Vol. 136, No. 1
JOURNAL OF BACTERIOLOGY, Oct. 1978, p. 111-116 0021-9193/78/0136-0111$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Independence of Bacillus subtilis Spore Outgrowth from DNA Synthesis DALIA GINSBERG AND ALEX KEYNAN* Section of Developmental and Molecular Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Received for publication 25 July 1978
The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidixic acid and 6-(phydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhibition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors. In recent years, evidence has accumulated that induction of sporulation in Bacillus sp. can occur only during a limited stage in the cell cycle and is linked to DNA replication (11, 15). Evidence for a link between the DNA replication cycle and induction of differentiation has also been demonstrated in a variety of other differentiating cells (9; for reviews, see also 17). Both bacterial sporulation and outgrowth of germinated spores differ from vegetative growth in the control of gene expression and have been suggested as model systems for the study of cell differentiation. It would be of interest to know if spore outgrowth is also dependent on DNA replication. Halvorson et al. (16, 21) obtained evidence that outgrowth in Bacillus cereus occurs in the absence of DNA synthesis. After germination, RNA and protein syntheses begin immediately and net DNA synthesis occurs only much later (10, 16, 24). On the other hand, limited incorporation of radioactive precursors into DNA has been observed immediately after germination in some Bacillus species (12, 16, 19) and has been interpreted as either repair synthesis or limited DNA replication. It was also found that inhibitors of DNA synthesis interfered with outgrowth (6, 14, 21), but because of the possibility of nonspecific effects, no final conclusion could be reached on the need of DNA synthesis for outgrowth. It appears that the question of the dependence of outgrowth on DNA synthesis has not yet been
settled. We reexamined this problem using thymidine-requiring mutants and temperature-sensitive conditional DNA mutants of Bacillus subtilis. We also studied the effect of two inhibitors, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), which have been reported to inhibit DNA synthesis specifically and are not known to have any other detectable metabolic effect (1, 2, 9, 23). Two main conclusions are the result of this study. First, DNA replication is not essential for outgrowth. Second, the experiments with inhibitors indicate that their effect on outgrowing spores is different from their effect on vegetatively dividing cells, confirming the existence of fundamental differences in the physiology of outgrowing spores and vegetative cells (10). MATERIALS AND METHODS B. subtilis 168 trpC2 and its mutants, MB65 (temperature sensitive for the initiation of chromosome replication) and BC109 (a thymine auxotroph), were used throughout this study. The inhibition of DNA synthesis under restrictive conditions was verified in each experiment in which these mutants were used. Activated spore stocks were plated (on Lalanine-containing media) under restrictive conditions and compared with parent spore stocks. Only spore suspensions that did not form colonies under restrictive conditions were used. No DNA synthesis occurred during outgrowth under restrictive conditions when measured by Burton's method (3). The outgrowing cultures of mutants grown under restrictive conditions were examined microscopically in the presence of CsCl 111
112
GINSBERG AND KEYNAN
(20) for visualization of septa. Under restrictive conditions, these cells always appeared as long filaments with no septa, as described previously for cells growing out in the absence of DNA synthesis (20). Spores were obtained after 36 to 40 h of bacterial growth at 370C in Sterlini and Mandelstam resuspension medium (22) containing 2 g of glucose per liter. The spores were harvested, washed with distilled water, and centrifuged through 50% (vol/vol) Urografin (Schering Chemicals, Ltd.) to remove any residual germinated spores (20). The pellet, consisting of uniformly brightphase spores, was washed several times. It was then suspended in distilled water at a concentration of 2 x 10'0 to 4 x 10"' spores per ml and stored at -20°C. Heat activation. The spore suspension was heat activated by incubation for 30 min at 700C. Germination. Heat-activated spores were incubated at concentrations of 2 x 108 to 4 x 108 spores per ml in germination medium containing 1.0 g of Lalanine, 0.59 g of NaCl, 0.1 g of Na2SO4, and 0.07 g of KH2PO4 per liter (pH adjusted to 7.0). After agitation at 370C for 30 min, over 95% of the spores had germinated. Germination was not altered by the presence of 20 ug of NAL (Sigma) per ml or 20 ,ug of HPUra (a gift of N. Brown) per ml. Where indicated, spores were centrifuged after germination and then placed in outgrowth media. In other experiments, activated but not germinated spores were introduced directly into outgrowth media. In these experiments there was an initial fall in optical density of the culture, corresponding to the period when germination took place in the outgrowth media. In the context of this report, the word "outgrowth" is used to define the period between the end of germination (loss of all typical spore properties) and the first cell division. It describes the stage in the life cycle of the cell in which a germinated spore is transformed into a vegetative cell (10). Outgrowth was measured in this work as an increase in optical density or by incorporation of radioactive amino acids. Outgrowth. Heat-activated spores were suspended at a concentration of 2 x 108 to 4 x 108 spores per ml in the following three media. (i) Rich medium was brain heart infusion broth (Difco) containing 5 g of yeast extract (Difco) per liter. (ii) Semisynthetic medium contained 14 g of K2HPO4, 6 g of KH2PO4, 0.25 g of MgSO4 7H20, 0.017 g of MnSO4 H20, 2 g of (NH4)2SO4, 0.15 g of sodium glutamate, 5 g of yeast extract (Difco), 1 g of L-alanine, and 25 mg of tryptophan, per liter. (iii) Synthetic medium was a modified semisynthetic medium in which L-alanine and yeast extract were omitted and supplemented with glucose (5 g/liter) and Casamino Acids (Nutritional Biochemicals Corp.) (8.5 g/liter). Since germination did not occur in this synthetic medium, spores were first germinated in a germination medium, then centrifuged and resuspended in the synthetic outgrowth medium. All cultures were incubated under conditions of vigorous aeration on a gyratory shaker at 370C. Outgrowth was followed by measurement of optical density at 600 nm with a Spectronic 21 model MV spectrophotometer (Bausch and Lomb). Protein synthesis during outgrowth was measured as follows: a uniformly "C-labeled L-amino acid mixture (Amersham; 1 ,uCi/ml) was added to the outgrowth medium, samples were removed into 5% cold trichloroacetic acid
