Vol. 129, No. 3 Printed in U.S.A.
JouRNAL OF BACTERIOLOGY, Mar. 1977, p. 1192-1197 Copyright 0 1977 American Society for Microbiology
Chromosome Replication in Caulobacter crescentus Growing in a Nutrient Broth HIDEO IBA,* AKIO FUKUDA, AND YOSHIMI OKADA
Department ofBiophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo, Tokyo 113, Japan
Received for publication 14 October 1976
The pattern of chromosome replication in the Caulobacter crescentus cell cycle studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G,), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.
was
Caulobacter crescentus, a gram-negative, stalked bacterium, is characterized by dimorphism of cell types that occur in a defined sequence in the cell cycle (10). For the study of cell differentiation in this organism, it is of considerable interest to investigate the timing of deoxyribonucleic acid (DNA) replication in both swarmer- and stalked-cell cycles and relate it to chromosome segregation and differential expression of gene functions. Concerning timing of DNA replication, it was reported previously (3) that, in Caulobacter cells growing in minimal salts medium, active DNA synthesis occurred only during a limited period (S period) that was preceded by a presynthetic gap (G1 period) And followed by a postsynthetic gap (G2 period) before cell division. It is not known, however, whether DNA replication occurs at a specific limited period in the Caulobacter cell cycle in a fast-growth medium, and how many chromosomes exist in Caulobacter cells. In Escherichia coli, the mode of DNA replication, its relation to cell division cycle, and chromosome numbers depend on the growth rate (2, 5). We demonstrate in this paper that the Caulobacter cell cycle in a fast-growth nutrient broth consisted of three distinct periods (G,, S, and G2) in terms of DNA synthetic activity which are similar to those observed in a slow-growth minimal medium. The swarmer and stalked
cells contained similar amounts of DNA. By nuclear staining, the cells were also observed to possess possibly two chromosomes. These results are discussed with respect to cell division and cell differentiation. MATERIALS AND METHODS Bacterial strain and growth conditions. C. crescentus CB13Bla was used. Cells were grown at 30°C with reciprocal shaking. Nutrient broth (PYE medium) and glucose (0.2%)-supplemented minimal salts medium (M3 medium) were described previously (10). Cell viability was assayed by the top-agar (0.5%) overlay technique after appropriate dilution with PYE medium. Counting cell numbers. Samples were fixed with 2% formaldehyde and cooled on ice. For each determination, more than 500 cells were counted in a counting chamber under a phase-contrast microscope (Nikon model S-Ke) at x600 magnification. Cell synchrony. Synchronously growing C. crescentus cultures in PYE medium were obtained by the plate selection technique, described previously (3), with minor modifications. A 5-ml cell culture was grown at 30°C without shaking in a petri dish (8.5-cm diameter). When the cell density reached about 8 x 108 cells per ml, the plate was washed carefully with prewarmed PYE medium and incubated in PYE medium for 2 h with gentle shaking. The plate was washed again carefully and incubated in prewarmed PYE medium. The plate culture was decanted after an additional 7-min incubation. Synchronous growth was initiated by shaking the culture at 30°C.
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For determination of DNA content per cell, synchronous cultures were obtained by the late-logphase (retardation phase) method (7). Six tube cultures (5 ml each) containing swarmer cells were prepared, and synchronous growth was initiated by dilution with fresh PYE medium (360 ml). The samples for swarmer and stalked cells were obtained at 0 and 30 min, respectively, after dilution. Nuclear staining. Cell samples were poisoned with 25 mM NaN3, dried on a clean slide glass at room temperature, fixed with osmium tetroxide vapor, and stained by the procedure of Pi6chaud (9). The staining solution was freshly prepared for each sample by mixing 2 ml of eosine bluish (0.2 to 0.3 mg/ml of aqueous solution) and 0.2 ml of Giemsa solution, and was allowed to stand for ca. 8 min on the slide. Stained cells were examined under a light microscope (Nikon model S-Ke) at x 1,500 magnification. Flagella and stalks were not stained by this method. Radioactive labeling of DNA. DNA was labeled with [G-3Hldeoxyadenosine (17 Ci/mmol). Incorporation of the label into DNA was determined by the procedure of Degnen and Newton (3) with minor modifications. Determination of DNA content. DNA was extracted by the Schneider procedure (11) with minor modifications including alkaline hydrolysis of ribonucleic acid (RNA). One sample contained DNA from more than 6 x 109 cells. DNA content was determined by the modified diphenylamine method (1), with calf thymus DNA (Worthington Biochemicals Corp.) as the standard. Reagents. [G-3H]deoxyadenosine (17 Ci/mmol) was purchased from Japan Isotope Association. Eosine bluish and Giemsa solution were purchased from E. Merk Japan, Ltd.
