JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 647-655 0021-9193/78/0135-0647$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 135, No. 2
Printed in U.S.A.
Rate of Major Protein Synthesis During the Cell Cycle of 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 30 May 1978
The rate of major protein synthesis was examined during the synchronous differentiation of Caulobacter crescentus. Total cell proteins were pulse-labeled with [3S]methionine at different times in the swarmer cell cycle and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rates of synthesis of total cell proteins and of about one-half of the individual major proteins examined increased through GI and S periods but remained nearly constant during G2 period. The rates of synthesis of the other half of the individual major proteins either increased continuously throughout the swarmer cell cycle or doubled during S period. One stage-specific protein was also detected in late S period. For most of the major proteins examined, the rate of synthesis in the swarmer cell was less than that in the stalked cell. It seemed that, before the onset of G2 period, the Caulobacter cell was already able to synthesize each major protein at the additive rate of the two progeny cells. Compared to the stability of cellular proteins, the functional degradation rate of mRNA coding for individual major proteins was rapid, with half-lives of 0.4 to 5.8 min. It thus seems that the rate of major protein synthesis mainly reflects the transcriptional control of gene expression.
A gram-negative stalked bacterium, Caulobacter crescentus, exhibits dimorphism of cell types in the cell cycle (14, 15). A predivisional cell divides asymmetrically and produces a motile swarmer cell and a nonmotile stalked cell. The swarner cell differentiates into a stalked cell before the next round of cell division, whereas the stalked cell directly differentiates into a predivisional cell. The cell cycle of C. crescentus can be divided, in terms of DNA synthesis activity, into three distinct periods, namely G1, S, and G2 periods in both fast-growth and slow-growth media (4, 6). For the normal process of cell differentiation in this bacterium, DNA transcription is required throughout the cell cycle (12). Some stage-specific proteins have been detected among soluble proteins of C. crescentus CB15 (3). We have studied the cell cycle dependency of the absolute rate of synthesis of major cellular proteins in an attempt to monitor the changes of gene expression quantitatively in the Caulobacter cell. Several patterns of the cell cycle dependency were observed and quantitative differences in the gene expression were found between the two progeny cells. Furthermore, the half-life of mRNA coding for each major protein was estimated by the analysis of the residual rate of protein synthesis after the inhibition of
RNA synthesis. The mRNA was found to be unstable compared to the stability of cellular proteins. MATERIALS AND METHODS Bacterial strain and growth conditions. C. crescentus CB13 tdr-806B35R4 was used throughout this study and grown in glucose (0.2%) -supplemented minimal salts medium (HMG medium [14]) at 30°C with shaking. This strain is a revertant from an obligate thymidine auxotroph, C. crescentus CB13 tdr-806B35, and, unlike the CB13 wild-type strain, grows well in HMG medium. Details of these strains will be described elsewhere (A. Fukuda and H. Iba, manuscript in preparation). Cell synchronization. Newborn swarmer cells were obtained by plate selection technique as described previously (4, 6) except that glass plates were coated with poly-L-lysine (0.002%). Preparation of total cell proteins for gel electrophoresis. In a typical experiment, CB13 tdr806B35R4 culture (0.2 ml) was pulse-labeled for 12 min with 109 ACi of [3S]methionine per ml and chased for 3 min by the addition of 5 ml of HMG medium containing 200 ug of cold methionine per ml. The labeled culture was chilled in ice, and a sample (0.5 ml) was taken for the counting of incorporated radio-
activity. To the rest of the labeled culture was added 0.2 ml of C. crescentus CB13 tdr-806B35R4 grown in HMG medium (0.5 of the optical density at 660 nm) as a carrier. After centrifugation for 10 min at 15,000
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rpm, the cell pellet was suspended in 3 ml of 10 mM phosphate-5 mM MgCl2 (pH 6.8). After another centrifugation for 10 min at 15,000 rpm, the cell pellet was suspended in 0.2 ml of the gel sample buffer consisting of 0.0625 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue, and boiled for 2 min. For the examination of the turnover of cellular proteins, C. crescentus CB13 tdr-806B35R4 culture (0.4 ml) was labeled with 218 yiCi of [3S]methionine per ml for 12 min and chased by the addition of 5 ml of HMG medium containing 2 jug of cold methionine per ml. Two volumes of the culture were withdrawn at the time indicated. One volume (0.2 ml) was used for the counting of incorporated radioactivity, and the other volume (1 ml) was used to prepare a sample for gel electrophoresis as follows. The labeled culture (1 ml) was mixed with carrier cells as described above and with 3 ml of 10% trichloroacetic acid. The mixture was then chilled in ice for 30 min and centrifuged for 10 min at 15,000 rpm. The precipitates were washed twice with acetone, solubilized in 0.2 ml of the gel sample buffer as described above, and boiled for 2 min. Radioactivity measurement. The samples were heated in 10% trichloroacetic acid at 90°C for 20 min, chilled in ice for 30 min, and filtered onto glass fiber filter disks (Whatman GF/B, 2.4 cm in diameter). Alternatively, the samples for gel electrophoresis were soaked into Whatman 3MM filter paper disks (2.4 cm in diameter). These disks were washed with 20 ml of ice-cold 10% trichloroacetic acid and with 10 ml of methanol, and dried at 80°C for 20 min. The radioactivity was counted in the toluene-base scintillation fluid. Gel electrophoresis. SDS-polyacrylamide slab gel electrophoresis (17) was carried out as described by Laemmli (9). For the analysis of total cell proteins, 12% slab gels were used, and for more detailed analysis of high-molecular-weight regions, 7% slab gels were used. Marker proteins for the molecular weight estimation were bovine serum albumin (67,000), chymotrypsinogen A (bovine pancreas, 24,800), myoglobin (whale sperm, 17,000), cytochrome c (horse heart, 12,400); fB (155,000), ,P (165,000), a (101,000), and a (44,000) subunits of the C. crescentus CB13 DNAdependent RNA polymerase (2), and the C. crescentus CB13 flagellins A (26,000) and B (28,500). The RNA polymerase was a kind gift from Dr. Ikehara and Mr. Iida in this laboratory. Gels were stained with Coomassie brilliant blue, destained, and dried on filter
papers. Autoradiography and densitometry. Dried gels were exposed (usually 2 to 8 days) to Kodak X Omat R film (XR-5) with a series of radioactive ink spots with different concentrations of [uS]methionine. Gel columns and ink spots were scanned by microphotometer (Mitaka Kohki Co., Tokyo). The analysis of ink spots gave a standard curve of optical density versus specific radioactivity (counts per minute per square centimeter) in each film. Then the radioactivity in each protein band among different gel columns was estimated from the peak heights of the optical density. After correction for the yield of the preparation process, the absolute rate of individual protein synthesis was calculated.