J. BACTERIOL.
containing 0.5% casein hydrolysate, and the trichloroacetic acid precipitate was collected onto a 0.45-pm membrane filter (Millipore) and washed with the trichloroacetic acid-casein hydrolysate solution. The filter was placed in a scintillation vial and dried. It was then dissolved in Instagel (Packard Instrument Company) scintillation fluid, and the radioactivity was measured in a scintillation counter. DNA concentrations were estimated according to the method of Burton (3). The reasons for not estimating DNA by isotope incorporation were given in a previous publication (11).
RESULTS Outgrowth of mutants inhibited in DNA synthesis. Spores of two conditional DNA synthesis mutants of B. subtilis 168 were germinated in germination medium and then placed in miniimal medium for outgrowth under conditions restrictive to DNA synthesis. One strain, MB65, is a mutant that is temperature sensitive to chromosomal initiation, and the other, strain BS109, requires thymine. A minimal medium was chosen for these experiments, because (as will be shown later) in it outgrowth can be inhibited by NAL and HPUra. It was our intent to determine whether outgrowth could occur in this medium in the absence of DNA synthesis. The parent strain under these experimental conditions, or the mutants under permissive conditions, underwent a lag of a few minutes, followed by outgrowth. This could be measured by an increase in optical density or by incorporation of
radioactive amino acids. When outgrowth of these two mutants was followed under restrictive conditions (440C for mutant MB65, and in the absence of thymine for mutant BS109), their outgrowth pattem in the absence of DNA synthesis for the first 2 to 3 h was identical to that observed with the same strains under permissive conditions. Figure 1B (control) shows the data obtained with a thymidineless mutant. The same pattern (data not shown) was obtained with the temperature-sensitive strain under restrictive conditions. The microscopic appearance of the mutant cells grown under restrictive conditions was similar to that of the outgrowing parent culture or the mutant cells under permissive conditions, with the exception that no septa appeared. Septa were visible in the parent cells or in the mutants under perminsive conditions at approximately 120 min after initiation of outgrowth. In the strains inhibited in DNA synthesis, long curved cells with no septa were formed. The absence of septa formation has been described previously in outgrowing spores inhibited in DNA synthesis (20). These results were not media dependent. With these mutants, outgrowth occurred under restrictive conditions
VOL. 136, 1978
SPORE OUTGROWTH AND DNA SYNTHESIS
in the absence of DNA synthesis on each of the three media tested. Thus, it can be concluded that outgrowth of spores, measured as an increase in optical density or incorporation of radioactive amino acids, proceeds normally in these mutants in the absence of DNA synthesis. Effect of DNA synthesis inhibitors on outgrowth. The effect of NAL (20 fAg/ml) and HPUra (20 fAg/ml) on B. subtilis spore outgrowth was found to be medium dependent. In rich medium, a significant increase in optical density of the germinated spore suspension occurred in the presence or absence of either NAL or HPUra. In semisynthetic and synthetic media containing these inhibitors, no increase in optical density was detected (Fig. 2). No DNA synthesis occurred under these conditions in any of the three media. Microscopic observations of the cultures confirmed the optical density measurements. In rich medium the spores became swollen and elongated and emerged from the spore coat in both the presence and the absence of the inhibitors. In semisynthetic and synthetic media, in the presence of the inhibitors, the spores appeared swollen but not elongated and did not emerge from the spore coat. In semisynthetic or synthetic media in the absence of the inhibitors, spore outgrowth was similar to that of the controls in the rich medium. Protein synthesis in the presence or absence of inhibitor paralleled the rate of change in optical density (Fig. 1A and 2D). The outgrowing spore is considered to be different in its physiology from vegetative, multiplying cells (7, 10). It was interesting to learn whether or not this medium-dependent inhibition of spore outgrowth and protein synthesis was specific only for the spore outgrowth stage. Vegetative cells were grown in the three media
and exposed during logarithmic growth phase to NAL or HPUra. Growth of these vegetative cells, as measured by increased optical density or amino acid incorporation capacity, was not altered during the first 100 min after exposure to the inhibitors, although no DNA synthesis or cell division occurred during this time. The effect of the inhibitors was, therefore, specific for the stage of spore outgrowth only. At which stage the outgrowing spore became resistant to the inhibitor was another point of interest. Outgrowth was measured in germinated spores exposed to HPUra at various time intervals after germination had ceased (Fig. 1). The results show that after 30 min of outgrowth the cells were still sensitive to the inhibitor and became completely resistant 90 min after outgrowth. Identical results were obtained using NAL. No change occurred in the effectiveness of
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NAL or HPUra as DNA synthesis inhibitors; they halted net DNA synthesis whenever added to the culture. The time of sensitivity to the inhibitors corresponded to the period between the end of germination and the duplication of initial spore DNA during normal outgrowth. Experiments were performed to test for reversion of outgrowth inhibition in the semisynthetic medium by the addition of a variety of carbon sources. It was found that glucose could both prevent and reverse the inhibitory process, reversal occurring upon its addition at any time up to 90 min after beginning of outgrowth. Glucose