RESULTS DNA synthesis in the stalked-cell cycle. Cells of C. crescentus CB13 absorbed onto a glass plate were pulse-labeled for 10 min with [3H]deoxyadenosine (23 ,uCi/ml or 0.34 ,ug/ml) and chased by gentle washing of the plate with fresh PYE medium containing cold deoxyadenosine (20 Ag/ml). After every 8-min incubation, the plate culture was decanted, and fresh PYE medium containing cold deoxyadenosine was added. The decanted culture containing eluted swarmer cells was used for the viability assay and for the determination of radioactivity incorporated into DNA (Fig. 1). The cells attached to the plate through stalks represent various stages of the cell cycle and elute swarmer cells at a rather constant rate (Fig. 1). Predivisional cells first release motile swarmer cells. When these attached cells are pulse-labeled with [3H]deoxyadenosine, the 3H label in the DNA from swarmer cells released at intervals reflects the 3H incorporation into DNA or, in other words, DNA replication proceeded actively at each cell stage. The radioactivity indi-
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80 120 160 TIME ( min ) FIG. 1. Elution pattern of swarmer cells with radioactively labeled DNA. Cells attached to a glass petri dish were washed several times with PYE medium and pulse-labeled by the addition of PYE medium (3 ml) containing [3H]deoxyadenosine (23 XCil ml) for 10 min. After pulse-labeling, the attached cells were carefully washed with prewarmed PYE medium containing cold deoxyadenosine (20 pg/ml) and incubated at 30°C in 5 ml of the same medium with gentle shaking. After an 8-min incubation, the plate culture was decanted, a 0.05-ml fraction was used for the viability assay, and the remaining 4-ml fraction was used for the determination of radioactivity incorporated into DNA. Immediately after the plate culture was decanted, 5 ml of fresh PYE medium containing cold deoxyadenosine (20 ug/ml) was added. Similar procedures were repeated 20 times at 8-min intervals. The upper insertion is a schematic representation of the C. crescentus cell cycle. The dashed line represents an idealized pattern of DNA synthesis. Symbols: 0, 3H label incorporated into DNA; 0, swarmer cell eluted. 40
cates directly the rate of DNA synthesis in the present experiment (Fig. 1). For the first 35 min, the eluted swarmer cells contained only a minimal amount of radioactiv-
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IBA, FUKUDA, AND OKADA
ity in their DNA. High 3H counts were found in the swarmer DNA released at a rather constant rate during the next 50 min. The stalked-cell cycle requires about 85 min before cell division, indicating that the first low level of label release represents the period of low DNA synthesis (G2 period) before cell division, and the subsequent peak of label release represents the period of high DNA synthesis (S period). After 85 min, the pattern of label release seems to be repeated. The second peak of the S period was about half of the first peak. From these results, the times for the G2 and S periods in the stalked-cell cycle are estimated to be about 35 and 50 min, respectively. DNA synthesis in the swarmer-cell cycle. The timing of chromosome replication in C. crescentus growing in a nutrient broth was also examined in swarmer cells. Swarmer cells were obtained by the plate selection technique and were incubated at 300C with reciprocal shaking for synchronous growth. At intervals, portions were taken from the synchronous culture and labeled for 8 min with [3H]deoxyadenosine. Simultaneously synchronous growth was monitored by counting viable cells. Figure 2 shows a pattern of pulse-label incorporation into DNA during the synchronous cell cycle from swarmer cells. No appreciable incorporation of 3H label into DNA was detected for the first 30 min when the swarmer cells differentiate into stalked cells in PYE medium (7, 10), suggesting that the transition of swarmer cells to stalked cells does not involve DNA synthesis. After the initial 30 min, a sharp increase in the label incorporation was observed, indicating the initiation of DNA replication, and the level of label incorporation was maintained at a rather constant rate for the next 50 min. Pulse-label incorporation was first reduced and then increased from 80 to 115 min after the initiation of synchronous growth. From these results, the swarmer-cell cycle in PYE medium may be divided into three periods in terms of DNA synthesis: presynthetic (G1), synthetic (S), and postsynthetic (G2). The lengths of these periods were about 30, 50, and 35 min, respectively. An idealized pattern of DNA synthesis is also presented in Fig. 2. As described above, only the S and G2 periods were observed in the stalked-cell cycle (Fig. 1). The lengths of periods S and G2 in the swarmer-cell cycle and those in the stalked-cell cycle are quite similar. Postsynthetic period estimated by blocking DNA synthesis. The length of the postsynthetic period in fast-growing C. crescentus CB13 was estimated by blocking DNA synthesis. Estima-
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FIG. 2. DNA synthesis in synchronously growing C. crescentus in PYE medium. The cultures were obtained by the plate selection technique described in the text. Swarmer cells were obtained from two swarmer eluting plates (4 ml of culture each) after a 7-min incubation in PYE medium. The synchronous growth from swarmer cells was monitored by counting viable cells. At the times indicated, 0.1-ml portions were withdrawn and labeled with [3H]deoxyadenosine (167 XCiIml) for 8 min. The incorporation of radioactivity was stopped by the addition of 2 ml of ice-cold trichloroacetic acid (10%). Radioactivity incorporated into DNA was determined as described in the text. The upper insertion is a schematic representation of the C. crescentus cell cycle. The dashed line represents an idealized pattern of DNA synthesis. Symbols: 0, 3H label incorporated into DNA; *, growth curve.