J. BACTERIOL.
Chemicals. L-[3S]methionine (579 Ci/mmol), L'4C-amino acid mixture (algal type), and L-[3H]phenylalanine (10 Ci/mmol) were purchased from New England Nuclear Corp. Rifampin was purchased from Sigma Chemical Co. Streptolydigin was a kind gift from A. Ishihama.
RESULTS DNA synthesis in the C. crescentus CB13 tdr-806B35R4 cell cycle. Timing of DNA synthesis was examined in the cell cycle of C. crescentus CB13 tdr-806B35R4 growing in HMG medium as described previously (6). The swarmer cell cycle consisted of G1, S, and G2 periods of 60, 100, and 40 min, respectively, and the stalked cell cycle consisted of S and G2 periods of 100 and 40 min, respectively. The G1 period corresponds to the time for the swarmer cell to differentiate into the stalked cell as reported previously in C. crescentus CB13 wild type (6). Rate of major protein synthesis in the swarmer cell cycle. The rate of major protein synthesis was estimated in the cell cycle synchronized swarmer cells. At 20-min intervals, 0.2 ml of a synchronous culture was labeled with [3S]methionine (109 ,uCi or 0.022 ,ug/ml) for 12 min and chased for 3 min. The incorporation rate increased exponentially until the onset of synchronous cell division and thereafter was almost constant (Fig. 1). Similar patterns of incorporation were obtained when [3H]phenylalanine (82 jCi/ml), "4C-amino acid mixture (22 ,uCi/ml), or [3S]methionine at a lower concentration (35 ,uCi or 0.007 ,ug/ml) was used for the labeling of total cell protein. The incorporation of these labeled amino acids was linear within the pulselabeling time (12 min) under these conditions. These results indicate that the radioactivity incorporated in the 12-min pulse-labeling time is proportional to the rate of total protein synthesis and that the rate of total protein synthesis increases during G1 and S periods and remains almost constant during G2 period. The synthesizing activity of total cellular proteins is higher in the stalked cell than in the swarmer cell. The labeled total cell proteins were then separated by 12% SDS-polyacrylamide slab gel electrophoresis. About 45 major protein bands could be detected (Fig. 2), and 20 of them were quantitatively analyzed (see Materials and Methods). This method of analysis gave reproducible results in most of the major proteins with the exception of two protein bands (21K and 20K). For about one-half of the major proteins analyzed, the rate of synthesis in the cell cycle exhibited a pattern similar to that of total cell protein (Fig. 1). The rate of synthesis of the other half of the major proteins could be grouped
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
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TIME ( MIN ) FIG. 1. Rate of total protein synthesis in the C. crescentus cell cycle. A swarmer cell culture (8 ml) was prepared by the plate selection technique. At 20min uttervals during synchronous ceU growth, 0.2 ml of the culture was pulse-labeled with [3SJmethionine (0.022 pg or 109 ILCi/ml) for 12 min and chased for an additional 3 min by the addition of 5 ml of HMG medium containing 200 pg of methionine per ml. The radioactivity was measured in 0.5 ml of the chased culture. G1, S, and G2 represent the periods of the cell cycle. Symbols: 0, cell viability; 0, 3S radioactivity.
