The synchronization of Bacillus subtilis by the relaxation of a division control system

R. J . L. PAULTON Deparfrrlerlt of Bacferiology, Urriversify of Saskarcheivatz, Srrskrrtoon, Saskatchewan

Received August 3, 1970

PAULTON, R. J. L. 1971. The synchronization of Bacill~rs srrbfilis by the relaxation of a d~vision control system. Can. J. Microbiol. 17: 119-122.

Synchronously dividing cultures of Bacilllts sltbtilis were obtained by harvesting cells from the stationary phase and reincubating them in fresh medium. It was discovered that the first division period was not necessarily synchronous, but when the first synchronous division occurred it involved a reduction in septation. These findings suggested that cell synthesis was directed to more than the forthcoming division, and that synchronization involved a subsequent division and a change in the cell division control mechanism.

Introduction were incubated for about 16 h at 30°C without shaking until populations had exceeded I X 107 cells/ml, when

The 'stationary-phase' method for the syn- 100-ml aliquots were removed. Cells were then harvested chronization of bacterial cultures which has by centrifugation at 4000g for 5 min at 30°C and syn- been applied to ~ ~ ~ h ~ ~ i ~ h i ~ co[i (2, 5), Profeus chronous growth obtained by inoculating the cells into vulgaris (2), and Baci~us subtilis (4, 6, 7) takes 100 m! of fresh medium and reincubating them at 30°C

with shaking. advantage of the tendency of cells to During growth, cell numbers were determined using a themselves when entering the stationary phase of model B Coulter Counter and samples removed for a growth. The results of this method imply that tannic acid/crystal violet cell wall stain (7).

the generally unfavorable growth conditions pref- erentially inhibited the cell populations at par- Results ticular points in the cell cycle and that reincuba- When cultures of ill^^ subtilis were in- tion in fresh medium, ~lsually at a lower cell cubated with low aeration, a considerable re- concentration, alleviated this inhibition (2, 5). duction in the growth rate was observed after There is, however, considerable evidence from the organism had reached a concentration of studies of the cell wall (8) and chromosome (1, 1.5 x 107 cells/ml (Fig. IA). If cells were 3) that cell synthesis is directed to more than the harvested 4 h later, when the numbers had in- forthcoming division. Because a reduction in cell creased to 3 x 107 cells/ml, synchronoLls growth size and complexity is expected as a culture was obtained after reincubation in fresh medium. enters the stationary phase, it follows that this The growth pattern of culture C shows that the method of synchronization may involve the response to reincubation was an immediate inhibition of one or several division sequences. division period, so that the synchronization This communication describes some observa- method appear to have inhibited the cell tions on Bacillus subtilis in which synchroniza- population at a point in the cell cycle just before tion was attributed to the ability of this bacterium cell division. to organize and change from a division control Figure 1~ also shows the growth of two cul- system. tures, A and B, which were obtained at earlier

Materials and Methods times from the stationary phase of the parent culture. In these cases, similar growth to C was

The strain of Bacill~rs subfilis used in this investigation was obtained from The Imperial College, University of after they had reached lo7 lm1. London, England. The organism was grown in a lnininlal However, the pattern of cell division from the salts medium containing dipotassium hydrogen phos- time of harvesting until this concentration had phate (0.773, potassium dihydrogen phosphate (0.3%), been reached was such that the synchronization ammonium sulphate (O.l%), trisodium citrate (O.l%), magnesium sulphate (0.0273, and glucose (0.2%). Initial method could not be explained simply by in- gowth was obtained in 600-m1 batch ql,antities of the hibition of the cell population at a specific point synthetic medium contained in 5-liter flasks. Cultures in a division cycle.

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120 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 17. 1971

During the first 60 min of growth in culture A, the cell number increased exponentially until, at 3 X 107 cells/ml, the first synchronous divi- sion occurred. When cells were harvested 2 h later, as in culture B, the first division period in- volved a threefold increase in cell numbers. The ability of a culture to change from one division sequence to another, or to exhibit a threefold increase in numbers, as seen in A and B, respec- tively, was partly explained by observations of simultaneous changes in the number of division sites per cell during these initial growth periods. These observations are shown in Fig. 1B.

During logarithmic growth of the parent culture, 85% of the cell population were found to contain three septa and the rest seven. This septation pattern was found in A and B as they increased to 3 X 107 cells/ml and also initially in C . However, at this concentration all three cultures, in dividing synchronously, became corn- posed predominantly of single septate cells which were observed until just before the next division when cells containing three septa were evident.

The observations presented in Fig. 1 suggest that this synchronization method involved a

T i m e ( h o u r s )

T i m e ( h o u r s )

FIG. 1. The pattern of cell division and change in septation during the growth of synchronous cultures obtained from the stationary phase of a parent culture. Fig. 1A shows the increase in cell numbers (X) of a static culture entering the stationary phase, and three synchronous cultures: A ( .), B (o), and C (A) obtained after 0, 2, and 4 h incubation in the stationary phase. The change in septation during the establishment of the three synchronous cultures is shown in Fig. 1B.