tion of the postsynthetic period, by the time of residual division after blocking DNA synthesis, was first used for E. coli B/r (6) and, more recently, for C. crescentus CB15 growing in minimal M3 medium (4). C. crescentus CB13 was grown fresh to the early log phase in PYE medium and mixed with hydroxyurea to a final concentration of 4 mg/ ml. Both cell number and 3H label incorporated into DNA were determined. Hydroxyurea inhibited DNA synthesis within 7 min. However, the cell number increased continuously for 37 min and leveled off after the addition of this inhibitor. The residual cell division observed
CHROMOSOME REPLICATION IN CAULOBACTER
VOL. 129, 1977
for 30 min after the inhibition of DNA synthesis indicates the existence of a 30-min period between the completion of DNA replication and cell division in this medium. The length of this period is in good agreement with that for the G2 period estimated by determining the DNA synthesis rate (Fig. 1 and 2). DNA contents in swarmer and stalked cells. Cultures enriched in swarmer and stalked cells were obtained as described in Materials and Methods. Immediately after fractions were withdrawn from the cultures for determination of cell number and cell viability, the rest of each culture was poisoned with 25 mM NaN3 and cooled on ice. Then DNA was extracted and determined as described in Materials and Methods. Table 1 presents DNA content per swarmer and stalked cells (determined in two separate experiments). The DNA content per stalked cell was nearly equal to that per swarmer cell. This finding is in good agreement with the observation that no appreciable DNA synthesis was detected during the transition period from swarmer cell to stalked cell (Fig. 2). It should be noted here that the DNA content per swarmer cell is approximately twice as much as the DNA content of one E. coli chromosome (2.8 x 109 daltons or 4.67 x 10-9 ,g [8]). Number of nuclear bodies per cell and cell length. A synchronous culture in PYE medium was obtained by the plate selection technique, and three typical cell types, swarmer cells, stalked cells, and predivisional cells, were withdrawn from the culture at 0, 30, and 85 min, respectively, after the initiation of synchronous growth from swarmer cells. The cells were stained and observed directly under a light microscope for the counting of nuclear bodies per cell. Most swarmer cells (Fig. 3a) possessed two closely oriented nuclear bodies. Most stalked cells (Fig. 3b) possessed two separate nuclear bodies. The majority of predivisional cells (Fig. 3c) possessed two nuclear bodies in each of the TABLE 1. DNA content per cell of C. crescentus DNA content Cell type
Expt 1
Expt 2
Cell no.b Viabili- Celn. tya
no." Viabili- Cell eln. tya
1.03 0.89 0.91 1.04 Swarmer 1.07 1.02 1.05 0.94 Stalked a Micrograms of DNA per colony-forming unit x lo8.
b Micrograms of DNA per cell number x 108.
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1 2 3 4 NUCLEAR BODIES PER A CELL FIG. 3. Numbers of nuclear bodies. C; crescentus cells were grown synchronously in PYE medium by the plate selection technique. Cell samples enriched in swarmer cells, stalked cells, and predivisional cells were withdrawn from the synchronous culture at 0, 30, and 85 min, respectively, after the initiation of synchronous growth from swarmer cells. Cells were then stained by the Piechaud method. Alternatively, cells were grown in M3 medium to the log phase and stained by the same method. When two nuclear bodies were separated within ca. 0.4 ,um, they were considered closely oriented. The crossed portions of the histogram represent cells that contained two nuclear bodies separated roughly by more than 0.4 um. (a) Swarmer cells, PYE medium; 213 cells scored. (b) Stalked cells, PYE medium; 272 cells scored. (c) Predivisional cells, PYE medium; 187 cells scored. (d) Cells from an asynchronous culture of C. crescerntus, minimal salts medium 1
2 3 4
(M3); 195 cells scored.
separated cell halves. The two nuclear bodies in one half were oriented closely, like those in swarmer cells, and the two in the other half were more separated, like those in stalked cells. C. crescentus cells growing in minimal M3 medium (generation time, 4 to 5 h) were also
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IBA, FUKUDA, AND OKADA
stained by the same method (Fig. 3d). The number of nuclear bodies per cell ranged from two to four in various cell types of the asynchronous culture. The number of nuclear bodies per cell and the nuclear orientation in the morphologically different cells growing in the minimal salts medium seem to be quite similar to those observed for respective cell types growing in PYE medium. More than 25 cells of each cell type growing in PYE medium were scored from micrographs of stained cells for the determination of cell length. Excluding the stalk, the lengths of swarmer cells, stalked cells, and predivisional cells was found to be 1.13 + 0.10, 1.52 ± 0.11, and 2.32 + 0.29 ,um, respectively. As reported previously (12), stalked cells are longer than swarmer cells, indicating that swarmer cells involve elongation during transition to stalked cells. This elongation might cause the separate nuclear orientation in stalked cells.