into one of three distinct patterns (Fig. 3A, B, and C) as follows. A stage-specific protein (77K) was synthesized preferentially at late S period, or in the elongated stalked cell (Fig. 3A). The rate of synthesis of the paired as well as the separated and fi' subunits of RNA polymerase increased constantly during the whole swarmer cell cycle (Fig. 3B). The ratio of the subunit band densities (/f)/f) ranged from 0.74 to 0.96 and appeared to be independent of the cell cycle periods. Some proteins such as 64K protein almost doubled in synthetic rate of S period, whereas no appreciable increase in rate was observed in G1 and G2 periods (Fig. 30). As one round of the cell cycle proceeds to completion, the Caulobacter cell should be able to synthesize each major protein at the total rate of the two progeny cells. When the rates of protein synthesis in the cells at 0 and 60 min in the swarner cell cycle were taken as the cellular activities of the swarmer cell and the stalked cell, respectively, then for all of the major proteins examined except the stage-specific protein (77K), the time in the cell cycle at which the total rate of the two progeny cells was achieved ranged from 120 to 160 min. This period corresponds to the late S period. Functional degradation rate of mRNA. ,
649
The degradation rate of mRNA can be studied by analyzing the rate of the protein synthesis after the inhibition of RNA synthesis. Rifampin and streptolydigin were used to inhibit, respectively, the initiation (18) and elongation (16) of RNA synthesis. A drastic decrease of RNA synthesis in the C. crescentus CB13 tdr-806B35R4 cell was observed immediately after the addition of rifampin (10 ,ug/ml) or streptolydigin (500 ,ug/ml). RNA synthesis was inhibited to less than 2.5% of the initial level within 3 min. Before and after an exponentially growing culture of C. crescentus CB13 tdr-806B35R4 was treated with rifampin (10 Ag/ml) or streptolydigin (500 yg/ml), aliquots were taken from the culture at 1-min intervals, pulse-labeled with [3Slmethionine for 1 min, and chased for 3 min. The rate of total cell protein synthesis decreased drastically within 1 min after streptolydigin addition (Fig. 4), indicating that little mRNA was available after the transcription was inhibited at the level of elongation. The rate of total protein synthesis then decreased exponentially. From this slope, the half-life of functional mRNA was estimated to be 1.5 to 2.2 min. Upon rifampin addition, the rate of total cell protein synthesis decreased exponentially (Fig. 4). Probably because of residual elongation of RNA synthesis after the inhibition by rifampin, the rate of synthesis was higher than that upon streptolydigin addition. The half-life of mRNA for total proteins was estimated again to be 1.5 to 1.9 min. The half-life of mRNA coding for individual major proteins was estimated in a similar manner, within the limits of the gel analysis of the pulse-labeled total proteins (Fig. 5). Similar values were obtained with either rifampin or streptolydigin addition. The half-life of mRNA was 1.1 min for the 77K protein (Fig. 3A), 1.15 min for the 64K protein (Fig. 3C), and 5.8 min for the 24K protein; the latter was longest among the major proteins examined (Fig. 6B). The halflives ranged from 0.4 to 3.0 min for other proteins. The ,B subunit of RNA polymerase presented an interesting exception. The rate of synthesis of this protein did not decrease but rather increased about 50% in the initial 4 min after rifampin addition, and then decreased with a half-life of less than 0.4 min (Fig. 6A). The f' subunit of RNA polymerase, however, decreased exponentially with a half-life of 0.8 min without an initial shoot-up (Fig. 6A). This unusual increase of the fi subunit synthesis was not observed when streptolydigin was used as the inhibitor. In the presence of streptolydigin, the synthesis of the ,B and f' subunits decreased to 3 and 5% of the initial rate, respectively, within 1 min and then decreased exponentially with a
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FIG. 2. SDS-polyacrylamide slab gel electrophoresis ofpulse-labeled total cell proteins. Total cell proteins were pulse-labeled during the swarmer cell cycle as described in the legend of Fig. 1. Labeled protein samples (14 ,tI) were analyzed by 12% SDS-polyacrylamide slab gel electrophoresis. Dried gels were exposed to Kodak X Omat R film (XR-5) for 5 days. Numbers at the top represent the time (in minutes) after the initiation of the cell cycle from the swarmer cell.
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
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>One protein (92K) decreased rapidly with a coordinated increase of another protein (85K). The 92K protein might be a possible precursor of the A
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85K protein. The turnover of total cell proteins in an unsynchronous culture was similar to that of swarmer cel proteins (Fig. 7). However, the
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FIG. 3. Typical patterns of the rate of synthesis of major proteins in the C. crescentus cell cycle. Autoradiograms such as are shown in Fig. 2 were traced by microphotometer. The heights of optical density peaks for individual proteins were converted into specific radioactwity (counts per minute per square milimeter), and the rate of synthesis of the protein was calculated as described in Materials and Methods. The results of two separate experiments (0, 0) are presented. G0, S, and 02 represent the periods of the cell cycle. (A) 77K protein; (B) the paired M and ,fP subunits of DNA-dependent RNA polymerase; (C) 64K protein.
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x units per ml) was treated with 10 pg of rifampin per ml (0) or with 500 pg of streptolydigin per ml (0). Before and after the addition of
half-life of less than 2 min. Stability of cellular proteins. For the esti- inhibitor, 0.2-ml aliquots were taken from the culture, mation of major protein turnover, swarner cells pulse-labeledI with [5Slmethionine (0.044 pg or 218 min, and chased for 3 min by the were pulse-labeled with [S]methionine JCi/ml) for (0.4 mil) 4addition of 5 ml of HMG medium containing 200 pg for 12 min and then chased. The radioactivity m Of methionineper ml. The radioactivity was measured the labeled total cell protein decreased only in 0.5 ml of the chased culture. The rate of synthesis slowly, with about 20% loss during the 180-min after the treatment was plotted as the percentage to the rate before the treatment. chase (Fig. 7).
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FIG. 5. SDS-polyacrylamide slab gel electrophoresis of total cell proteins pulse-labeled in the presence of rifampin. Total proteins were pulse-labeled as described in the legend of Fig. 4. The labeled proteins (14 p1 each) were separated on 12% (lower columns) and 7% (upper insertions) gels. Dried gels were exposed to Kodak X Omat R film (XR-5) for 6 days. The numbers at the top indicate the time ofpulse-labeling (minutes). Rifampin (10 pg/ml) was added at 0 min.
