Requirement of Deoxyribonucleic Acid Synthesis for Microcycle

Vol. 120, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Dec. 1974, p. 1331-1338 Copyright 0 1974 American Society for Microbiology

Requirement of Deoxyribonucleic Acid Synthesis for Microcycle Sporulation in Bacillus megaterium MYRON MYCHAJLONKA AND RALPH A. SLEPECKY

Department of Biology, Syracuse University, Syracuse, New York 13210 Received for publication 23 September 1974

Bacillus megaterium cells have been examined during outgrowth for their macromolecular content, ability to undergo microcycle sporulation, the time of their growth division, the time of deoxyribonucleic acid (DNA) replication initiation, and their ability to synthesize DNA after transfer to sporulation medium. The increase in total DNA content of the cells increased discontinuously beginning at 90 min. Thymidine incorporation became insensitive to chloramphenicol between 90 and 105 min of outgrowth. At 90 min the cells acquired the ability to undergo microcycle sporulation and the degree of sporulation depended on the time spent in outgrowth, with maximal sporulation occurring at 180 min. During outgrowth, cells underwent one synchronous growth division beginning at 225 min and ending at 270 min. Outgrowing cells were not able to continue DNA synthesis after transfer to sporulation medium. The data suggest that DNA replication starts before cells are able to undergo microcycle sporulation; however, the initiation of replication may not be the only requirement for microcycle sporulation.

After bacterial spore germination, the outgrowing cells can proceed under proper conditions to the first growth division. After further divisions in batch culture or upon transfer of stationary-phase cells to replacement media the cells can sporulate. It is possible to manipulate the cells during outgrowth so that they will proceed directly to sporulation before the first growth division. This conversion of an outgrowing cell to a sporulating cell without any intervening cell division, termed microcycle sporulation, was first described by Vinter and Slepecky (38) who induced outgrowing cells of Bacillus cereus and Bacillus megaterium to sporulate by diluting the outgrowth medium with saline. Microcycle sporulation in B. megaterium was later shown to be cytologically identical to the sporulation which occurs in this organism in batch culture (7). In addition to medium dilution, microcycle sporulation may occur in media specifically designed to allow germination, outgrowth, and microcycle sporulation (17, 23). Microcycle sporulation may be induced in a complete growth medium by treating the outgrowing cells at a particular time with D-cyclOserine and vancomycin (30). Early work has suggested that outgrowing cells are best able to undergo microcycle sporulation when the shift from growth conditions to sporulation conditions occurs during a time when cells were synthesizing deoxyribonucleic

acid (DNA) (38). In media designed to allow outgrowth and microcycle sporulation, the DNA content was seen to increase during the course of microcycle sporulation, suggesting an involvement of the DNA replication cycle (17, 23). A microcycle sporulation system offers a way to study the relationship of DNA replication to the sporulation cycle as has been shown in the prokaryotic cell division cycle (2, 3, 6, 12). We have established and characterized a microcycle sporulation system based on medium replacement during outgrowth. We have determined the following: the germination kinetics; the growth kinetics after transfer to growth medium; the timing and synchrony of the first cell division after outgrowth; the onset of the ability of outgrowing cells to undergo microcycle sporulation; the pattern of RNA, protein, and DNA synthesis in outgrowth; and that outgrowing cells are unable to continue DNA synthesis after transfer to sporulation medium. MATERIALS AND METHODS Organism. The organism used was B. megaterium ATCC 19213. Preparation of spores. A flask containing 50 ml of nutrient broth (Difco) was inoculated with 0.1 ml of a stock spore suspension and incubated overnight, and 0.1-ml samples were spread on nutrient agar (Difco) supplemented with MnSO4.H20 (7 mg/liter). After 72 h at 30 C, spores were harvested and cleaned as

