JOURNAL OF BACTERIOLOGY, JUlY 1977, p. 369-371 Copyright © 1977 American Society for Microbiology
Vol. 131, No. 1 Printed in U.S.A.
Gamma-Ray Sensitivity During Synchronous Cell Differentiation in Caulobacter crescentus HIDEO IBA,* AKIO FUKUDA, AND YOSHIMI OKADA Department of Biophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo,
Tokyo 113, Japan Received for publication 24 March 1977
Gamma-ray sensitivity of Caulobacter crescentus during its cell cycle was examined. Survival curves of the swarmer and stalked cells were similar and exponential in shape, whereas that of the predivisional cell was sigmoidal, with an extrapolation number of 1.8. A gram-negative stalked bacterium, Caulobacter crescentus, is characterized by dimorphism of cell types that occur in a defined sequence in the cell cycle (4, 5). The cell cycle of this bacterium is divided into three distinct periods with respect to the activity of deoxyribonucleic acid synthesis in either slow-growth minimal medium (1) or in fast-growth nutrient broth (3). In nutrient broth (PYE medium), the swarmercell cycle consists of G1, S, and G2 periods of about 30, 50, and 35 min, respectively, and the stalked-cell cycle consists of S and G2 periods of about 50 and 35 min, respectively. It was reported previously (3) that swarmer and stalked cells grown either in minimal M3 medium or PYE medium have two identical chromosomes, and predivisional cells have four chromosomes. The existence of multichromosome in this bacterium, irrespective of growth conditions, might imply that each chromosome is essential for Caulobacter cell growth and differentiation. In this report, we have studied the target number of lethal damage by gammaray irradiation in typical Caulobacter cell types in an attempt to get further information about the functions of "two chromosomes." Cell cycle dependency of gamma-ray sensitivity was also examined. Swarmer cells of C. crescentus CB13Bla were obtained by the plate selection technique (1) with minor modifications (3), and grown synchronously in a nutrient broth (PYE medium; 4) at 300C with reciprocal shaking. At the time indicated, 0. 1-ml portions were withdrawn from the synchronous culture (1 x 107 to 2 x 107 cells/ ml) and diluted 50-fold with ice-cold 10 mM phosphate buffer (pH 6.8). The diluted culture was fractionated into 0.5-ml glass vials (1.2-cm diameter). One sample vial was used to assay viability (colony-forming units) by plating cells on PYE plates, with the usual top-agar overlay
technique after appropriate dilution with the same buffer. The other sample vials were simultaneously irradiated at 00C with 6OCo gamma-ray at 2 krad/min for various lengths of time to give a desired dose to each sample. Immediately after irradiation, irradiation samples were similarly plated to assay viability. Three plates were used to assay the viability of each sample and placed in a 300C incubator immediately after plating. Changes in irradiation sensitivity of synchronously growing C. crescentus were examined by exposing cells to a constant gamma-ray dose of 6.0 krad (Fig. 1). There was no appreciable change of sensitivity during the first 30 min or during the G, period, indicating that the swarmer and stalked cells are similar in sensitivity to gamma-ray irradiation. During the periods from early S to G2, surviving fractions increased progressively. Upon cell division, surviving fractions then sharply decreased, nearly to those observed during the G, period. Progressive increase of resistance to ionizing radiation is also observed during the cell division cycle in Escherichia coli B/r (2). The pattern of ionizing radiation sensitivity during the Caulobacter cell cycle also resembles that of Chinese hamster cells which have a short G1 period (6), except that C. crescentus remains rather resistant during the G2 period. Figure 2 shows gamma-ray survival curves for three typical cell types of C. crescentus. Swarmer, stalked, and predivisional cells were obtained at 0, 30, and 85 min from a synchronously growing culture similar to the one shown in Fig. 1. The gamma-ray survival curve for swarmer cells was almost exponential for three decades of inactivation, and the mean lethal dose (Do) was 3.0 krad. The survival curve for stalked cells was nearly identical to that for swarmer cells, and the mean lethal dose was 369
370
J. BACTZIOL.
NOTES
3.2 krad. The gamma-ray survival curve for predivisional cells was sigmoidal, with an extrapolation number of 1.8. The limiting slope was less than those of the other cell types, resulting in an increased mean lethal dose of 4.1 krad. This increase in the mean lethal dose might be attributed to differences in target size and/or physiology, such as enhanced repair activity, during the S and G2 periods. It is possible to interpret the doubling in the extrapolation number accompanying chromosome replication in terms of the target theory (one-hit, one-target model or one-hit, two-target model). The swarmer and stalked cells would then possess one target, whereas the
G_
,
G
,
0S
C.) x12.0
5
EJ
-40Z
12 16 20 DOSE ( krad ) FIG. 2. Gamma-ray survival curves for three typical cell types of C. crescentus. Swarmer, stalked, and predivisional cells were obtained at 0, 30, and 85 min durBg the synchronous growth from swarmer cells in PYE medium and irradiated with gamma-ray at varying dosages. Symbols: *, survival curve for swarmer cells; 0, survival curve for stalked cells; A, survival curve for predivisional cells.