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
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FIG. 6. Rate of synthesis of individual major proteins after rifampin addition. The autoradiograms shown in Fig. 5 were traced by microphotometer. The rates of synthesis of individual major proteins were calculated as described in Materials and Methods. The results offive major proteins are presented. The rate before the addition of rifampin (an average of the rate at -2 to -1 min and -1 to 0 min) is taken as 100)%. Symbols: (A) 0, the (8 subunit of RNA polymerase; 0, the I? subunit of RNA polymerase. (B) 0, 77Kprotein; *, 64K protein; O, 24K protein. -4
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60 120 180 240 TIME AFTER C HA SE (MIN ) FDIG. 7. Turnover of total cell proteins. A swarmer cell culture (7.0 x 106 colony-forming units per ml) prelpared by the plate selection technique was pulselabeeled with [3S]methionine (0.044 pg or 218 /LCi/ml) for 12 min and chased by the addition of 5 ml of HMfG medium containing 2 ug of methionine per ml At the time indicated, 0.2 ml of the chased culture was withdrawn for radioactivity measurement (0). The protein turnover in the unsynchronous culture (1.0 x 16' colony-forming units per ml, 0) is also pre.sented. 0
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DISCUSSION The absolute rate of protein synthesis is a criterion by which the degree of gene expression may be estimated. We have described in this paper the absolute rate of protein synthesis in C. crescentus in an attempt to examine the changes in the gene expression during its synchronous cell differentiation. The analysis of rate of synthesis was limited to major proteins which could clearly be separated by a one-dimensional SDS-polyacrylamide gel electrophoresis. To avoid possible artifacts, cell proteins were not fractionated prior to the gel analysis. Several typical patterns of rate of major protein synthesis were observed during the swarmer cel cycle (Fig. 1 and 3). The stage-specific 77K protein was synthesized preferentially at late S period (Fig. 3A). This protein seems to correspond to a 76K protein which was identified as a cell division-specific protein among soluble proteins of C. crescentus CB15 (3). In our anal-
ysi of total cell proteins, other stage-specific proteins were not detected. Another interesting pattern of synthesis is that of a 64K protein (Fig. 3C). The stepwise increase of the protein synthesis during S period might reflect gene dosage effect upon chromosome replication. The gene stage-specific protein (77K, Fig. 3A), which was dosage effect alone, however, cannot explain the nott synthesized in the swarmer cell, was de- pattern of synthesis of the ,B and ff subunits of RNA polymerase in this bacterium (Fig. 3B), as graLded more than 80% per generation.
654
J. BACTrERIOL.
IBA, FUKUDA, AND OKADA
C)
15 50 180
proteins was found to be rather slow (Fig. 7 and
8) as suggested previously in the soluble proteins of C. crescentus CB15 (3). In contrast, the functional half-life of mRNA for each major protein estimated from the residual rate of synthesis of the protein after inhibition of RNA synthesis was found to be short (0.4 to 5.8 min), as also reported in E. coli (1). The functional half-life for the total mRNA in C. crescentus CB13 was estimated to be 1.5 to 2.2 min, which is similar to the chemical half-life of the bulk mRNA from C. crescentus CB15 (13). It is most likely that the absolute rate of individual major protein synthesis reflects the amount of mRNA available and that the patterns of protein synthesis described above are mainly attributable to the transcriptional control of gene expression. -- .....Although the possibility is not rigorously excluded that each band in the gel consists of more than a single major protein, some common features were found in the rate of major protein synthesis examined. For example, two types of cells in the Caulobacter cell cycle seem to differ in their activity in the synthesis of individual major proteins. The activity in the swarmer cell is less than that in the stalked cell (Fig. 1 and 3B). Thus these types of cells should have quantitative differences in gene expression. This difference is apparently expressed immediately before or at early G2 period, since the Caulobacter cell was able to synthesize individual major proteins at the total rate of the two types of cells at that time (Fig. 1 and 3). The asymmetry of the gene expression may be achieved upon chromosomal separation into respective cell counterparts. This notion is consistent with our previous observation that the elongated stalked cell (at early G2 period) has two targets against gammaFIG. 8. SDS-polyacri ylamide slab gel electropho- ray inactivation (7). resis of pulse-chased cellular protems. A swarmer ACKNOWLEDGMENT cell culture was pulse-chased as described in the legend of Fig. 7. At intervals, 1 ml of the chased This work was supported by a grant from the Scientific culture was withdrawn for slab gel electrophoresis. Research Fund of the Ministry of Education, Science and Labeled proteins (14 #l each) were separated on 7% Culture of Japan. gel. The numbers at the top represent the time after LITERATURE CrTED chase (minutes).
(33.d
92K 85K
64K
also suggested in Escherichia coli (8, 10). The existence of a novel control of the ,B subunit synthesis might be expected from the observation in Fig. 6A that the absolute rate of f8 subunit synthesis increased after the addition of rifampin which inhibits RNA synthesis by binding to the ,B subunit (5). Streptolydigin, which also binds to the ,8 subunit (5), did not have a similar effect. The differential effect of rifampin and streptolydigin treatment on ft subunit synthesis was also reported in E. coli (11). The degradation rate of most cellular major
1. Barnsley, P. G. H., and B. H. Sells. 1977. Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins of Esche-
richia coli. Mol. Gen. Genet. 153:121-127. L. K., and 1. Shapiro. 1973. Deoxyribonucleic acid-dependent ribonucleic acid polymerase of Caulo-
2. Bendis,
bacter crescentus. J. Bacteriol. 115:848-857. 3. Cheung, K. K., and A. Newton. 1977. Patterns of protein synthesis during the development in Caulobacter crescentus. Dev. Biol. 56:417-425. 4. Degnen, S. T., and A. Newton. 1972. Chromosome replication during development in Caulobacter crescentus. J. Mol. Biol. 64:671-680. 5. Heil, A., and W. Zilling. 1970. Reconstitution of bacterial DNA-dependent RNA polymerase from isolated subunits as a tool for elucidation of the role of the subunits
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PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
in transcription. FEBS Lett. 11:165-168. 6. Iba, H., A. Fukuda, and Y. Okada. 1977. Chromosome replication in Caulobacter crescentus growing in a nutrient broth. J. Bacteriol. 129:1192-1197. 7. Iba, H., A. Fukuda, and Y. Okada. 1977. Gamma-ray sensitivity during synchronous cell differentiation in Caulobacter crescentus. J. Bacteriol. 131:369-371. 8. Iwakura, Y., K. Ito, and A. Ishihama. 1974. Biosynthesis of RNA polymerase in Escherichia coli. 1. Control of RNA polymerase content at various growth rates. Mol. Gen. Genet. 133:1-23. 9. Laemmli, U. K. '1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Matzura, H., B. S. Hansen, and J. Zeuthen. 1973. Biosynthesis of the fi and ff subunits of RNA polymerase in Escherichia coli. J. Mol. Biol. 74:9-20. 11. Nakamura, Y., and,T. Yura. 1976. Effects of rifampicin on synthesis and functional activity of DNA-dependent RNA polymerase in Escherichia coli. Mol. Gen. Genet.