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MYCHAJLONKA AND SLEPECKY

described earlier (15). Spores were suspended in distilled water containing 200 Ag of lysozyme per ml (Calbiochem) and incubated 1 h at 37 C. Spores were then washed successively with sodium dodecyl sulfate (17.3 mM), NaCl (1.0 and 0.14 M), and three times with distilled water. The spore suspension was diluted with distilled water to an optical density of 400 Klett units on a Klett Summerson photoelectric colorimeter with a no. 54 filter. The final spore suspension was distributed into 5.0-ml volumes and stored at -20 C until used. Media and growth conditions. The growth medium was the sucrose salts medium (SS) of Slepecky and Foster (33), a defined minimal salts medium containing sucrose which was routinely passed through a membrane filter (0.45 Am pore size; Millipore Corp.) before use. The replacement sporulation medium (R) contained sodium acetate (5.0 mM), MgSO4.7H,O (0.5 mM), CaCl2 (0.3 mM), and potassium phosphate buffer (10.0 mM, pH 6.9). All growth and sporulation was carried out at 30 C on a New Brunswick gyratory water bath shaker (180 rpm). For each experiment, a sufficient quantity of spores was thawed and then heat-shocked at 60 C for 60 min. After cooling, the spore suspension was diluted with an equal volume of prewarmed, double-strength germination medium to give final concentrations of L-alanine (100 ,ug/ml), inosine (100 ug/ml), and potassium phosphate buffer (0.055 M, pH 7.0), and a final volume of 50 to 220 ml depending on the experiment. After 3 h the germinated spores were removed from the germination medium and suspended in growth medium. In some experiments the cells were removed from growth medium and placed into sporulation medium. All cultures were incubated in Erlenmeyer flasks of sufficient size such that the volume of culture was never greater than 20% of flask capacity. To effect transfer from one medium to another, samples of culture were filtered by membrane filtration and washed on the filter with the transfer medium. Cells were removed from the filter by vigorously agitating (Vortex Genie; Scientific Industries Inc.) the filter containing adsorbed cells in 5.0 ml of the transfer medium in a large, sterile screw-cap tube; the cells were then transferred to Erlenmeyer flasks and incubated. The final volume of the resuspension medium was in all cases the same as the volume of the culture sample which was filtered. The turbidity of a sample before filtration was found to be the same as the turbidity of a suspended sample after filtration, an indication that cells were not being lost during filtration. Measurement of bacterial growth. Germination of spores and growth of the cells was followed turbidimetrically as described previously. To determine cell number, 1.0-ml samples of a culture were diluted 1:10 into 50% (wt/vol) trichloroacetic acid, and samples of this dilution were transferred with a wire loop to a Petroff-Hausser counting chamber (C. A. Hausser and Son). Duplicate counts of about 300 cells were made for each dilution. Ability to undergo microcycle sporulation. At intervals during growth in SS medium, 10-ml samples of cells were taken from growth medium, filtered,

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washed with sporulation medium, suspended in sporulation medium, and incubated at 30 C with gyratory shaking. The total number of germinated and refractile forms was determined using a Petroff-Hausser counting chamber and a phase-contrast microscope at zero time and the number of refractile forms was determined after 12 h of incubation. The number of phase-bright forms present at the beginning of incubation in sporulation medium was subtracted from the phase-bright forms present after incubation. This corrected number of refractile forms present at the end of incubation is expressed as a percentage of phase-dark forms present at the start of incubation in sporulation medium. Measurement of DNA, RNA, and protein synthesis. Ten samples of culture were taken at intervals, 1.0 ml of 50% trichloroacetic acid was added, and the samples were chilled in an ice bath overnight. Samples were centrifuged and the pellet was washed with ice cold 5% trichloroacetic acid. The pellet was then extracted three times with perchloric acid (0.5 N) at 70 C. The extracts were pooled and assayed for DNA by the method of Burton (1) using indicator-grade diphenylamine and redistilled glacial acetic acid. In assessing DNA synthesis in replacement sporulation medium, the cultures were assayed for total DNA content 180 min after transfer of the last sample to sporulation medium. The extracts were assayed for ribonucleic acid (RNA) by the orcinol method (13, 28). A spectrum of the colored product of the orcinol reaction from the last experimental tube (250 min) was identical with the spectrum obtained using pure RNA, indicating the absence of any hexose interference in the assay system (13). The pellet was dissolved in 1.0 N NaOH, and protein was assayed by the method of Lowry et al. (20). Isotopic methods. The incorporation of labeled thymidine was used as an indicator of DNA synthesis. Cold thymidine (Mann Research Labs) (5 Mg/ml), deoxyadenosine (CalBiochem) (500 Mg/ml) and [methyl-'HJthymidine (ICN, Pharmaceuticals, Inc.) (specific activity 57.8 Ci/mmol) was added to a culture at zero time. Samples (1 ml) of the culture was added to an equal volume of 2.0 N NaOH containing 50 Mg of unlabeled thymidine per ml. After 12 to 18 h of incubation, the samples were neutralized with 1.0 ml of 2.0 N HCI and 0.5 ml of carrier calf thymus DNA (Calbiochem) (150 Mg/ml) was added. Finally, 0.5 ml of 50% trichloroacetic acid was added, and the contents of the tube were mixed and held on an ice bath for several hours. The precipitate was collected on a membrane filter which had been presoaked with 5% trichloroacetic acid containing thymidine (50 Mg/ml). The precipitate was washed three times with 5% trichloroacetic acid containing thymidine. The filters were dried, covered with scintillation fluid (2,5-diphenyloxazole, 0.4% [wt/voll and 1,4-bis(5-phenyloxazolyl)benzene 0.01%, Packard Instrument Co.; in toluene, Baker and Adamson), and counted in a scintillation counter (Beckman LS-100). Chloramphenicol - insensitive incorporation. Cells were treated with [3H Jthymidine, cold thymidine, and deoxyadenosine immediately after transfer to growth medium. At intervals, samples of the