114
J. BACTERIOL.
GINSBERG AND KEYNAN
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could be replaced by fructose and sucrose but not by a-methyl glucoside or ribose. Addition of glucose did not affect the inhibition of DNA synthesis by HPUra or NAL, the inhibition occurring both in its presence and absence. The data describing DNA inhibition, in the presence of HPUra with or without glucose, are given in Fig. 3, curves 1 and 2. Although no DNA was
synthesized under both these conditions, the culture with glucose (curve 1) showed the same increase in optical density as the culture without inhibitors in which DNA synthesis was normal (Fig. 3). Reversal of the outgrowth inhibition, as well as renewed DNA synthesis, occurred when NAL or HPUra was removed by centrifugation and two washings with growth media. Changes in the sensitivity of outgrowing spores to NAL and HPUra in the absence of DNA synthesis. As stated above, spores
outgrowing in synthetic or semisynthetic media are inhibited by NAL and HPUra, and this inhibition decreases as a function of outgrowth time. The cell becomes completely resistant to these inhibitors at such time when, under normal outgrowth, chromosomal replication occurs. When the spores of the two mutants, MB65 and BC109, were outgrown in synthetic or semisynthetic media under conditions restrictive to DNA synthesis, outgrowth proceeded normally (as described above) but was prevented by the addition of NAL or HPUra. The time sequences of the "escape" from the inhibitory effects of HPUra or NAL in these mutants were identical to the parent strain and were the same under restrictive or permissive conditions. Thus, the development of resistance to these inhibitors during outgrowth is not DNA synthesis dependent (Fig. 1B), and covers roughly the same pe-
SPORE OUTGROWTH AND DNA SYNTHESIS
VOL. 136, 1978
12/
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2 21;
30 60 90 120 150 180 Time of outgrowth (min) FIG. 3. DNA synthesis during outgrowth and its inhibition by HPUra in the presence and absence of glucose. Heat-shocked spores of B. subtilis were suspended in semisynthetic medium, and net DNA synthesis was followed. HPUra (A) was added at 0 time with (1) or without (2) 0.5% glucose and at 90 min without glucose (3). Spores suspended in outgrowth medium lacking HPUra and glucose served as control (0). 0
riod during which, under normal conditions, the germinated spore is transformed into a vegetative cell. DISCUSSION
One objective of this work was to investigate the dependence of outgrowth of B. subtilis 168 spores on DNA synthesis. Our experimental conclusion is that outgrowth, measured as increase in protein synthesis, increase in optical density, or microscopic elongation of cells, is not dependent on net DNA synthesis. The most convincing evidence comes from the experiments using conditional mutants which, while blocked in DNA synthesis, nevertheless continued outgrowth normally. In those experiments in which outgrowth was inhibited by NAL or HPUra in synthetic or semisynthetic media, the inhibition of outgrowth, but not of DNA synthesis, could be reversed by the addition of glucose. In the presence of glucose and NAL or HPUra, outgrowth in the synthetic or semisynthetic media continued normally in the absence of DNA synthesis. These findings are consistent with the observations of Halvorson et al. (6, 21) in B. cereus, as well as those of Riva et al. (18) and Buu and Sonenshein (4) that outgrowth proceeds in the absence of DNA synthesis. The experiments carried out with the inhibitors confirm the differences in the physiology of
115
the outgrowing spore from that of the vegetative cell. It was surprising to see the differential effect of two inhibitors of DNA synthesis, NAL and HPUra, on macromolecular synthesis in two different developmental states. Protein synthesis, which is not known to be affected by these inhibitors during normal vegetative growth, was inhibited during outgrowth of spores. The effect of the inhibitors is therefore specific for the stage of outgrowth. This may serve as additional evidence in support of the notion that controls, or metabolic needs during the transformation of germinated spores into vegetative cells, are different from those which control cell proliferations. A selective inhibitory effect similar to that reported here has been demonstrated by McDonald (14) using the drug chloroquin and by Orrego (personal communication) using novobiocin. The recent isolation by Galizzi et al. (7) of mutants specifically blocked in DNA synthesis during outgrowth, but not during vegetative growth, is additional proof of the existence of different control patterns of macromolecular synthesis between outgrowth and vegetative growth. Although germinated spores can grow and elongate in the absence of DNA synthesis, the resulting cells are not necessarily identical in composition and control mechanisms to normal cells grown under uninhibited conditions. Siccardi et al. (20) have demonstrated that such cells will not develop division septa, and we have found that they differ greatly in their respiratory system and somewhat in their ability to synthesize some proteins (Ginsberg and Keynan, unpublished data). The outgrowing spore, in the presence and absence of DNA synthesis, would appear to be a good model for the investigation of the dependence of specific gene expression on DNA replication(s) during outgrowth (5). One additional conclusion determined from the experiments detailed here is that HPUra and NAL, under the described nutritional conditions, have some inhibitory effect on outgrowing spores other than their known effect on the blocking of DNA synthesis. The mechanism of this outgrowth-specific inhibition is under investigation. ACKNOWLEDGMENTS This work was supported by a grant from the Stiftung Wolkswagenwerk and by the Mathilda and Terence Kennedy Charitable Trust. We are grateful for helpful discussions with J. Mandelstam, H. 0. Halvorson, and M. Young, and we thank I. Mahler for help with the preparation of this manuscript.