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145:227-237. 12. Newton, A. 1972. Role of transcription in the temporal control of development in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 69:447-451. 13. Ohta, N., M. Sanders, and A. Newton. 1977. Characterization of unstable poly (A)-RNA in Caulobacter crescentus. Biochim. Biophys. Acta 366:149-158. 14. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 15. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407. 16. Siddhikol, C., J. W. Erbstoeszer, and B. Weisblunu 1969. Mode of action of streptolydigin. J. Bacteriol.
99:151-155. 17. Studier, F. W. 1973. Analysis on bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79:237-248. 18. Wehrli, W., and M. Staehelin. 1971. Actions of the rifamycins. Bacteriol. Rev. 35:290-309.
Vol. 135, No. 2
Printed in U.S.A.
Rate of Major Protein Synthesis During the Cell Cycle of 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 30 May 1978
The rate of major protein synthesis was examined during the synchronous differentiation of Caulobacter crescentus. Total cell proteins were pulse-labeled with [3S]methionine at different times in the swarmer cell cycle and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rates of synthesis of total cell proteins and of about one-half of the individual major proteins examined increased through GI and S periods but remained nearly constant during G2 period. The rates of synthesis of the other half of the individual major proteins either increased continuously throughout the swarmer cell cycle or doubled during S period. One stage-specific protein was also detected in late S period. For most of the major proteins examined, the rate of synthesis in the swarmer cell was less than that in the stalked cell. It seemed that, before the onset of G2 period, the Caulobacter cell was already able to synthesize each major protein at the additive rate of the two progeny cells. Compared to the stability of cellular proteins, the functional degradation rate of mRNA coding for individual major proteins was rapid, with half-lives of 0.4 to 5.8 min. It thus seems that the rate of major protein synthesis mainly reflects the transcriptional control of gene expression.
A gram-negative stalked bacterium, Caulobacter crescentus, exhibits dimorphism of cell types in the cell cycle (14, 15). A predivisional cell divides asymmetrically and produces a motile swarmer cell and a nonmotile stalked cell. The swarner cell differentiates into a stalked cell before the next round of cell division, whereas the stalked cell directly differentiates into a predivisional cell. The cell cycle of C. crescentus can be divided, in terms of DNA synthesis activity, into three distinct periods, namely G1, S, and G2 periods in both fast-growth and slow-growth media (4, 6). For the normal process of cell differentiation in this bacterium, DNA transcription is required throughout the cell cycle (12). Some stage-specific proteins have been detected among soluble proteins of C. crescentus CB15 (3). We have studied the cell cycle dependency of the absolute rate of synthesis of major cellular proteins in an attempt to monitor the changes of gene expression quantitatively in the Caulobacter cell. Several patterns of the cell cycle dependency were observed and quantitative differences in the gene expression were found between the two progeny cells. Furthermore, the half-life of mRNA coding for each major protein was estimated by the analysis of the residual rate of protein synthesis after the inhibition of
RNA synthesis. The mRNA was found to be unstable compared to the stability of cellular proteins. MATERIALS AND METHODS Bacterial strain and growth conditions. C. crescentus CB13 tdr-806B35R4 was used throughout this study and grown in glucose (0.2%) -supplemented minimal salts medium (HMG medium [14]) at 30°C with shaking. This strain is a revertant from an obligate thymidine auxotroph, C. crescentus CB13 tdr-806B35, and, unlike the CB13 wild-type strain, grows well in HMG medium. Details of these strains will be described elsewhere (A. Fukuda and H. Iba, manuscript in preparation). Cell synchronization. Newborn swarmer cells were obtained by plate selection technique as described previously (4, 6) except that glass plates were coated with poly-L-lysine (0.002%). Preparation of total cell proteins for gel electrophoresis. In a typical experiment, CB13 tdr806B35R4 culture (0.2 ml) was pulse-labeled for 12 min with 109 ACi of [3S]methionine per ml and chased for 3 min by the addition of 5 ml of HMG medium containing 200 ug of cold methionine per ml. The labeled culture was chilled in ice, and a sample (0.5 ml) was taken for the counting of incorporated radio-
activity. To the rest of the labeled culture was added 0.2 ml of C. crescentus CB13 tdr-806B35R4 grown in HMG medium (0.5 of the optical density at 660 nm) as a carrier. After centrifugation for 10 min at 15,000
647
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IBA, FUKUDA, AND OKADA