J. BACTERIOL.
dium and M3 medium contain two nuclear bodies, whereas predivisional cells contain four nuclear bodies (Fig. 3). We might exclude the possibility that two lobes of DNA replicative intermediates were observed by nuclear staining, since the majority of the swarmer cells observed were similar in nuclear body number (Fig. 3a), and since active DNA synthesis does not occur in swarmer cells (Fig. 2). Furthermore, the DNA contents in swarmer and stalked cells are quite similar and nearly twice as much as that of one E. coli chromosome (Table 1). From these results, and since it has been reported (13) that the kinetic complexity of Caulobacter DNA (determined by Cot analysis) is no greater than that of E. coli DNA, it is thus likely that swarmer and stalked cells contain two identical chromosomes. During period S, two chromosomes would be replicated into four in predivisional cells. The existence of two chromosomes in cells grown in both fast-growth PYE medium and slow-growth minimal M3 DISCUSSION medium might imply that each chromosome is The C. crescentus CB13 cell cycle in PYE essential for the differential expression of gene nutrient broth may be conveniently divided functions for Caulobacter cell differentiation. into three distinct periods with respect to the ACKNOWLEDGMENTS activity of DNA synthesis. These periods are presynthetic (GI), synthetic (S), and postsynWe wish to thank T. Horiuchi for his kind advice conthetic (G2), which last for 30, 50 and 35 min, cerning nuclear staining. This work was supported by a grant from the Scientific respectively, in the swarmer-cell cycle (Fig. 2). Research Fund of the Ministry of Education Science and The stalked-cell cycle, on the other hand, con- Culture, Japan. sists of only S and GI periods of 50 and 35 min, LITERATURE CITED respectively (Fig. 1). The distinct periods, in terms of DNA synthe- 1. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetsis, in the Caulobacter cell cycle were first desric estimation of deoxyribonucleic acid. Biochem. J. ignated by Degnen and Newton, who also found 62:315-323. G,, S, and G2 periods in the C. crescentus CB15 2. Cooper, S., and C. E. Helmstetter. 1968. Chromosome cell cycle in minimal M3 medium (3). Although replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. different strains were used in these experiS. T., and A. Newton. 1972. Chromosome repliments, it is noteworthy that the lengths of pe- 3. Degnen, cation during development in Caulobacter crescentus. riod G2 are quite similar in fast-growth PYE J. Mol. Biol. 64:671-680. medium and slow-growth M3 medium (35 and 4. Degnen, S. T., and A. Newton. 1972. Dependence of cell division on the completion of chromosome replication 30 min, respectively [3]), and that period G, in Caulobacter crescentus. J. Bacteriol. 100:852-856. exists even at the fastest growth rate in PYE 5. Gudas, L. J., and A. B. Pardee. 1974. Deoxyribonucleic medium (Fig. 2). In M3 medium, periods G, and acid synthesis during the division cycle of Escherichia S are longer (65 and 85 min, respectively (3)). coli: a comparison of strains B/r, K-12, 15, and 15Tunder conditions of slow growth. J. Bacteriol. In E. coli when the division time is less than 117:1216-1223. 1 h in different media, the time (C value) for a C. E., and 0. Pierucci. 1968. Cell division replication point to traverse the genome and 6. Helmstetter, during inhibition of deoxyribonucleic acid synthesis the time (D value) between the arrival of the in Escherichia coli. J. Bacteriol. 95:1627-1633. replication point at the terminus and cell divi- 7. Iba, H., A. Fukuda, and Y. Okada. 1975. Synchronous cell differentiation in Caulobacter crescentus. Jpn. J. sion remain relatively constant (2). The rather Microbiol. 19:441-446. constant length of period G2 in the two differ- 8. Lehninger, A. L. 1975. DNA and the structure of the ment media for Caulobacter is analogous to the genetic material, p. 859-890. In A. L. Lehninger (ed.), Biochemistry, 2nd ed. Worth Publishers, Inc., postreplication time (D value) in E. coli. HowNew York. ever, the length of period S increases in the 9. Pi6chaud, M. 1954. La coloration sans hydrolyse du slow-growth medium. noyau des bacteries. Ann. Inst. Pasteur (Paris) Nuclear staining indicates that both 86:787-793. swarmer and stalked cells growing in PYE me- 10. Poindexter, J. S. 1964. Biological properties and classi-
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fication of the Caulobacter group. Bacteriol. Rev. 28:231-295. 11. Schneider, W. C. 1957. Determination of nucleic acids by pentose analysis, p. 680-684. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York.
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12. Terrana, B., and A. Newton. 1975. Pattern of unequal cell division and development in Caulobacter cre8centus. Dev. Biol. 44:380-385. 13. Wood, N. B., A. V. Rake, and L. Shapiro. 1976. Structure of Caulobacter deoxyribonucleic acid. J. Bacteriol. 126:1305-1315.