LITERATURE CITED 1. Brown, N. C. 1971. Inhibition of bacterial DNA replica-
tion by 6-(p-hydroxyphenylazol)-uracil: differential effect on repair and semi-conservative synthesis in Bacilluss subtilis. J. Mol. Biol. 59:1-16.
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2. Brown, N. C., C. L Wisseman, and T. Matsushita. 1972. Inhibition of bacterial DNA replication by 6-(phydroxyphenylazo)-uracil. Nature (London) New Biol.
13.
237:72-74. 3. Burton, K. 1956. A study of the conditions and mecha-
14.
nisms of diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J. 62:315-323. 4. Buu, A., and A. L. Sonenshein. 1975. Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis. J. Bacteriol. 124:190-200. 5. Cascino, A., S. Riva, and P. Geiduschek. 1970. DNA ligation and a coupling of T4 late transcription to replication. Cold Spring Harbor Symp. Quant. Biol. 35:213-220. 6. Dawes, I. W., and H. 0. Halvorson. 1972. Membrane synthesis during outgrowth of bacterial spores, p. 449-455. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 7. Gaizzi, A., M. Albertini, L Baldi, E. Ferrari, E. Isneghi, and M. T. Zambelli. 1978. Genetic studies of Bacillus subtilis spore germination and outgrowth, p. 150-157. In G. M. Chambliss and J. C. Vary (ed.), Spores VII. American Society fot Microbiology, Washington, D.C. 8. Goss, W. A., T. M. Cook, and W. H. Deitz. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074. 9. Haber, J. E., and H. 0. Halvorson. 1972. Cell cycle dependence of sporulation in Saccharomyces cerevisiae. J. Bacteriol. 109:1027-1033. 10. Keynan, A. 1973. The transformation of bacterial endospores into vegetative cells, p. 85-123. In J. M. Ashworth and J. E. Smith (ed.), Microbial differentiation. 23rd Symposium of the Society for General Microbiology. Cambridge University Press, London. 11. Keynan, A., A. A. Berns, G. Dunn, M. Young, and J. Mandelstam. 1976. Resporulation of outgrowing Bacillus subtilis spores. J. Bacteriol. 128:8-14. 12. lammi, C. J., and J. C. Vary. 1972. DNA synthesis during outgrowth of Bacillus megaterium QM B1551 spores, p. 277-286. In H. 0. Halvorson, R. Hanson, and
15. 16.
L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. Lewin, B. 1977. Gene expression, vol. 3, p. 594-604. John Wiley & Sons, New York. McDonald, W. C. 1967. Inhibition of spore outgrowth in Bacillus subtilis by chloroquin. Can. J. Microbiol. 13:611. Mandel8tam, J., and S. A. Higgs. 1974. Induction of sporulation during synchronized chromosome replication in Bacillus subtilis. J. Bacteriol. 120:38-42. Rana, R. S., and H. 0. Halvorson. 1972. Nature of DNA synthesis and its relationship to protein synthesis during outgrowth of Bacillus cereus T. J. Bacteriol.
109:606-615. 17. Reinet, J., and H. Holtzer (ed.). 1975. Results and problems in cell differentiation, vol. 7. Springer Verlag, New York. 18. Riva, S., C. Van Sluis, G. Mastromei, C. Attolini, G. Mazza, M. Polsinelli, and A. Falaschi. 1975. A new mutant of Bacillus subtilis altered in the initiation of chromosome replication. Mol. Gen. Genet. 137:185-202. 19. Setlow, P. 1973. DNA synthesis and deoxynucleotide metabolism during bacterial spore germination. J. Bacteriol. 114:1099-1107. 20. Siccardi, A. G., A. Galizzi, G. Mazza, A. CHlvio, and A. M. Altertini. 1975. Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells. J. Bacteriol. 121:13-19. 21. Steinberg, W., and H. 0. Halvorson. 1968. Timing of enzyme synthesis during outgrowth of spores of Bacillus cereus. II. Relationship between ordered enzyme synthesis and DNA replication. J. Bacteriol.
95:479-489. 22. Sterlini, J. M., and J. Mandelstam. 1969. Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. Biochem. J.
113:29-37.
23. Winchell, E. B., and H. S. Rosenkranx. 1970. Nalidixic acid and the metabolism of Escherichia coli. J. Bacteriol. 104:1168-1175. 24. Woese, C. R., and J. R. Forro. 1960. Correlation between RNA and DNA metabolism during spore germination. J. Bacteriol. 80:811-817.