rpm, the cell pellet was suspended in 3 ml of 10 mM phosphate-5 mM MgCl2 (pH 6.8). After another centrifugation for 10 min at 15,000 rpm, the cell pellet was suspended in 0.2 ml of the gel sample buffer consisting of 0.0625 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue, and boiled for 2 min. For the examination of the turnover of cellular proteins, C. crescentus CB13 tdr-806B35R4 culture (0.4 ml) was labeled with 218 yiCi of [3S]methionine per ml for 12 min and chased by the addition of 5 ml of HMG medium containing 2 jug of cold methionine per ml. Two volumes of the culture were withdrawn at the time indicated. One volume (0.2 ml) was used for the counting of incorporated radioactivity, and the other volume (1 ml) was used to prepare a sample for gel electrophoresis as follows. The labeled culture (1 ml) was mixed with carrier cells as described above and with 3 ml of 10% trichloroacetic acid. The mixture was then chilled in ice for 30 min and centrifuged for 10 min at 15,000 rpm. The precipitates were washed twice with acetone, solubilized in 0.2 ml of the gel sample buffer as described above, and boiled for 2 min. Radioactivity measurement. The samples were heated in 10% trichloroacetic acid at 90°C for 20 min, chilled in ice for 30 min, and filtered onto glass fiber filter disks (Whatman GF/B, 2.4 cm in diameter). Alternatively, the samples for gel electrophoresis were soaked into Whatman 3MM filter paper disks (2.4 cm in diameter). These disks were washed with 20 ml of ice-cold 10% trichloroacetic acid and with 10 ml of methanol, and dried at 80°C for 20 min. The radioactivity was counted in the toluene-base scintillation fluid. Gel electrophoresis. SDS-polyacrylamide slab gel electrophoresis (17) was carried out as described by Laemmli (9). For the analysis of total cell proteins, 12% slab gels were used, and for more detailed analysis of high-molecular-weight regions, 7% slab gels were used. Marker proteins for the molecular weight estimation were bovine serum albumin (67,000), chymotrypsinogen A (bovine pancreas, 24,800), myoglobin (whale sperm, 17,000), cytochrome c (horse heart, 12,400); fB (155,000), ,P (165,000), a (101,000), and a (44,000) subunits of the C. crescentus CB13 DNAdependent RNA polymerase (2), and the C. crescentus CB13 flagellins A (26,000) and B (28,500). The RNA polymerase was a kind gift from Dr. Ikehara and Mr. Iida in this laboratory. Gels were stained with Coomassie brilliant blue, destained, and dried on filter
papers. Autoradiography and densitometry. Dried gels were exposed (usually 2 to 8 days) to Kodak X Omat R film (XR-5) with a series of radioactive ink spots with different concentrations of [uS]methionine. Gel columns and ink spots were scanned by microphotometer (Mitaka Kohki Co., Tokyo). The analysis of ink spots gave a standard curve of optical density versus specific radioactivity (counts per minute per square centimeter) in each film. Then the radioactivity in each protein band among different gel columns was estimated from the peak heights of the optical density. After correction for the yield of the preparation process, the absolute rate of individual protein synthesis was calculated.
J. BACTERIOL.
Chemicals. L-[3S]methionine (579 Ci/mmol), L'4C-amino acid mixture (algal type), and L-[3H]phenylalanine (10 Ci/mmol) were purchased from New England Nuclear Corp. Rifampin was purchased from Sigma Chemical Co. Streptolydigin was a kind gift from A. Ishihama.
RESULTS DNA synthesis in the C. crescentus CB13 tdr-806B35R4 cell cycle. Timing of DNA synthesis was examined in the cell cycle of C. crescentus CB13 tdr-806B35R4 growing in HMG medium as described previously (6). The swarmer cell cycle consisted of G1, S, and G2 periods of 60, 100, and 40 min, respectively, and the stalked cell cycle consisted of S and G2 periods of 100 and 40 min, respectively. The G1 period corresponds to the time for the swarmer cell to differentiate into the stalked cell as reported previously in C. crescentus CB13 wild type (6). Rate of major protein synthesis in the swarmer cell cycle. The rate of major protein synthesis was estimated in the cell cycle synchronized swarmer cells. At 20-min intervals, 0.2 ml of a synchronous culture was labeled with [3S]methionine (109 ,uCi or 0.022 ,ug/ml) for 12 min and chased for 3 min. The incorporation rate increased exponentially until the onset of synchronous cell division and thereafter was almost constant (Fig. 1). Similar patterns of incorporation were obtained when [3H]phenylalanine (82 jCi/ml), "4C-amino acid mixture (22 ,uCi/ml), or [3S]methionine at a lower concentration (35 ,uCi or 0.007 ,ug/ml) was used for the labeling of total cell protein. The incorporation of these labeled amino acids was linear within the pulselabeling time (12 min) under these conditions. These results indicate that the radioactivity incorporated in the 12-min pulse-labeling time is proportional to the rate of total protein synthesis and that the rate of total protein synthesis increases during G1 and S periods and remains almost constant during G2 period. The synthesizing activity of total cellular proteins is higher in the stalked cell than in the swarmer cell. The labeled total cell proteins were then separated by 12% SDS-polyacrylamide slab gel electrophoresis. About 45 major protein bands could be detected (Fig. 2), and 20 of them were quantitatively analyzed (see Materials and Methods). This method of analysis gave reproducible results in most of the major proteins with the exception of two protein bands (21K and 20K). For about one-half of the major proteins analyzed, the rate of synthesis in the cell cycle exhibited a pattern similar to that of total cell protein (Fig. 1). The rate of synthesis of the other half of the major proteins could be grouped
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
S
GI
G2°
E
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TIME ( MIN ) FIG. 1. Rate of total protein synthesis in the C. crescentus cell cycle. A swarmer cell culture (8 ml) was prepared by the plate selection technique. At 20min uttervals during synchronous ceU growth, 0.2 ml of the culture was pulse-labeled with [3SJmethionine (0.022 pg or 109 ILCi/ml) for 12 min and chased for an additional 3 min by the addition of 5 ml of HMG medium containing 200 pg of methionine per ml. The radioactivity was measured in 0.5 ml of the chased culture. G1, S, and G2 represent the periods of the cell cycle. Symbols: 0, cell viability; 0, 3S radioactivity.