JouRNAL OF BACTERIOLOGY, Mar. 1977, p. 1192-1197 Copyright 0 1977 American Society for Microbiology
Chromosome Replication in Caulobacter crescentus Growing in a Nutrient Broth HIDEO IBA,* AKIO FUKUDA, AND YOSHIMI OKADA
Department ofBiophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo, Tokyo 113, Japan
Received for publication 14 October 1976
The pattern of chromosome replication in the Caulobacter crescentus cell cycle studied by examining the rate of deoxyribonucleic acid (DNA) synthesis during synchronous growth in a fast-growth nutrient broth. As reported previously for the cell cycle in a slow-growth minimal medium (Degnen and Newton, 1972), the Caulobacter cell cycle (at the fastest available growth rate) in nutrient broth consisted of three distinct periods in terms of DNA synthetic activity. The swarmer-cell cycle consisted of a presynthetic period (G,), synthetic period (S), and postsynthetic period (G2) of 30, 50, and 35 min, respectively, whereas the stalked-cell cycle consisted of S and G2 periods of 50 and 35 min, respectively. Synchronously growing cells in the nutrient broth were stained to visualize nuclear bodies. Two nuclear bodies could be discerned in both swarmer and stalked cells, and four could be discerned in predivisional cells. DNA content per cell was determined chemically and found to be about the same in swarmer and stalked cells; it was equivalent to roughly twice the value expected from the kinetic complexity reported previously (Wood et al., 1976) for Caulobacter DNA.
was
Caulobacter crescentus, a gram-negative, stalked bacterium, is characterized by dimorphism of cell types that occur in a defined sequence in the cell cycle (10). For the study of cell differentiation in this organism, it is of considerable interest to investigate the timing of deoxyribonucleic acid (DNA) replication in both swarmer- and stalked-cell cycles and relate it to chromosome segregation and differential expression of gene functions. Concerning timing of DNA replication, it was reported previously (3) that, in Caulobacter cells growing in minimal salts medium, active DNA synthesis occurred only during a limited period (S period) that was preceded by a presynthetic gap (G1 period) And followed by a postsynthetic gap (G2 period) before cell division. It is not known, however, whether DNA replication occurs at a specific limited period in the Caulobacter cell cycle in a fast-growth medium, and how many chromosomes exist in Caulobacter cells. In Escherichia coli, the mode of DNA replication, its relation to cell division cycle, and chromosome numbers depend on the growth rate (2, 5). We demonstrate in this paper that the Caulobacter cell cycle in a fast-growth nutrient broth consisted of three distinct periods (G,, S, and G2) in terms of DNA synthetic activity which are similar to those observed in a slow-growth minimal medium. The swarmer and stalked
cells contained similar amounts of DNA. By nuclear staining, the cells were also observed to possess possibly two chromosomes. These results are discussed with respect to cell division and cell differentiation. MATERIALS AND METHODS Bacterial strain and growth conditions. C. crescentus CB13Bla was used. Cells were grown at 30°C with reciprocal shaking. Nutrient broth (PYE medium) and glucose (0.2%)-supplemented minimal salts medium (M3 medium) were described previously (10). Cell viability was assayed by the top-agar (0.5%) overlay technique after appropriate dilution with PYE medium. Counting cell numbers. Samples were fixed with 2% formaldehyde and cooled on ice. For each determination, more than 500 cells were counted in a counting chamber under a phase-contrast microscope (Nikon model S-Ke) at x600 magnification. Cell synchrony. Synchronously growing C. crescentus cultures in PYE medium were obtained by the plate selection technique, described previously (3), with minor modifications. A 5-ml cell culture was grown at 30°C without shaking in a petri dish (8.5-cm diameter). When the cell density reached about 8 x 108 cells per ml, the plate was washed carefully with prewarmed PYE medium and incubated in PYE medium for 2 h with gentle shaking. The plate was washed again carefully and incubated in prewarmed PYE medium. The plate culture was decanted after an additional 7-min incubation. Synchronous growth was initiated by shaking the culture at 30°C.
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For determination of DNA content per cell, synchronous cultures were obtained by the late-logphase (retardation phase) method (7). Six tube cultures (5 ml each) containing swarmer cells were prepared, and synchronous growth was initiated by dilution with fresh PYE medium (360 ml). The samples for swarmer and stalked cells were obtained at 0 and 30 min, respectively, after dilution. Nuclear staining. Cell samples were poisoned with 25 mM NaN3, dried on a clean slide glass at room temperature, fixed with osmium tetroxide vapor, and stained by the procedure of Pi6chaud (9). The staining solution was freshly prepared for each sample by mixing 2 ml of eosine bluish (0.2 to 0.3 mg/ml of aqueous solution) and 0.2 ml of Giemsa solution, and was allowed to stand for ca. 8 min on the slide. Stained cells were examined under a light microscope (Nikon model S-Ke) at x 1,500 magnification. Flagella and stalks were not stained by this method. Radioactive labeling of DNA. DNA was labeled with [G-3Hldeoxyadenosine (17 Ci/mmol). Incorporation of the label into DNA was determined by the procedure of Degnen and Newton (3) with minor modifications. Determination of DNA content. DNA was extracted by the Schneider procedure (11) with minor modifications including alkaline hydrolysis of ribonucleic acid (RNA). One sample contained DNA from more than 6 x 109 cells. DNA content was determined by the modified diphenylamine method (1), with calf thymus DNA (Worthington Biochemicals Corp.) as the standard. Reagents. [G-3H]deoxyadenosine (17 Ci/mmol) was purchased from Japan Isotope Association. Eosine bluish and Giemsa solution were purchased from E. Merk Japan, Ltd.