u
30< 20 z 0
60
10 cr In 0
120 180 TIME ( mn) FIG. 1. Gamma-ray sensitivity to 6 krad for the Caulobacter cells during synchronous cell differentiation. Surviving fractions of the culture are presented in a percentage to the cell viability before irradiation. The upper insertions are a schematic representation of the Caulobacter cell cycle and designated cell periods. Symbols: 0, cell viability in synchronous growth; *, surviving fractions after irradiation. CFU, colony-forming units. 0
predivisional cell, which has completed the chromosome replication, possesses two targets. Thus, the target number does not appear to correspond to the chromosome number in this organism, and the two chromosomes in the swarmer cell and the stalked cell seem to act as a single target. This finding supports the possibility that differential expression of each of the two chromosomes is essential for Caulobacter cell growth and differentiation or, alternatively, the target theory may not be directly applicable to the lethal damage of this organism by gamma-ray irradiation. We thank Kenshi Suzuki for his helpful discussion. This work was supported in part by a grant from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan.
VOL. 131, 1977
NOTES LITERATURE CITED
1. Degnen, S. T., and A. Newton. 1972. Chromosome replication during development in Caulobacter crescentus. J. Mol. Biol. 64:671-680. 2. Heimatetter, C. E., and R. B. Uretz. 1963. X-ray and ultraviolet sensitivity of synchronously dividing Escherichia coli. Biophys. J. 3:35-47. 3. Iba, H., A. Fukuda, and Y. Okada. 1977. Chromosome replication in Caulobacter crescentus growing in a
371
nutrient broth. J. Bacteriol. 129:1192-1197. 4. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev.
28:231-295. 5. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407. 6. Sinclair, W. K., and R. A. Morton. 1966. X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat. Res. 29:450-474.
Vol. 131, No. 1 Printed in U.S.A.
Gamma-Ray Sensitivity During Synchronous Cell Differentiation in Caulobacter crescentus HIDEO IBA,* AKIO FUKUDA, AND YOSHIMI OKADA Department of Biophysics and Biochemistry, Faculty of Sciences, University of Tokyo, Hongo,
Tokyo 113, Japan Received for publication 24 March 1977
Gamma-ray sensitivity of Caulobacter crescentus during its cell cycle was examined. Survival curves of the swarmer and stalked cells were similar and exponential in shape, whereas that of the predivisional cell was sigmoidal, with an extrapolation number of 1.8. A gram-negative stalked bacterium, Caulobacter crescentus, is characterized by dimorphism of cell types that occur in a defined sequence in the cell cycle (4, 5). The cell cycle of this bacterium is divided into three distinct periods with respect to the activity of deoxyribonucleic acid synthesis in either slow-growth minimal medium (1) or in fast-growth nutrient broth (3). In nutrient broth (PYE medium), the swarmercell cycle consists of G1, S, and G2 periods of about 30, 50, and 35 min, respectively, and the stalked-cell cycle consists of S and G2 periods of about 50 and 35 min, respectively. It was reported previously (3) that swarmer and stalked cells grown either in minimal M3 medium or PYE medium have two identical chromosomes, and predivisional cells have four chromosomes. The existence of multichromosome in this bacterium, irrespective of growth conditions, might imply that each chromosome is essential for Caulobacter cell growth and differentiation. In this report, we have studied the target number of lethal damage by gammaray irradiation in typical Caulobacter cell types in an attempt to get further information about the functions of "two chromosomes." Cell cycle dependency of gamma-ray sensitivity was also examined. Swarmer cells of C. crescentus CB13Bla were obtained by the plate selection technique (1) with minor modifications (3), and grown synchronously in a nutrient broth (PYE medium; 4) at 300C with reciprocal shaking. At the time indicated, 0. 1-ml portions were withdrawn from the synchronous culture (1 x 107 to 2 x 107 cells/ ml) and diluted 50-fold with ice-cold 10 mM phosphate buffer (pH 6.8). The diluted culture was fractionated into 0.5-ml glass vials (1.2-cm diameter). One sample vial was used to assay viability (colony-forming units) by plating cells on PYE plates, with the usual top-agar overlay