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DNA SYNTHESIS AND MICROCYCLE SPORULATION

VOL. 120, 1974

culture were transferred to flasks and chloramphenicol (Sigma Chem. Co.) was added (final concentration 50 gg/ml). After the addition of chloramphenicol, a 1.0-ml sample of culture was taken for the determination of thymidine incorporation into DNA. The remainder of the sample was incubated at 30 C. At 385 min after the start of outgrowth, all of the chloramphenicol-treated cultures were sampled for thymidine incorporation. The amount of thymidine incorporated at the time of chloramphenicol addition was subtracted from the amount incorporated in the presence of chloramphenicol.

RESULTS The germination of heat-shocked spores was initially rapid (Fig. 1). Spores were retained in germination medium for 3 h to insure maximum germination. At the end of that time, 90 to 95% of the spores present has lost their refractility as judged by microscope examination. The germination medium was not a growth medium and germinated spores would not proceed beyond phase darkening and slight swelling even when incubated overnight. The germinated spores would resume their development only if placed in complete growth medium. Figure 2 shows that there was a lag after cells were placed in growth medium before the turbidity of the culture began to increase exponentially. This lag was reproducible from experiment to experiment. The time required to double the optical density of the culture was approximately 3 h giving a specific growth rate based on the increase in turbidity of 0.23. In our outgrowing system, as in others (14, 36), the germinated spore first swelled to approximately double its size before starting elongation, and then divided after elongation was complete. The rate of increase of total RNA and protein

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content of the outgrowing cells was determined (Fig. 3). Although RNA content seemed to increase immediately after transfer to growth medium, the increase was not exponential until approximately 40 min after the start of incubation in growth medium. This lag was of the same duration as the lag seen before the turbidity began to increase. The specific growth rate of the outgrowing culture, based on the increase in total RNA content, was 0.55. There was an exponential increase in total protein content throughout the outgrowth period. The specific growth rate of the outgrowing culture based on the rate of increase of total protein content was 0.39. The specific growth rate of the outgrowing was different for each of the parameters measured-cell mass (turbidity), total RNA, or total protein-indicating that the outgrowing system was in unbalanced growth (22). The cells divided at the end of outgrowth. The first cell division after outgrowth (Fig. 4) began 225 min after the start of incubation in growth medium and was complete by 270 min. There was a doubling of cell number during this interval and the cell number doubling occurred 100 in the stepwise fashion expected of synchronous growth (32). o Figure 5 shows the ability of outgrowing cells to undergo microcycle sporulation after replacement of the growth medium with sporulation " 60 medium. In place of the sucrose found in growth medium, sporulation medium contained low levels of acetate as a carbon source and no 40 nitrogen source. Cells were unable to divide in medium when sucrose was replaced with growth 20 . the level of acetate found in sporulation medium (9). Cells did not divide in sporulation 60 80 20 40 100 120 0 medium when nitrogen, NH,, was added, nor MINUTES SPENT IN GERMINANTS cells divide in sporulation medium when would FIG. 1. Germination of spores of B. megaterium in phosphate buffer containing inosine and L-alanine. sucrose was substituted for acetate. However, The ordinate is expressed as a percentage of initial cells grew and divided only when sporulation medium was supplemented with a nitrogen optical density.