RESULTS DNA synthesis in the stalked-cell cycle. Cells of C. crescentus CB13 absorbed onto a glass plate were pulse-labeled for 10 min with [3H]deoxyadenosine (23 ,uCi/ml or 0.34 ,ug/ml) and chased by gentle washing of the plate with fresh PYE medium containing cold deoxyadenosine (20 Ag/ml). After every 8-min incubation, the plate culture was decanted, and fresh PYE medium containing cold deoxyadenosine was added. The decanted culture containing eluted swarmer cells was used for the viability assay and for the determination of radioactivity incorporated into DNA (Fig. 1). The cells attached to the plate through stalks represent various stages of the cell cycle and elute swarmer cells at a rather constant rate (Fig. 1). Predivisional cells first release motile swarmer cells. When these attached cells are pulse-labeled with [3H]deoxyadenosine, the 3H label in the DNA from swarmer cells released at intervals reflects the 3H incorporation into DNA or, in other words, DNA replication proceeded actively at each cell stage. The radioactivity indi-
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80 120 160 TIME ( min ) FIG. 1. Elution pattern of swarmer cells with radioactively labeled DNA. Cells attached to a glass petri dish were washed several times with PYE medium and pulse-labeled by the addition of PYE medium (3 ml) containing [3H]deoxyadenosine (23 XCil ml) for 10 min. After pulse-labeling, the attached cells were carefully washed with prewarmed PYE medium containing cold deoxyadenosine (20 pg/ml) and incubated at 30°C in 5 ml of the same medium with gentle shaking. After an 8-min incubation, the plate culture was decanted, a 0.05-ml fraction was used for the viability assay, and the remaining 4-ml fraction was used for the determination of radioactivity incorporated into DNA. Immediately after the plate culture was decanted, 5 ml of fresh PYE medium containing cold deoxyadenosine (20 ug/ml) was added. Similar procedures were repeated 20 times at 8-min intervals. The upper insertion is a schematic representation of the C. crescentus cell cycle. The dashed line represents an idealized pattern of DNA synthesis. Symbols: 0, 3H label incorporated into DNA; 0, swarmer cell eluted. 40
cates directly the rate of DNA synthesis in the present experiment (Fig. 1). For the first 35 min, the eluted swarmer cells contained only a minimal amount of radioactiv-
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ity in their DNA. High 3H counts were found in the swarmer DNA released at a rather constant rate during the next 50 min. The stalked-cell cycle requires about 85 min before cell division, indicating that the first low level of label release represents the period of low DNA synthesis (G2 period) before cell division, and the subsequent peak of label release represents the period of high DNA synthesis (S period). After 85 min, the pattern of label release seems to be repeated. The second peak of the S period was about half of the first peak. From these results, the times for the G2 and S periods in the stalked-cell cycle are estimated to be about 35 and 50 min, respectively. DNA synthesis in the swarmer-cell cycle. The timing of chromosome replication in C. crescentus growing in a nutrient broth was also examined in swarmer cells. Swarmer cells were obtained by the plate selection technique and were incubated at 300C with reciprocal shaking for synchronous growth. At intervals, portions were taken from the synchronous culture and labeled for 8 min with [3H]deoxyadenosine. Simultaneously synchronous growth was monitored by counting viable cells. Figure 2 shows a pattern of pulse-label incorporation into DNA during the synchronous cell cycle from swarmer cells. No appreciable incorporation of 3H label into DNA was detected for the first 30 min when the swarmer cells differentiate into stalked cells in PYE medium (7, 10), suggesting that the transition of swarmer cells to stalked cells does not involve DNA synthesis. After the initial 30 min, a sharp increase in the label incorporation was observed, indicating the initiation of DNA replication, and the level of label incorporation was maintained at a rather constant rate for the next 50 min. Pulse-label incorporation was first reduced and then increased from 80 to 115 min after the initiation of synchronous growth. From these results, the swarmer-cell cycle in PYE medium may be divided into three periods in terms of DNA synthesis: presynthetic (G1), synthetic (S), and postsynthetic (G2). The lengths of these periods were about 30, 50, and 35 min, respectively. An idealized pattern of DNA synthesis is also presented in Fig. 2. As described above, only the S and G2 periods were observed in the stalked-cell cycle (Fig. 1). The lengths of periods S and G2 in the swarmer-cell cycle and those in the stalked-cell cycle are quite similar. Postsynthetic period estimated by blocking DNA synthesis. The length of the postsynthetic period in fast-growing C. crescentus CB13 was estimated by blocking DNA synthesis. Estima-
J. BACTERIOL.
0
4x
1
E
3D U 0
2>ECLo~~~~~~~~~~~~~~~~~c I-
A I_5 tn
I-
z
0
z 0n
0
30
60
90
120
150
TIME ( min
FIG. 2. DNA synthesis in synchronously growing C. crescentus in PYE medium. The cultures were obtained by the plate selection technique described in the text. Swarmer cells were obtained from two swarmer eluting plates (4 ml of culture each) after a 7-min incubation in PYE medium. The synchronous growth from swarmer cells was monitored by counting viable cells. At the times indicated, 0.1-ml portions were withdrawn and labeled with [3H]deoxyadenosine (167 XCiIml) for 8 min. The incorporation of radioactivity was stopped by the addition of 2 ml of ice-cold trichloroacetic acid (10%). Radioactivity incorporated into DNA was determined as described in the text. The upper insertion is a schematic representation of the C. crescentus cell cycle. The dashed line represents an idealized pattern of DNA synthesis. Symbols: 0, 3H label incorporated into DNA; *, growth curve.
