reached 1 to 2 absorbance units, 5-ml portions were removed and added to cuvettes, each containing an appropriate antibiotic. Mitomycin C (MIT), cerulenin, and potassium penicillin G were obtained from Calbiochem, San Diego, Calif.; cycloserine and vancomycin hydrochloride were obtained from Eli Lilly & Co., Indianapolis, Ind.; chloramphenicol (CAP) was obtained from Sigma Chemical Co., St. Louis, Mo.; and sodium fosfomycin was obtained from Merck Sharp, & Dohme Research Laboratories, Rahway, N.J. These cultures were incubated at 37 C and treated the same as the control culture. Samples were removed for 4 to 6 h after the addition of antibiotic. Also, an overnight sample was taken from each antibiotic culture and from the control culture. The formaldehyde-fixed cells were diluted into sterile.9% sodium chloride (Abbott Laboratories, North Chicago, Ill.) and counted in an Electrozone/Cello- Downloaded from http://iai.asm.org/ on May 9, 218 by guest 28

VOL. 41, 1983 x E w-i 32 161 8 4 I I I A( I- 1 2 3 O/N TIME (min) FIG. 1. Increases in cell number of S. mutans GS-5 in the presence of no antibiotic (), CAP (2,ug/ml) (), and MIT (.5,ug/ml) (A). The intercepts between the dashed lines and the control culture line represent the numbers of residual cell divisions. The doubling time of this culture was 8 min. scope (model 112 LTH; Particle Data, Inc., Elmhurst, Ill.). The percentage of lysis was estimated from loss of absorbance by using the equation % lysis = highest absorbance - final absorbance highest absorbance EFFECTS OF CELL WALL INHIBITORS ON S. MUTANS 29 x 1 Since fully lysed cell suspensions may exhibit some absorbance, the equation may underestimate the extent of cell lysis.. /--_ -A - a--w- -e- ~~~~~~~. --o I DIV Cw1 CER MI T II I C DJ 2, RESULTS Figure 1 shows the effects of the addition of MIT or CAP on the cell number increase of S. mutans. As is the case with Streptococcus faecium, a greater cell number increase is obtained after addition of MIT than after addition of CAP. Also, the overnight point in MIT-treated cultures is lower than the 27-min point (Fig. 1) due to slight lysis of the culture. From the data shown in Fig. 1, it is also possible to obtain the fraction of the cell population which can divide after inhibition of protein and DNA synthesis, respectively (2). The fraction dividing after the addition of MIT represents cells in the DI phase (the time between completion of chromosome replication and division) of the cell cycle; the fraction dividing after the addition of CAP represents cells in the D2 phase (time required for daughter cell separation) of the cell division cycle (2). In each instance, r, the fraction of the population capable of division, is obtained from the equation ln N/No r =- ln 2 where N equals the maximal cell number obtained after antibiotic treatment and No equals the number of cells at the time the antibiotic was added. When r is multiplied by the doubling time of the culture, residual minutes of divisions are obtained that indicate the time of the division block in minutes before division (Fig. 2). In the experiment shown in Fig. 1, the doubling time was 8 min, and the timing of the MIT and CAP blocks was 65 and 45 min, respectively. The respective r values were.81 and.55. In cultures dividing with doubling times of 4 to 8 min, there is a linear increase in the D1 and D2 times with increasing doubling time (T. A. 1 8 6 4 2 MINUTES BEFORE DIVISION FIG. 2. Cell division cycle of S. mutans GS-5 based on the effects of antibiotics on residual divisions. This model shows a culture with a doubling time of 8 min. The top arrows represent two successive divisions (DIV), 8 min apart. In this example, C was initiated 35 min before the first division. D1 was 65 min, and D2 was 45 min. The timing before cell division of the action of low concentrations of cell wall antibiotics (CW1) (obtained from Table 1) is indicated on the left. The values were obtained by multiplying r by a doubling time of 8 min. Cerulenin (CER) affects cell division about 1 min before completion of C. MIT defines completion of C and onset of Dl. CAP defines completion of D1 and onset of D2. High concentrations of several cell wall antibiotics (CW2) seem to inhibit cell division at this time. CAP CW2 D2 DIV Downloaded from http://iai.asm.org/ on May 9, 218 by guest