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90 min, outgrowing cells began to acquire the ability to undergo microcycle. The cells reached a maximum in this ability at 180 min and this maximum was seen 45 min before the first signs of cell division were observed. The incubation period in sporulation medium is long (12 h) to assure that all of the cells which can sporulate will sporulate. Because of the long incubation time, the percentage of sporulation is based on the number of phase-dark forms seen at the beginning of incubation in sporulation medium. Our method of counting gives us a background of apparent sporulation with samples transferred to sporulation medium at 90 min or earlier. In none of these samples was there any evidence of refractile forms present inside of sporangia, suggesting that the background levels of spores did not arise from microcycle sporulation. There appeared to be no increase of the total DNA content for the first 90 min of incubation in growth medium (Fig. 6). At 90 min the total DNA content began to increase in an apparently linear fashion. Beginning at 180 min the rate of increase of the total DNA content increased at a new higher rate about 3.5 times the rate of DNA increase between 90 and 180 min. These data suggested that chromosome replication began at 90 min and that another

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FIG. 5. Ability of outgrowing cells to undergo misource, NH4+, and when acetate was replaced crocycle. At intervals after transfer to growth medium (SS), samples of culture were transferred to sporulawith sucrose. tion medium (R). The sporulating ability was scored Outgrowing cells transferred to sporulation as described in Materials and Methods. The ordinate medium at 90 min or earlier were relatively shows the percentage of phase-dark forms which inefficient at undergoing microcycle sporulation proceed through sporulation during 12 h of incubation on transfer to sporulation medium (Fig. 5). At in sporulation medium.

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dichotomous round began at 180 min. The time at which the total DNA content began to increase was the same time that the cells began to be able to microcycle (Fig. 5). The pattern of chloramphenicol-insensitive thymidine incorporation was determined to test whether the discontinuities in the pattern of total DNA increase represented periods of replication initiation. The initiation of DNA replication is known to be sensitive to chloramphenicol inhibition. A round of replication, once initiated, is able to proceed to completion in the presence of chloramphenicol; however, no subsequent replication rounds may be started (21, 22). Figure 7 shows a sharp increase in the chloramphenicol-insensitive thymidine incorporation between 90 and 105 min of incubation in growth medium. The stepwise increase in chloramphenicol-insensitive thymidine incorporation was followed by a plateau until 150 min, suggesting that the 90-min mark represented the initiation of a round of replication. The data also suggest that a second dichotomous round of replication began between 150 and 195 min, a result also predicted by the discontinuity of the increase in total DNA at 180 min (Fig. 6). If DNA replication, once started, could proceed to completion in sporulation medium, then the total DNA content of outgrowing cells transferred to an incubated in sporulation medium would be expected to show a stepwise increase in DNA content for those samples transferred after 90 min. No such stepwise pattern was seen (Fig. 8). Furthermore, the amount of DNA found after incubation in sporulation medium was not significantly different from the DNA content of the sample. before incubation in sporulation medium. Outgrowing cells were apparently not able to continue DNA synthesis after transfer to sporulation medium.

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DISCUSSION The most striking aspect of cell division (8). The first step in the sporulation cycle control is its close relationship to the DNA presumably would be the initiation of chromoreplication cycle (2, 6, 12, 39). Donachie et al. some replication. The onset of the ability of outgrowing cells to have proposed that the cell division cycle starts with the initiation of chromosome replication complete microcycle sporulation may corre(6). A point of view advanced recently suggests spond to some outgrowth event implicating that that the early stages of sporulation may be a event as a requirement for sporulation. In the modified type of cell division (8, 16, 31). Such a system described, all components are chemiview allows one to construct models of sporula- cally defined, the outgrowth process is ordered tion in which the location of the forespore (19, 34, 35), and the replication of the chromoseptum and the timing of its appearance are some may be highly synchronous as has been determined by the same controls operative in found in B. subtilis (29, 40, 41). Thus microcycell division modified by step-down conditions cle sporogenesis may be an ideal tool with which