tion of the postsynthetic period, by the time of residual division after blocking DNA synthesis, was first used for E. coli B/r (6) and, more recently, for C. crescentus CB15 growing in minimal M3 medium (4). C. crescentus CB13 was grown fresh to the early log phase in PYE medium and mixed with hydroxyurea to a final concentration of 4 mg/ ml. Both cell number and 3H label incorporated into DNA were determined. Hydroxyurea inhibited DNA synthesis within 7 min. However, the cell number increased continuously for 37 min and leveled off after the addition of this inhibitor. The residual cell division observed
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for 30 min after the inhibition of DNA synthesis indicates the existence of a 30-min period between the completion of DNA replication and cell division in this medium. The length of this period is in good agreement with that for the G2 period estimated by determining the DNA synthesis rate (Fig. 1 and 2). DNA contents in swarmer and stalked cells. Cultures enriched in swarmer and stalked cells were obtained as described in Materials and Methods. Immediately after fractions were withdrawn from the cultures for determination of cell number and cell viability, the rest of each culture was poisoned with 25 mM NaN3 and cooled on ice. Then DNA was extracted and determined as described in Materials and Methods. Table 1 presents DNA content per swarmer and stalked cells (determined in two separate experiments). The DNA content per stalked cell was nearly equal to that per swarmer cell. This finding is in good agreement with the observation that no appreciable DNA synthesis was detected during the transition period from swarmer cell to stalked cell (Fig. 2). It should be noted here that the DNA content per swarmer cell is approximately twice as much as the DNA content of one E. coli chromosome (2.8 x 109 daltons or 4.67 x 10-9 ,g [8]). Number of nuclear bodies per cell and cell length. A synchronous culture in PYE medium was obtained by the plate selection technique, and three typical cell types, swarmer cells, stalked cells, and predivisional cells, were withdrawn from the culture at 0, 30, and 85 min, respectively, after the initiation of synchronous growth from swarmer cells. The cells were stained and observed directly under a light microscope for the counting of nuclear bodies per cell. Most swarmer cells (Fig. 3a) possessed two closely oriented nuclear bodies. Most stalked cells (Fig. 3b) possessed two separate nuclear bodies. The majority of predivisional cells (Fig. 3c) possessed two nuclear bodies in each of the TABLE 1. DNA content per cell of C. crescentus DNA content Cell type
Expt 1
Expt 2
Cell no.b Viabili- Celn. tya
no." Viabili- Cell eln. tya
1.03 0.89 0.91 1.04 Swarmer 1.07 1.02 1.05 0.94 Stalked a Micrograms of DNA per colony-forming unit x lo8.
b Micrograms of DNA per cell number x 108.
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V_ C)
~~d)
z
50LU
1 2 3 4 NUCLEAR BODIES PER A CELL FIG. 3. Numbers of nuclear bodies. C; crescentus cells were grown synchronously in PYE medium by the plate selection technique. Cell samples enriched in swarmer cells, stalked cells, and predivisional cells were withdrawn from the synchronous culture at 0, 30, and 85 min, respectively, after the initiation of synchronous growth from swarmer cells. Cells were then stained by the Piechaud method. Alternatively, cells were grown in M3 medium to the log phase and stained by the same method. When two nuclear bodies were separated within ca. 0.4 ,um, they were considered closely oriented. The crossed portions of the histogram represent cells that contained two nuclear bodies separated roughly by more than 0.4 um. (a) Swarmer cells, PYE medium; 213 cells scored. (b) Stalked cells, PYE medium; 272 cells scored. (c) Predivisional cells, PYE medium; 187 cells scored. (d) Cells from an asynchronous culture of C. crescerntus, minimal salts medium 1
2 3 4
(M3); 195 cells scored.
separated cell halves. The two nuclear bodies in one half were oriented closely, like those in swarmer cells, and the two in the other half were more separated, like those in stalked cells. C. crescentus cells growing in minimal M3 medium (generation time, 4 to 5 h) were also
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stained by the same method (Fig. 3d). The number of nuclear bodies per cell ranged from two to four in various cell types of the asynchronous culture. The number of nuclear bodies per cell and the nuclear orientation in the morphologically different cells growing in the minimal salts medium seem to be quite similar to those observed for respective cell types growing in PYE medium. More than 25 cells of each cell type growing in PYE medium were scored from micrographs of stained cells for the determination of cell length. Excluding the stalk, the lengths of swarmer cells, stalked cells, and predivisional cells was found to be 1.13 + 0.10, 1.52 ± 0.11, and 2.32 + 0.29 ,um, respectively. As reported previously (12), stalked cells are longer than swarmer cells, indicating that swarmer cells involve elongation during transition to stalked cells. This elongation might cause the separate nuclear orientation in stalked cells.