JOURNAL OF BACTERIOLOGY, Oct. 1978, p. 111-116 0021-9193/78/0136-0111$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Independence of Bacillus subtilis Spore Outgrowth from DNA Synthesis DALIA GINSBERG AND ALEX KEYNAN* Section of Developmental and Molecular Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel Received for publication 25 July 1978
The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidixic acid and 6-(phydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhibition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors. In recent years, evidence has accumulated that induction of sporulation in Bacillus sp. can occur only during a limited stage in the cell cycle and is linked to DNA replication (11, 15). Evidence for a link between the DNA replication cycle and induction of differentiation has also been demonstrated in a variety of other differentiating cells (9; for reviews, see also 17). Both bacterial sporulation and outgrowth of germinated spores differ from vegetative growth in the control of gene expression and have been suggested as model systems for the study of cell differentiation. It would be of interest to know if spore outgrowth is also dependent on DNA replication. Halvorson et al. (16, 21) obtained evidence that outgrowth in Bacillus cereus occurs in the absence of DNA synthesis. After germination, RNA and protein syntheses begin immediately and net DNA synthesis occurs only much later (10, 16, 24). On the other hand, limited incorporation of radioactive precursors into DNA has been observed immediately after germination in some Bacillus species (12, 16, 19) and has been interpreted as either repair synthesis or limited DNA replication. It was also found that inhibitors of DNA synthesis interfered with outgrowth (6, 14, 21), but because of the possibility of nonspecific effects, no final conclusion could be reached on the need of DNA synthesis for outgrowth. It appears that the question of the dependence of outgrowth on DNA synthesis has not yet been
settled. We reexamined this problem using thymidine-requiring mutants and temperature-sensitive conditional DNA mutants of Bacillus subtilis. We also studied the effect of two inhibitors, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), which have been reported to inhibit DNA synthesis specifically and are not known to have any other detectable metabolic effect (1, 2, 9, 23). Two main conclusions are the result of this study. First, DNA replication is not essential for outgrowth. Second, the experiments with inhibitors indicate that their effect on outgrowing spores is different from their effect on vegetatively dividing cells, confirming the existence of fundamental differences in the physiology of outgrowing spores and vegetative cells (10). MATERIALS AND METHODS B. subtilis 168 trpC2 and its mutants, MB65 (temperature sensitive for the initiation of chromosome replication) and BC109 (a thymine auxotroph), were used throughout this study. The inhibition of DNA synthesis under restrictive conditions was verified in each experiment in which these mutants were used. Activated spore stocks were plated (on Lalanine-containing media) under restrictive conditions and compared with parent spore stocks. Only spore suspensions that did not form colonies under restrictive conditions were used. No DNA synthesis occurred during outgrowth under restrictive conditions when measured by Burton's method (3). The outgrowing cultures of mutants grown under restrictive conditions were examined microscopically in the presence of CsCl 111
112
GINSBERG AND KEYNAN
(20) for visualization of septa. Under restrictive conditions, these cells always appeared as long filaments with no septa, as described previously for cells growing out in the absence of DNA synthesis (20). Spores were obtained after 36 to 40 h of bacterial growth at 370C in Sterlini and Mandelstam resuspension medium (22) containing 2 g of glucose per liter. The spores were harvested, washed with distilled water, and centrifuged through 50% (vol/vol) Urografin (Schering Chemicals, Ltd.) to remove any residual germinated spores (20). The pellet, consisting of uniformly brightphase spores, was washed several times. It was then suspended in distilled water at a concentration of 2 x 10'0 to 4 x 10"' spores per ml and stored at -20°C. Heat activation. The spore suspension was heat activated by incubation for 30 min at 700C. Germination. Heat-activated spores were incubated at concentrations of 2 x 108 to 4 x 108 spores per ml in germination medium containing 1.0 g of Lalanine, 0.59 g of NaCl, 0.1 g of Na2SO4, and 0.07 g of KH2PO4 per liter (pH adjusted to 7.0). After agitation at 370C for 30 min, over 95% of the spores had germinated. Germination was not altered by the presence of 20 ug of NAL (Sigma) per ml or 20 ,ug of HPUra (a gift of N. Brown) per ml. Where indicated, spores were centrifuged after germination and then placed in outgrowth media. In other experiments, activated but not germinated spores were introduced directly into outgrowth media. In these experiments there was an initial fall in optical density of the culture, corresponding to the period when germination took place in the outgrowth media. In the context of this report, the word "outgrowth" is used to define the period between the end of germination (loss of all typical spore properties) and the first cell division. It describes the stage in the life cycle of the cell in which a germinated spore is transformed into a vegetative cell (10). Outgrowth was measured in this work as an increase in optical density or by incorporation of radioactive amino acids. Outgrowth. Heat-activated spores were suspended at a concentration of 2 x 108 to 4 x 108 spores per ml in the following three media. (i) Rich medium was brain heart infusion broth (Difco) containing 5 g of yeast extract (Difco) per liter. (ii) Semisynthetic medium contained 14 g of K2HPO4, 6 g of KH2PO4, 0.25 g of MgSO4 7H20, 0.017 g of MnSO4 H20, 2 g of (NH4)2SO4, 0.15 g of sodium glutamate, 5 g of yeast extract (Difco), 1 g of L-alanine, and 25 mg of tryptophan, per liter. (iii) Synthetic medium was a modified semisynthetic medium in which L-alanine and yeast extract were omitted and supplemented with glucose (5 g/liter) and Casamino Acids (Nutritional Biochemicals Corp.) (8.5 g/liter). Since germination did not occur in this synthetic medium, spores were first germinated in a germination medium, then centrifuged and resuspended in the synthetic outgrowth medium. All cultures were incubated under conditions of vigorous aeration on a gyratory shaker at 370C. Outgrowth was followed by measurement of optical density at 600 nm with a Spectronic 21 model MV spectrophotometer (Bausch and Lomb). Protein synthesis during outgrowth was measured as follows: a uniformly "C-labeled L-amino acid mixture (Amersham; 1 ,uCi/ml) was added to the outgrowth medium, samples were removed into 5% cold trichloroacetic acid