3 KRAL AND DANEO-MOORE TABLE 1. Residual cell divisions after addition of antibiotics to cultures of S. mutans GS-5 ranked by mean r value Conc No. of Mean Antibiotic Conc experi- relative SD (~gm) ments r a CAP 2 17 1. Vancomycin 5 3.86.292 Cycloserine 1, 1.91 MIT.5 8 1.4.138 Cerulenin 1 9 1.62.29 Cycloserine 1-2 2 2.46.1 Vancomycin.5 3 2.61.122 Fosfomycin 1-1, 4 2.67.313 a In each case, the r values have been divided by the r value obtained in a CAP-treated culture. A typical r value for CAP is.55 (Fig. 1). Kral, A. M. Cuffini, and L. Daneo-Moore, manuscript in preparation). The cultures used in this study doubled every 7 to 9 min. Consequently, to normalize for this variation and to account for minor variations in D1 and D2 due to the occasional presence of chains, the actual r values obtained in any single experiment were divided by the value obtained for a CAP-treated culture, which was given a relative r value of 1.. Table 1 shows the results of such a treatment. It can be seen that the addition of relatively high concentrations of vancomycin and cycloserine gave relative r values close to those obtained for CAP. These values indicate that the addition of high concentrations of these two inhibitors of cell wall synthesis arrests divisions at the same stage as CAP (i.e., at or about the D2 phase of the cell division cycle). Table 1 also shows that 1-fold lower concentrations of these two antibiotics and concentrations offosfomycin between 1 and 1,,ug/ml permitted considerably more residual divisions than either CAP or MIT. Interestingly, the residual divisions obtained from all of these antibiotics clustered around a mean relative r value of 2.55. Cerulenin, an inhibitor of fatty acid synthesis, appeared to affect cell division somewhat earlier than the onset of D1 (defined by the r values for MIT). This finding is in agreement with findings for S. faecium (1). These observations can be summarized in a diagram of the cell cycle (Fig. 2), which, for simplicity, is based on a culture with a doubling time of 8 min, where the D2 time is 45 min and the D1 time is 65 min (Fig. 1). The chromosome replication (C) time for S. mutans is similar to that of S. faecium (6), or about 5 min (Kral et al., manuscript in preparation). From the data in Fig. 1 and Table 1, it would appear that low concentrations of vancomycin and cycloserine and all concentrations of fosfomycin tested permit cell divisions for 115, or C + D1, minutes. We have labeled the two stages of the cell cycle affected by cell wall antibiotics CW1 and CW2 (Fig. 2). In S. mutans, CW1 appears to coincide with the initiation of chromosome replication. CW2 appears to coincide with the onset of D2. The effects of various concentrations of penicillin G on the extent of residual cell divisions were examined. As shown in Fig. 3, exposure of cultures to various concentrations of penicillin G provided two plateaus of residual cell division, one with a relative r value of about 2.5 between.1 and.25,ug/ml, and one with a relative r value of about 1. at.3,ug/ml and above. When examined in the context of the data in Table 1 and the model in Fig. 2, these two plateaus correspond to CW1 and CW2, respectively. The autolytic enzyme system of S. mutans has not been characterized. Nevertheless, in several of our experiments, we observed limited lysis of cultures after prolonged incubation with some of these antibiotics. Figure 4 shows the results of an experiment in which incubation with penicillin G resulted in up to 75% lysis of the culture after overnight incubation. In other more typical experiments, maximal lysis was on the order of 2% under the conditions described (T. A. Kral, unpublished data). In either case, the lysis observed occurred in a range of penicillin G concentrations between the two plateaus described in the experiment mentioned above (Fig. 3). These data, as well as the observation of about 5% lysis after incubation of cultures overnight 3. 2. 1. INFECT. IMMUN. X4 ) _) I I I I I I I C.5.1.2.4.8.16.32.64 PENICILLIN G CONCENTRATION (pg/ml) FIG. 3. Effects of various concentrations of penicillin G on minutes of residual cell divisions in S. mutans GS-5. Numbers on the ordinate represent the minutes of residual cell division obtained at each penicillin G concentration divided by minutes of division obtained at the highest penicillin G concentration. Data for this figure were obtained from seven individual experiments. The bars represent the standard deviation of the mean. The arrow indicates the relative r value for CAP. Downloaded from http://iai.asm.org/ on May 9, 218 by guest

VOL. 41, 1983 1 EFFECTS OF CELL WALL INHIBITORS ON S. MUTANS 31 C/) 8 z a: lu cl.1.2.4.8.16.32 PENICILLIN G CONCENTRATION (pg/ml) FIG. 4. Extent of overnight lysis of S. mutans GS-5 in the presence of various concentrations of penicillin G. in the presence of MIT, suggest that, as is the case in S. faecium (7), autolytic activity is at its maximum during the latter part of the C time and before the onset of D1 (Fig. 2). Limited lysis after overnight incubation has also been observed in some of these experiments with cycloserine and fosfomycin. Lysis was not observed in all expeirments, even under what appeared to be identical conditions. DISCUSSION The data presented here suggest that the addition of inhibitors of cell wall biosynthesis to exponential-phase cultures of S. mutans has