J. BACTERIOL.
dium and M3 medium contain two nuclear bodies, whereas predivisional cells contain four nuclear bodies (Fig. 3). We might exclude the possibility that two lobes of DNA replicative intermediates were observed by nuclear staining, since the majority of the swarmer cells observed were similar in nuclear body number (Fig. 3a), and since active DNA synthesis does not occur in swarmer cells (Fig. 2). Furthermore, the DNA contents in swarmer and stalked cells are quite similar and nearly twice as much as that of one E. coli chromosome (Table 1). From these results, and since it has been reported (13) that the kinetic complexity of Caulobacter DNA (determined by Cot analysis) is no greater than that of E. coli DNA, it is thus likely that swarmer and stalked cells contain two identical chromosomes. During period S, two chromosomes would be replicated into four in predivisional cells. The existence of two chromosomes in cells grown in both fast-growth PYE medium and slow-growth minimal M3 DISCUSSION medium might imply that each chromosome is The C. crescentus CB13 cell cycle in PYE essential for the differential expression of gene nutrient broth may be conveniently divided functions for Caulobacter cell differentiation. into three distinct periods with respect to the ACKNOWLEDGMENTS activity of DNA synthesis. These periods are presynthetic (GI), synthetic (S), and postsynWe wish to thank T. Horiuchi for his kind advice conthetic (G2), which last for 30, 50 and 35 min, cerning nuclear staining. This work was supported by a grant from the Scientific respectively, in the swarmer-cell cycle (Fig. 2). Research Fund of the Ministry of Education Science and The stalked-cell cycle, on the other hand, con- Culture, Japan. sists of only S and GI periods of 50 and 35 min, LITERATURE CITED respectively (Fig. 1). The distinct periods, in terms of DNA synthe- 1. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetsis, in the Caulobacter cell cycle were first desric estimation of deoxyribonucleic acid. Biochem. J. ignated by Degnen and Newton, who also found 62:315-323. G,, S, and G2 periods in the C. crescentus CB15 2. Cooper, S., and C. E. Helmstetter. 1968. Chromosome cell cycle in minimal M3 medium (3). Although replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. different strains were used in these experiS. T., and A. Newton. 1972. Chromosome repliments, it is noteworthy that the lengths of pe- 3. Degnen, cation during development in Caulobacter crescentus. riod G2 are quite similar in fast-growth PYE J. Mol. Biol. 64:671-680. medium and slow-growth M3 medium (35 and 4. Degnen, S. T., and A. Newton. 1972. Dependence of cell division on the completion of chromosome replication 30 min, respectively [3]), and that period G, in Caulobacter crescentus. J. Bacteriol. 100:852-856. exists even at the fastest growth rate in PYE 5. Gudas, L. J., and A. B. Pardee. 1974. Deoxyribonucleic medium (Fig. 2). In M3 medium, periods G, and acid synthesis during the division cycle of Escherichia S are longer (65 and 85 min, respectively (3)). coli: a comparison of strains B/r, K-12, 15, and 15Tunder conditions of slow growth. J. Bacteriol. In E. coli when the division time is less than 117:1216-1223. 1 h in different media, the time (C value) for a C. E., and 0. Pierucci. 1968. Cell division replication point to traverse the genome and 6. Helmstetter, during inhibition of deoxyribonucleic acid synthesis the time (D value) between the arrival of the in Escherichia coli. J. Bacteriol. 95:1627-1633. replication point at the terminus and cell divi- 7. Iba, H., A. Fukuda, and Y. Okada. 1975. Synchronous cell differentiation in Caulobacter crescentus. Jpn. J. sion remain relatively constant (2). The rather Microbiol. 19:441-446. constant length of period G2 in the two differ- 8. Lehninger, A. L. 1975. DNA and the structure of the ment media for Caulobacter is analogous to the genetic material, p. 859-890. In A. L. Lehninger (ed.), Biochemistry, 2nd ed. Worth Publishers, Inc., postreplication time (D value) in E. coli. HowNew York. ever, the length of period S increases in the 9. Pi6chaud, M. 1954. La coloration sans hydrolyse du slow-growth medium. noyau des bacteries. Ann. Inst. Pasteur (Paris) Nuclear staining indicates that both 86:787-793. swarmer and stalked cells growing in PYE me- 10. Poindexter, J. S. 1964. Biological properties and classi-
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CHROMOSOME REPLICATION IN CAULOBACTER
fication of the Caulobacter group. Bacteriol. Rev. 28:231-295. 11. Schneider, W. C. 1957. Determination of nucleic acids by pentose analysis, p. 680-684. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 3. Academic Press Inc., New York.
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12. Terrana, B., and A. Newton. 1975. Pattern of unequal cell division and development in Caulobacter cre8centus. Dev. Biol. 44:380-385. 13. Wood, N. B., A. V. Rake, and L. Shapiro. 1976. Structure of Caulobacter deoxyribonucleic acid. J. Bacteriol. 126:1305-1315.