J. BACTERIOL.
containing 0.5% casein hydrolysate, and the trichloroacetic acid precipitate was collected onto a 0.45-pm membrane filter (Millipore) and washed with the trichloroacetic acid-casein hydrolysate solution. The filter was placed in a scintillation vial and dried. It was then dissolved in Instagel (Packard Instrument Company) scintillation fluid, and the radioactivity was measured in a scintillation counter. DNA concentrations were estimated according to the method of Burton (3). The reasons for not estimating DNA by isotope incorporation were given in a previous publication (11).
RESULTS Outgrowth of mutants inhibited in DNA synthesis. Spores of two conditional DNA synthesis mutants of B. subtilis 168 were germinated in germination medium and then placed in miniimal medium for outgrowth under conditions restrictive to DNA synthesis. One strain, MB65, is a mutant that is temperature sensitive to chromosomal initiation, and the other, strain BS109, requires thymine. A minimal medium was chosen for these experiments, because (as will be shown later) in it outgrowth can be inhibited by NAL and HPUra. It was our intent to determine whether outgrowth could occur in this medium in the absence of DNA synthesis. The parent strain under these experimental conditions, or the mutants under permissive conditions, underwent a lag of a few minutes, followed by outgrowth. This could be measured by an increase in optical density or by incorporation of
radioactive amino acids. When outgrowth of these two mutants was followed under restrictive conditions (440C for mutant MB65, and in the absence of thymine for mutant BS109), their outgrowth pattem in the absence of DNA synthesis for the first 2 to 3 h was identical to that observed with the same strains under permissive conditions. Figure 1B (control) shows the data obtained with a thymidineless mutant. The same pattern (data not shown) was obtained with the temperature-sensitive strain under restrictive conditions. The microscopic appearance of the mutant cells grown under restrictive conditions was similar to that of the outgrowing parent culture or the mutant cells under permissive conditions, with the exception that no septa appeared. Septa were visible in the parent cells or in the mutants under perminsive conditions at approximately 120 min after initiation of outgrowth. In the strains inhibited in DNA synthesis, long curved cells with no septa were formed. The absence of septa formation has been described previously in outgrowing spores inhibited in DNA synthesis (20). These results were not media dependent. With these mutants, outgrowth occurred under restrictive conditions
VOL. 136, 1978
SPORE OUTGROWTH AND DNA SYNTHESIS
in the absence of DNA synthesis on each of the three media tested. Thus, it can be concluded that outgrowth of spores, measured as an increase in optical density or incorporation of radioactive amino acids, proceeds normally in these mutants in the absence of DNA synthesis. Effect of DNA synthesis inhibitors on outgrowth. The effect of NAL (20 fAg/ml) and HPUra (20 fAg/ml) on B. subtilis spore outgrowth was found to be medium dependent. In rich medium, a significant increase in optical density of the germinated spore suspension occurred in the presence or absence of either NAL or HPUra. In semisynthetic and synthetic media containing these inhibitors, no increase in optical density was detected (Fig. 2). No DNA synthesis occurred under these conditions in any of the three media. Microscopic observations of the cultures confirmed the optical density measurements. In rich medium the spores became swollen and elongated and emerged from the spore coat in both the presence and the absence of the inhibitors. In semisynthetic and synthetic media, in the presence of the inhibitors, the spores appeared swollen but not elongated and did not emerge from the spore coat. In semisynthetic or synthetic media in the absence of the inhibitors, spore outgrowth was similar to that of the controls in the rich medium. Protein synthesis in the presence or absence of inhibitor paralleled the rate of change in optical density (Fig. 1A and 2D). The outgrowing spore is considered to be different in its physiology from vegetative, multiplying cells (7, 10). It was interesting to learn whether or not this medium-dependent inhibition of spore outgrowth and protein synthesis was specific only for the spore outgrowth stage. Vegetative cells were grown in the three media
and exposed during logarithmic growth phase to NAL or HPUra. Growth of these vegetative cells, as measured by increased optical density or amino acid incorporation capacity, was not altered during the first 100 min after exposure to the inhibitors, although no DNA synthesis or cell division occurred during this time. The effect of the inhibitors was, therefore, specific for the stage of spore outgrowth only. At which stage the outgrowing spore became resistant to the inhibitor was another point of interest. Outgrowth was measured in germinated spores exposed to HPUra at various time intervals after germination had ceased (Fig. 1). The results show that after 30 min of outgrowth the cells were still sensitive to the inhibitor and became completely resistant 90 min after outgrowth. Identical results were obtained using NAL. No change occurred in the effectiveness of
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NAL or HPUra as DNA synthesis inhibitors; they halted net DNA synthesis whenever added to the culture. The time of sensitivity to the inhibitors corresponded to the period between the end of germination and the duplication of initial spore DNA during normal outgrowth. Experiments were performed to test for reversion of outgrowth inhibition in the semisynthetic medium by the addition of a variety of carbon sources. It was found that glucose could both prevent and reverse the inhibitory process, reversal occurring upon its addition at any time up to 90 min after beginning of outgrowth. Glucose
114
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GINSBERG AND KEYNAN