technique after appropriate dilution with the same buffer. The other sample vials were simultaneously irradiated at 00C with 6OCo gamma-ray at 2 krad/min for various lengths of time to give a desired dose to each sample. Immediately after irradiation, irradiation samples were similarly plated to assay viability. Three plates were used to assay the viability of each sample and placed in a 300C incubator immediately after plating. Changes in irradiation sensitivity of synchronously growing C. crescentus were examined by exposing cells to a constant gamma-ray dose of 6.0 krad (Fig. 1). There was no appreciable change of sensitivity during the first 30 min or during the G, period, indicating that the swarmer and stalked cells are similar in sensitivity to gamma-ray irradiation. During the periods from early S to G2, surviving fractions increased progressively. Upon cell division, surviving fractions then sharply decreased, nearly to those observed during the G, period. Progressive increase of resistance to ionizing radiation is also observed during the cell division cycle in Escherichia coli B/r (2). The pattern of ionizing radiation sensitivity during the Caulobacter cell cycle also resembles that of Chinese hamster cells which have a short G1 period (6), except that C. crescentus remains rather resistant during the G2 period. Figure 2 shows gamma-ray survival curves for three typical cell types of C. crescentus. Swarmer, stalked, and predivisional cells were obtained at 0, 30, and 85 min from a synchronously growing culture similar to the one shown in Fig. 1. The gamma-ray survival curve for swarmer cells was almost exponential for three decades of inactivation, and the mean lethal dose (Do) was 3.0 krad. The survival curve for stalked cells was nearly identical to that for swarmer cells, and the mean lethal dose was 369
370
J. BACTZIOL.
NOTES
3.2 krad. The gamma-ray survival curve for predivisional cells was sigmoidal, with an extrapolation number of 1.8. The limiting slope was less than those of the other cell types, resulting in an increased mean lethal dose of 4.1 krad. This increase in the mean lethal dose might be attributed to differences in target size and/or physiology, such as enhanced repair activity, during the S and G2 periods. It is possible to interpret the doubling in the extrapolation number accompanying chromosome replication in terms of the target theory (one-hit, one-target model or one-hit, two-target model). The swarmer and stalked cells would then possess one target, whereas the
G_
,
G
,
0S
C.) x12.0
5
EJ
-40Z
12 16 20 DOSE ( krad ) FIG. 2. Gamma-ray survival curves for three typical cell types of C. crescentus. Swarmer, stalked, and predivisional cells were obtained at 0, 30, and 85 min durBg the synchronous growth from swarmer cells in PYE medium and irradiated with gamma-ray at varying dosages. Symbols: *, survival curve for swarmer cells; 0, survival curve for stalked cells; A, survival curve for predivisional cells.
u
30< 20 z 0
60
10 cr In 0
120 180 TIME ( mn) FIG. 1. Gamma-ray sensitivity to 6 krad for the Caulobacter cells during synchronous cell differentiation. Surviving fractions of the culture are presented in a percentage to the cell viability before irradiation. The upper insertions are a schematic representation of the Caulobacter cell cycle and designated cell periods. Symbols: 0, cell viability in synchronous growth; *, surviving fractions after irradiation. CFU, colony-forming units. 0
predivisional cell, which has completed the chromosome replication, possesses two targets. Thus, the target number does not appear to correspond to the chromosome number in this organism, and the two chromosomes in the swarmer cell and the stalked cell seem to act as a single target. This finding supports the possibility that differential expression of each of the two chromosomes is essential for Caulobacter cell growth and differentiation or, alternatively, the target theory may not be directly applicable to the lethal damage of this organism by gamma-ray irradiation. We thank Kenshi Suzuki for his helpful discussion. This work was supported in part by a grant from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan.
VOL. 131, 1977
NOTES LITERATURE CITED
1. Degnen, S. T., and A. Newton. 1972. Chromosome replication during development in Caulobacter crescentus. J. Mol. Biol. 64:671-680. 2. Heimatetter, C. E., and R. B. Uretz. 1963. X-ray and ultraviolet sensitivity of synchronously dividing Escherichia coli. Biophys. J. 3:35-47. 3. Iba, H., A. Fukuda, and Y. Okada. 1977. Chromosome replication in Caulobacter crescentus growing in a
371
nutrient broth. J. Bacteriol. 129:1192-1197. 4. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev.
28:231-295. 5. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407. 6. Sinclair, W. K., and R. A. Morton. 1966. X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat. Res. 29:450-474.