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PAULTON: SYNCHRONIZATION OF BACILLUS SUBTILIS 121

change in the division control system. In the parent culture, where most of the cells contained three septa and the rest seven, a prerequisite to cell division was the increase in the number of successive division sites from two to three. At a concentration of 1.5 X 107 cells/ml, the cells of this culture were potentially synchronized, not for the forthcoming division, which was random, but for that following. Therefore, reincubation of these cells in fresh medium, as in A, produced one further random division whilst maintaining the existing septation pattern until a concentra- tion of 3 X 107 cells/ml was reached. Then the entire population divided simultaneously with- out producing future division sites.

Before culture B was harvested, the cells were exposed for 2 h to an inhibition of division during which time only one-half of the population divided. The rest were apparently arrested just before cell division. Reincubation allowed them to divide almost immediately, which increased the cell numbers to 3 X 107 cells/ml when the total population again divided synchronously. Cells that were obtained from the stationary phase at a concentration of 3 X 107 cells/ml were capable of an immediate synchronous division and a reduction in septation, as shown by culture C.

This reduction in septation has also been observed in asynchronous cultures which were obtained by incubation with shaking from the lower concentration of 103 cells/ml. In this case, before the concentration of 3 X 107 cells /ml was reached, the average number of septa per cell was found to be about 3.5, which did not differ from that observed with static cultures. How- ever, in the next 60 min, as the cells divided asynchronously, this value was reduced by one- half. I t would thus appear that this reduction in cell complexity is a characteristic of growth of this bacterium a t particular cell concentrations, as is the existence of several growth phase in batch culture (9).

The synchronization method, involving initial growth in static cultures followed by reincuba- tion with increased aeration, appears to have manipulated the cell population to a condition where a simultaneous reduction in cell com- plexity took place. Subsequently, the cells were a t the same stage of preparedness for the next division.

Discussion I t has recently been established that during

asynchronous logarithmic growth of B. subtilis, the septation pattern can be predicted from the growth rate (8). With a generation time of 60 min, as in this present study, the population would be expected to contain cells with either three or seven septa. This contrasted with observations on synchronous cultures, also with a generation time of 60 min, in which cells con- tained one septum throughout the cell cycle, excepting periods just before cell division when triseptate cells were observed (7). This present study was designed to account for this dis- crepancy and required a reexamination of the 'stationary-phase' method for the synchroniza- tion of bacterial cells.

The explanation for the production of multi- septate cells by B. subtilis (8) was similar to that proposed for multifork replication of the chro- mosome in E. coli (1, 3). In both cases, the organisms were grown at varying rates in re- sponse to the nutrition of the medium, and it became apparent that there were restrictions on the rate of individual syntheses. In E. coli, growing at 37OC with a generation time of between one and three doublings per hour, the time between the initiation of chromosome synthesis and the separation of the replicated chromosomes at division was found to be 63 min. Similarly, in B. subtilis grown a t 30°C, the time between the appearance of cell septa and their involvement in division was 138 min, regardless of the growth rate. The ability of these organisms to grow a t rates in excess of a 63- o r a 138-min generation time, respectively, was attributed to the organization of multiple sites for chromosome and septum synthesis.

The measurements in both of the above studies were performed a t particular cell con- centrations and it was implied that growth was logarithmic. However, continuous changes in cell complexity have been recorded throughout the logarithmic phase (9). As a culture reaches the maximum population afforded by the growth conditions a reduction in growth rate and cell complexity is expected. The reduced growth rate would not necessitate the 'overlap' in cell synthesis recorded for cell wall (8) and chromo- some (1, 3) synthesis. Consequently, if syn- chronization occurs, the inhibition may be

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1 22 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 17. 1971

directed towards, depending on the organism and growth rate, several division sequences.

Acknowledgments

The assistance of Mr. W. D. Hoppe and Mrs. Marilyn Haskins is greatly appreciated. This work forms part of an investigation supported by the Medical Research Council, Ottawa, Canada.

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2. CUTLER, R. G., and J. E. EVANS. 1966. Synchroniza- tion of bacteria by a stationary-phase method. J. Bacteriol. 91 : 469476.

3. HELMSTEITER, C . E., S. COOPER, 0. PLERUCCI, and E. REVELAS. 1968. On the bacterial life sequence. Symp. Quan. Biol. 33: 809-822.

4. MASTERS, M., P. L. KUEMPEL, and A. B. PARDEE. 1964. Enzyme synthesis in synchronous cultures of bacteria. Biochem. Biophys. Res. Commun. 15: 3842.

5. NISHI, A., S. OKAMURA, and T. YANAGITA. 1967. Shift of cell-age distribution pattern in the later phases of Escherichia coli culture. J . Gen. Appl. Microbial. 13: 103-119.

6. PAULTON, R. J. L. 1968. Cell wall formation in Bacillrrs srrbtilis. Ph.D. Thesis, University of London, London, England.

7. PAULTON, R. J. L. 1970. Cell septation duringsynchro- nous growth of Bacill~is srrbtilis. Nature (London), 227: . .

517-fi8. 8. PAULTON, R. J. L. 1970. Analysis of the multiseptate

potential of Bacillrrs srrbtilis. J. Bacteriol. 104: 762- 767

9. ToeNN~es, G., L. ISZARD, N. B. ROGERS, and G. D. SHOCKMAN. 1961. Cell multiplication studied with an electronic particle counter. J. Bacteriol. 82: 857-866.

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