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MINUTES IN SS BEFORE TRANSFER TO R FIG. 8. Total DNA of outgrowing cells of B. megaterium in SS medium and after transfer to sporulation medium (R). Samples of an outgrowing culture were assayed for total DNA content at the times indicated as described previously (top panel). Other samples were membrane filtered, washed on the filter with sporulation medium, and resuspended in sporulation medium (R). All of the sporulating subcultures were assayed for DNA content (bottom panel) 180 min after the last sample was transferred to sporulation

medium. to study the relationship of the DNA replication cycle with the sporulation cycle. No obvious relationship between the exponential increase in cell mass, total RNA, and protein observed during outgrowth and cell ability to initiate microcycle sporulation was seen. Total RNA increased exponentially only after a lag of 40 min. This lag is of the same duration as that seen prior to the increase in turbidity or cell mass. The specific growth rate measured by each of these three parameters is different, indicating unbalanced growth, characteristic of cells adapting to a new environment (22). After outgrowth, the cells divide, doubling their number in a stepwise synchronous fashion starting at 225 min and ending at 270 min. Cell division in this system occurs after the time at which cells may be induced to sporulate on transfer to a sporulation medium. The outgrow-

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ing system is synchronous with respect to the cell division cycle, indicating that the progression of outgrowth events is ordered at least to the first cell division. At a point, then, in this progression, the cells acquire the ability to complete microcycle after transfer to sporulation medium. At the same time that cells began to acquire their ability to microcycle (90 min), the total DNA content began to increase. These data suggest that DNA replication does not start until 90 min after outgrowth has begun. They also suggest that, later on in outgrowth, DNA replication may be dichotomous. We have tested the possibility that the increase of total DNA content at 90 min represents an initiation of chromosome replication using chloramphenicol inhibition. If the 90-min mark represents a replication initiation point, then there should be a stepwise increase in the amount of chloramphenicol-insensitive thymidine incorporation at that time. Our data show such a step and, in agreement with the total DNA data, suggest that chromosome replication is initiated in our system at 90 min. The onset of the ability to complete microcycle sporulation occurs at the same time in outgrowth as the start of DNA replication, suggesting that the two events are related and that the cell must start DNA replication before it is able to complete microcycle after transfer to sporulation medium. This involvement of the replication cycle in microcycle seems analogous to the involvement of the DNA replication cycle in the division cycle. Initiation of chromosome replication appears to be the starting point of those events which lead to cell division-the same may be true of the relationship of initiation of replication and the sporulation septation of microcycle sporulation. If the start of replication were the only event that outgrowing cells had to achieve before they were able to complete microcycle, then one would expect to see a stepwise increase in the ability to form spores upon transfer to sporulation medium after 90 min. Such a stepwise increase was not seen, so it would appear that the initiation of replication is not sufficient to allow cells to complete microcycle. Perhaps a round of replication must run to completion before a cell may microcycle. Outgrowing cells cannot continue DNA synthesis after transfer to sporulation medium. This result leaves open the possibility that completion of a replication round must occur before the outgrowing cells may microcycle. Another possibility is that the cells, in addition to initiating

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replication, must reach a particular stage of outgrowth, e. g., start elongation, before they are able to microcycle. That high levels in cell mass, total RNA, and protein were achieved before the time the cells could switch to the sporulation mode suggests that some minimum level of RNA and protein, or possibly specific types, may be required. Other results suggest that sporulation induction exhibits a cell cycle dependency (4, 5, 24, 26; J. Mandelstam and S. A. Higgs. Abstracts of communications, 540th Meet. Biochem. Soc., University of Oxford, 1973, p. 2-3). There are, however, some differences in the two systems: (i) ours is an outgrowing system; (ii) our growth medium is minimal and defined and gives a slow growth rate; (iii) DNA synthesis does not continue after resuspension of cells in the defined sporulation medium. Nevertheless, both approaches demonstrate that the replication cycle must be initiated before cells are able to undergo sporulation. ACKNOWLEDGMENTS This investigation was supported by Grant P2B3347 from the National Science Foundation to Ralph Slepecky. LITERATURE CITED

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