into one of three distinct patterns (Fig. 3A, B, and C) as follows. A stage-specific protein (77K) was synthesized preferentially at late S period, or in the elongated stalked cell (Fig. 3A). The rate of synthesis of the paired as well as the separated and fi' subunits of RNA polymerase increased constantly during the whole swarmer cell cycle (Fig. 3B). The ratio of the subunit band densities (/f)/f) ranged from 0.74 to 0.96 and appeared to be independent of the cell cycle periods. Some proteins such as 64K protein almost doubled in synthetic rate of S period, whereas no appreciable increase in rate was observed in G1 and G2 periods (Fig. 30). As one round of the cell cycle proceeds to completion, the Caulobacter cell should be able to synthesize each major protein at the total rate of the two progeny cells. When the rates of protein synthesis in the cells at 0 and 60 min in the swarner cell cycle were taken as the cellular activities of the swarmer cell and the stalked cell, respectively, then for all of the major proteins examined except the stage-specific protein (77K), the time in the cell cycle at which the total rate of the two progeny cells was achieved ranged from 120 to 160 min. This period corresponds to the late S period. Functional degradation rate of mRNA. ,
649
The degradation rate of mRNA can be studied by analyzing the rate of the protein synthesis after the inhibition of RNA synthesis. Rifampin and streptolydigin were used to inhibit, respectively, the initiation (18) and elongation (16) of RNA synthesis. A drastic decrease of RNA synthesis in the C. crescentus CB13 tdr-806B35R4 cell was observed immediately after the addition of rifampin (10 ,ug/ml) or streptolydigin (500 ,ug/ml). RNA synthesis was inhibited to less than 2.5% of the initial level within 3 min. Before and after an exponentially growing culture of C. crescentus CB13 tdr-806B35R4 was treated with rifampin (10 Ag/ml) or streptolydigin (500 yg/ml), aliquots were taken from the culture at 1-min intervals, pulse-labeled with [3Slmethionine for 1 min, and chased for 3 min. The rate of total cell protein synthesis decreased drastically within 1 min after streptolydigin addition (Fig. 4), indicating that little mRNA was available after the transcription was inhibited at the level of elongation. The rate of total protein synthesis then decreased exponentially. From this slope, the half-life of functional mRNA was estimated to be 1.5 to 2.2 min. Upon rifampin addition, the rate of total cell protein synthesis decreased exponentially (Fig. 4). Probably because of residual elongation of RNA synthesis after the inhibition by rifampin, the rate of synthesis was higher than that upon streptolydigin addition. The half-life of mRNA for total proteins was estimated again to be 1.5 to 1.9 min. The half-life of mRNA coding for individual major proteins was estimated in a similar manner, within the limits of the gel analysis of the pulse-labeled total proteins (Fig. 5). Similar values were obtained with either rifampin or streptolydigin addition. The half-life of mRNA was 1.1 min for the 77K protein (Fig. 3A), 1.15 min for the 64K protein (Fig. 3C), and 5.8 min for the 24K protein; the latter was longest among the major proteins examined (Fig. 6B). The halflives ranged from 0.4 to 3.0 min for other proteins. The ,B subunit of RNA polymerase presented an interesting exception. The rate of synthesis of this protein did not decrease but rather increased about 50% in the initial 4 min after rifampin addition, and then decreased with a half-life of less than 0.4 min (Fig. 6A). The f' subunit of RNA polymerase, however, decreased exponentially with a half-life of 0.8 min without an initial shoot-up (Fig. 6A). This unusual increase of the fi subunit synthesis was not observed when streptolydigin was used as the inhibitor. In the presence of streptolydigin, the synthesis of the ,B and f' subunits decreased to 3 and 5% of the initial rate, respectively, within 1 min and then decreased exponentially with a
650
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FIG. 2. SDS-polyacrylamide slab gel electrophoresis ofpulse-labeled total cell proteins. Total cell proteins were pulse-labeled during the swarmer cell cycle as described in the legend of Fig. 1. Labeled protein samples (14 ,tI) were analyzed by 12% SDS-polyacrylamide slab gel electrophoresis. Dried gels were exposed to Kodak X Omat R film (XR-5) for 5 days. Numbers at the top represent the time (in minutes) after the initiation of the cell cycle from the swarmer cell.
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
-
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'
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2
* * / ffi t v
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ing the ,B and f,' subunits of RNA polymerase, similarly were found to be stable (Fig. 8). One less-stable protein (64K, Fig. 3C) was detected which turned over
.
about 50% before cell division.
>One protein (92K) decreased rapidly with a coordinated increase of another protein (85K). The 92K protein might be a possible precursor of the A
t
0
Most of the individual major proteins, includ-
G2
/ Ch
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651
85K protein. The turnover of total cell proteins in an unsynchronous culture was similar to that of swarmer cel proteins (Fig. 7). However, the
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120 180 240 TIME (MIN )
FIG. 3. Typical patterns of the rate of synthesis of major proteins in the C. crescentus cell cycle. Autoradiograms such as are shown in Fig. 2 were traced by microphotometer. The heights of optical density peaks for individual proteins were converted into specific radioactwity (counts per minute per square milimeter), and the rate of synthesis of the protein was calculated as described in Materials and Methods. The results of two separate experiments (0, 0) are presented. G0, S, and 02 represent the periods of the cell cycle. (A) 77K protein; (B) the paired M and ,fP subunits of DNA-dependent RNA polymerase; (C) 64K protein.