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could be replaced by fructose and sucrose but not by a-methyl glucoside or ribose. Addition of glucose did not affect the inhibition of DNA synthesis by HPUra or NAL, the inhibition occurring both in its presence and absence. The data describing DNA inhibition, in the presence of HPUra with or without glucose, are given in Fig. 3, curves 1 and 2. Although no DNA was
synthesized under both these conditions, the culture with glucose (curve 1) showed the same increase in optical density as the culture without inhibitors in which DNA synthesis was normal (Fig. 3). Reversal of the outgrowth inhibition, as well as renewed DNA synthesis, occurred when NAL or HPUra was removed by centrifugation and two washings with growth media. Changes in the sensitivity of outgrowing spores to NAL and HPUra in the absence of DNA synthesis. As stated above, spores
outgrowing in synthetic or semisynthetic media are inhibited by NAL and HPUra, and this inhibition decreases as a function of outgrowth time. The cell becomes completely resistant to these inhibitors at such time when, under normal outgrowth, chromosomal replication occurs. When the spores of the two mutants, MB65 and BC109, were outgrown in synthetic or semisynthetic media under conditions restrictive to DNA synthesis, outgrowth proceeded normally (as described above) but was prevented by the addition of NAL or HPUra. The time sequences of the "escape" from the inhibitory effects of HPUra or NAL in these mutants were identical to the parent strain and were the same under restrictive or permissive conditions. Thus, the development of resistance to these inhibitors during outgrowth is not DNA synthesis dependent (Fig. 1B), and covers roughly the same pe-
SPORE OUTGROWTH AND DNA SYNTHESIS
VOL. 136, 1978
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2 21;
30 60 90 120 150 180 Time of outgrowth (min) FIG. 3. DNA synthesis during outgrowth and its inhibition by HPUra in the presence and absence of glucose. Heat-shocked spores of B. subtilis were suspended in semisynthetic medium, and net DNA synthesis was followed. HPUra (A) was added at 0 time with (1) or without (2) 0.5% glucose and at 90 min without glucose (3). Spores suspended in outgrowth medium lacking HPUra and glucose served as control (0). 0
riod during which, under normal conditions, the germinated spore is transformed into a vegetative cell. DISCUSSION
One objective of this work was to investigate the dependence of outgrowth of B. subtilis 168 spores on DNA synthesis. Our experimental conclusion is that outgrowth, measured as increase in protein synthesis, increase in optical density, or microscopic elongation of cells, is not dependent on net DNA synthesis. The most convincing evidence comes from the experiments using conditional mutants which, while blocked in DNA synthesis, nevertheless continued outgrowth normally. In those experiments in which outgrowth was inhibited by NAL or HPUra in synthetic or semisynthetic media, the inhibition of outgrowth, but not of DNA synthesis, could be reversed by the addition of glucose. In the presence of glucose and NAL or HPUra, outgrowth in the synthetic or semisynthetic media continued normally in the absence of DNA synthesis. These findings are consistent with the observations of Halvorson et al. (6, 21) in B. cereus, as well as those of Riva et al. (18) and Buu and Sonenshein (4) that outgrowth proceeds in the absence of DNA synthesis. The experiments carried out with the inhibitors confirm the differences in the physiology of
115
the outgrowing spore from that of the vegetative cell. It was surprising to see the differential effect of two inhibitors of DNA synthesis, NAL and HPUra, on macromolecular synthesis in two different developmental states. Protein synthesis, which is not known to be affected by these inhibitors during normal vegetative growth, was inhibited during outgrowth of spores. The effect of the inhibitors is therefore specific for the stage of outgrowth. This may serve as additional evidence in support of the notion that controls, or metabolic needs during the transformation of germinated spores into vegetative cells, are different from those which control cell proliferations. A selective inhibitory effect similar to that reported here has been demonstrated by McDonald (14) using the drug chloroquin and by Orrego (personal communication) using novobiocin. The recent isolation by Galizzi et al. (7) of mutants specifically blocked in DNA synthesis during outgrowth, but not during vegetative growth, is additional proof of the existence of different control patterns of macromolecular synthesis between outgrowth and vegetative growth. Although germinated spores can grow and elongate in the absence of DNA synthesis, the resulting cells are not necessarily identical in composition and control mechanisms to normal cells grown under uninhibited conditions. Siccardi et al. (20) have demonstrated that such cells will not develop division septa, and we have found that they differ greatly in their respiratory system and somewhat in their ability to synthesize some proteins (Ginsberg and Keynan, unpublished data). The outgrowing spore, in the presence and absence of DNA synthesis, would appear to be a good model for the investigation of the dependence of specific gene expression on DNA replication(s) during outgrowth (5). One additional conclusion determined from the experiments detailed here is that HPUra and NAL, under the described nutritional conditions, have some inhibitory effect on outgrowing spores other than their known effect on the blocking of DNA synthesis. The mechanism of this outgrowth-specific inhibition is under investigation. ACKNOWLEDGMENTS This work was supported by a grant from the Stiftung Wolkswagenwerk and by the Mathilda and Terence Kennedy Charitable Trust. We are grateful for helpful discussions with J. Mandelstam, H. 0. Halvorson, and M. Young, and we thank I. Mahler for help with the preparation of this manuscript.