one of three effects. Low concentrations of these antibiotics appear to prevent cell division at a time coincident with the onset of the C time. High concentrations of the same inhibitors appear to inhibit cell division much later in the cell cycle, coincident with or near D2. In S. faecium, D2 is coincident with septum closure (4). Finally, intermediate antibiotic concentrations may lead to limited cell lysis. These results differ from observations with S. faecium 979 and Escherichia coli (3, 6, 7). In these two organisms, low concentrations of inhibitors of cell wall synthesis inhibit cell division at a later time in the cell cycle, about halfway into the C time. However, high concentrations of cell wall antibiotics appear to affect cell division at about the same time in all three organisms. It was interesting to find that, for penicillin G, the concentrations required to inhibit division at the time of onset of C were identical to the concentrations of pencillin G that failed to substantially inhibit surface growth, DNA, RNA, or protein synthesis (5). In contrast, cells treated with high concentrations of penicillin G exhibited inhibition of DNA, RNA, and protein synthesis, and cells appeared to be unable to increase their surface (5). Finally, the observations of limited lysis seen in this study after overnight incubation are consistent with a timing for maximal autolytic activity late in the C time. This timing of maximal autolytic activity would be identical to that observed for S. faecium (6). However, the limited and slow lysis of S. mutans observed here suggests that the level of cellular autolytic activity is low. The low level of autolytic activity as well as an effect of cell wall inhibitors on an event early during the C time may explain the relative tolerance of S. mutans to inhibitors of cell wall biosynthesis. In the related organism, S. faecium, autolytic activity is initiated about 25 min after the initiation of the C time and terminated after the completion of chromosome replication (6, 7). Assuming that the low level of autolytic activity in S. mutans also coincides with a period somewhere within the C time, S. mutans cultures exposed to low concentrations of cell wall inhibitors would be blocked before the initiation of autolytic activity, whereas cultures exposed to high concentrations of cell wall inhibitors would be blocked after termination of autolytic activity (Fig. 2). The limited lysis observed at intermediate concentrations of penicillin G would represent cells trapped between these two cell division blocks. Further studies will be required to substantiate the relevance of our suggested mechanism of antibiotic tolerance to other aspects of surface biosynthetic reactions in tolerant oral streptococci. ACKNOWLEDGMENTS This research was supported by U.S. Department of Health, Education and Welfare grant DE-518. T.A.K. was supported by National Research Service Award fellowship DE-5227. Downloaded from http://iai.asm.org/ on May 9, 218 by guest

32 KRAL AND DANEO-MOORE We thank G. D. Shockman for stimulating discussions and criticism. LITERATURE CITED 1. Carson, D., and L. Daneo-Moore. 1978. Effects of cerulenin on Streptococcus faecalis macromolecular synthesis and cell division. J. Bacteriol. 133:472-476. 2. Daneo-Moore, L., and G. D. Shockman. 1977. The bacterial cell surface in growth and division, p. 597-715. In G. Poste (ed.), Cell surface reviews, vol. 4. Elsevier/North Holland, Amsterdam. 3. Hakenbeck, R., and W. Messer. 1977. Activity of murein hydrolases in synchronized cultures of Escherichia coli. J. Bacteriol. 129:1239-1244. 4. Higgins, M. L., and L. Daneo-Moore. 198. Effect of macromolecular synthesis and lytic capacity on surface growth of Streptococcus faecalis. J. Bacteriol. 141:938-945. INFECT. IMMUN. 5. Higgins, M. L., T. D. McDowell, U. B. Sleytr, M. Mychajlonka, and G. D. Shockman. 198. Effects of penicillin on macromolecular synthesis and surface growth of a tolerant streptococcus as studied by computer reconstruction methods. J. Bacteriol. 144:1168-1173. 6. Hinks, R. P., L. Daneo-Moore, and G. D. Shockman. 1978. Cellular autolytic activity in synchronized populations of Streptococcusfaecium. J. Bacteriol. 133:822-829. 7. Shockman, G. D., L. Daneo-Moore, T. D. McDowell, and W. Wong. 1981. Function and structure of the cell wall-its importance in the life and death of bacteria, p. 31-66. In M. Salton and G. D. Shockman (ed.), P-Lactam antibiotics. Academic Press, Inc., New York. 8. Terleckyj, B., N. P. Wi^lett, and G. D. Shockman. 1975. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect. Immun. 11:649-655. 9. Tonasz, A. 1979. The mechanisms of the irreversible antimicrobial effects of penicillins: how the beta-lactam antibiotics kill and lyse bacteria. Annu. Rev. Microbiol. 33:113-137. Downloaded from http://iai.asm.org/ on May 9, 218 by guest