LITERATURE CITED 1. Brown, N. C. 1971. Inhibition of bacterial DNA replica-
tion by 6-(p-hydroxyphenylazol)-uracil: differential effect on repair and semi-conservative synthesis in Bacilluss subtilis. J. Mol. Biol. 59:1-16.
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2. Brown, N. C., C. L Wisseman, and T. Matsushita. 1972. Inhibition of bacterial DNA replication by 6-(phydroxyphenylazo)-uracil. Nature (London) New Biol.
13.
237:72-74. 3. Burton, K. 1956. A study of the conditions and mecha-
14.
nisms of diphenylamine reaction for the colorimetric estimation of DNA. Biochem. J. 62:315-323. 4. Buu, A., and A. L. Sonenshein. 1975. Nucleic acid synthesis and ribonucleic acid polymerase specificity in germinating and outgrowing spores of Bacillus subtilis. J. Bacteriol. 124:190-200. 5. Cascino, A., S. Riva, and P. Geiduschek. 1970. DNA ligation and a coupling of T4 late transcription to replication. Cold Spring Harbor Symp. Quant. Biol. 35:213-220. 6. Dawes, I. W., and H. 0. Halvorson. 1972. Membrane synthesis during outgrowth of bacterial spores, p. 449-455. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. 7. Gaizzi, A., M. Albertini, L Baldi, E. Ferrari, E. Isneghi, and M. T. Zambelli. 1978. Genetic studies of Bacillus subtilis spore germination and outgrowth, p. 150-157. In G. M. Chambliss and J. C. Vary (ed.), Spores VII. American Society fot Microbiology, Washington, D.C. 8. Goss, W. A., T. M. Cook, and W. H. Deitz. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074. 9. Haber, J. E., and H. 0. Halvorson. 1972. Cell cycle dependence of sporulation in Saccharomyces cerevisiae. J. Bacteriol. 109:1027-1033. 10. Keynan, A. 1973. The transformation of bacterial endospores into vegetative cells, p. 85-123. In J. M. Ashworth and J. E. Smith (ed.), Microbial differentiation. 23rd Symposium of the Society for General Microbiology. Cambridge University Press, London. 11. Keynan, A., A. A. Berns, G. Dunn, M. Young, and J. Mandelstam. 1976. Resporulation of outgrowing Bacillus subtilis spores. J. Bacteriol. 128:8-14. 12. lammi, C. J., and J. C. Vary. 1972. DNA synthesis during outgrowth of Bacillus megaterium QM B1551 spores, p. 277-286. In H. 0. Halvorson, R. Hanson, and
15. 16.
L. L. Campbell (ed.), Spores V. American Society for Microbiology, Washington, D.C. Lewin, B. 1977. Gene expression, vol. 3, p. 594-604. John Wiley & Sons, New York. McDonald, W. C. 1967. Inhibition of spore outgrowth in Bacillus subtilis by chloroquin. Can. J. Microbiol. 13:611. Mandel8tam, J., and S. A. Higgs. 1974. Induction of sporulation during synchronized chromosome replication in Bacillus subtilis. J. Bacteriol. 120:38-42. Rana, R. S., and H. 0. Halvorson. 1972. Nature of DNA synthesis and its relationship to protein synthesis during outgrowth of Bacillus cereus T. J. Bacteriol.
109:606-615. 17. Reinet, J., and H. Holtzer (ed.). 1975. Results and problems in cell differentiation, vol. 7. Springer Verlag, New York. 18. Riva, S., C. Van Sluis, G. Mastromei, C. Attolini, G. Mazza, M. Polsinelli, and A. Falaschi. 1975. A new mutant of Bacillus subtilis altered in the initiation of chromosome replication. Mol. Gen. Genet. 137:185-202. 19. Setlow, P. 1973. DNA synthesis and deoxynucleotide metabolism during bacterial spore germination. J. Bacteriol. 114:1099-1107. 20. Siccardi, A. G., A. Galizzi, G. Mazza, A. CHlvio, and A. M. Altertini. 1975. Synchronous germination and outgrowth of fractionated Bacillus subtilis spores: tool for the analysis of differentiation and division of bacterial cells. J. Bacteriol. 121:13-19. 21. Steinberg, W., and H. 0. Halvorson. 1968. Timing of enzyme synthesis during outgrowth of spores of Bacillus cereus. II. Relationship between ordered enzyme synthesis and DNA replication. J. Bacteriol.
95:479-489. 22. Sterlini, J. M., and J. Mandelstam. 1969. Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. Biochem. J.
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23. Winchell, E. B., and H. S. Rosenkranx. 1970. Nalidixic acid and the metabolism of Escherichia coli. J. Bacteriol. 104:1168-1175. 24. Woese, C. R., and J. R. Forro. 1960. Correlation between RNA and DNA metabolism during spore germination. J. Bacteriol. 80:811-817.