\
\
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5 V TIME (MI N) FIG. 4. Rate of total protein synthesis after the inhibition of RNA synthesis. An exponentially growC. cresentus CB13 tdr-806B35R4 (3.0 ing culre offC rwnu C1 d-0B54(. 0
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x units per ml) was treated with 10 pg of rifampin per ml (0) or with 500 pg of streptolydigin per ml (0). Before and after the addition of
half-life of less than 2 min. Stability of cellular proteins. For the esti- inhibitor, 0.2-ml aliquots were taken from the culture, mation of major protein turnover, swarner cells pulse-labeledI with [5Slmethionine (0.044 pg or 218 min, and chased for 3 min by the were pulse-labeled with [S]methionine JCi/ml) for (0.4 mil) 4addition of 5 ml of HMG medium containing 200 pg for 12 min and then chased. The radioactivity m Of methionineper ml. The radioactivity was measured the labeled total cell protein decreased only in 0.5 ml of the chased culture. The rate of synthesis slowly, with about 20% loss during the 180-min after the treatment was plotted as the percentage to the rate before the treatment. chase (Fig. 7).
652
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FIG. 5. SDS-polyacrylamide slab gel electrophoresis of total cell proteins pulse-labeled in the presence of rifampin. Total proteins were pulse-labeled as described in the legend of Fig. 4. The labeled proteins (14 p1 each) were separated on 12% (lower columns) and 7% (upper insertions) gels. Dried gels were exposed to Kodak X Omat R film (XR-5) for 6 days. The numbers at the top indicate the time ofpulse-labeling (minutes). Rifampin (10 pg/ml) was added at 0 min.
PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
VOL. 135, 1978
0
3
6 T I M E
0
3 ( M I N
6
653
9
)
FIG. 6. Rate of synthesis of individual major proteins after rifampin addition. The autoradiograms shown in Fig. 5 were traced by microphotometer. The rates of synthesis of individual major proteins were calculated as described in Materials and Methods. The results offive major proteins are presented. The rate before the addition of rifampin (an average of the rate at -2 to -1 min and -1 to 0 min) is taken as 100)%. Symbols: (A) 0, the (8 subunit of RNA polymerase; 0, the I? subunit of RNA polymerase. (B) 0, 77Kprotein; *, 64K protein; O, 24K protein. -4
5 OE 0
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._ _ _
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2
60 120 180 240 TIME AFTER C HA SE (MIN ) FDIG. 7. Turnover of total cell proteins. A swarmer cell culture (7.0 x 106 colony-forming units per ml) prelpared by the plate selection technique was pulselabeeled with [3S]methionine (0.044 pg or 218 /LCi/ml) for 12 min and chased by the addition of 5 ml of HMfG medium containing 2 ug of methionine per ml At the time indicated, 0.2 ml of the chased culture was withdrawn for radioactivity measurement (0). The protein turnover in the unsynchronous culture (1.0 x 16' colony-forming units per ml, 0) is also pre.sented. 0
I
DISCUSSION The absolute rate of protein synthesis is a criterion by which the degree of gene expression may be estimated. We have described in this paper the absolute rate of protein synthesis in C. crescentus in an attempt to examine the changes in the gene expression during its synchronous cell differentiation. The analysis of rate of synthesis was limited to major proteins which could clearly be separated by a one-dimensional SDS-polyacrylamide gel electrophoresis. To avoid possible artifacts, cell proteins were not fractionated prior to the gel analysis. Several typical patterns of rate of major protein synthesis were observed during the swarmer cel cycle (Fig. 1 and 3). The stage-specific 77K protein was synthesized preferentially at late S period (Fig. 3A). This protein seems to correspond to a 76K protein which was identified as a cell division-specific protein among soluble proteins of C. crescentus CB15 (3). In our anal-
ysi of total cell proteins, other stage-specific proteins were not detected. Another interesting pattern of synthesis is that of a 64K protein (Fig. 3C). The stepwise increase of the protein synthesis during S period might reflect gene dosage effect upon chromosome replication. The gene stage-specific protein (77K, Fig. 3A), which was dosage effect alone, however, cannot explain the nott synthesized in the swarmer cell, was de- pattern of synthesis of the ,B and ff subunits of RNA polymerase in this bacterium (Fig. 3B), as graLded more than 80% per generation.
654
J. BACTrERIOL.
IBA, FUKUDA, AND OKADA
C)
15 50 180
proteins was found to be rather slow (Fig. 7 and
8) as suggested previously in the soluble proteins of C. crescentus CB15 (3). In contrast, the functional half-life of mRNA for each major protein estimated from the residual rate of synthesis of the protein after inhibition of RNA synthesis was found to be short (0.4 to 5.8 min), as also reported in E. coli (1). The functional half-life for the total mRNA in C. crescentus CB13 was estimated to be 1.5 to 2.2 min, which is similar to the chemical half-life of the bulk mRNA from C. crescentus CB15 (13). It is most likely that the absolute rate of individual major protein synthesis reflects the amount of mRNA available and that the patterns of protein synthesis described above are mainly attributable to the transcriptional control of gene expression. -- .....Although the possibility is not rigorously excluded that each band in the gel consists of more than a single major protein, some common features were found in the rate of major protein synthesis examined. For example, two types of cells in the Caulobacter cell cycle seem to differ in their activity in the synthesis of individual major proteins. The activity in the swarmer cell is less than that in the stalked cell (Fig. 1 and 3B). Thus these types of cells should have quantitative differences in gene expression. This difference is apparently expressed immediately before or at early G2 period, since the Caulobacter cell was able to synthesize individual major proteins at the total rate of the two types of cells at that time (Fig. 1 and 3). The asymmetry of the gene expression may be achieved upon chromosomal separation into respective cell counterparts. This notion is consistent with our previous observation that the elongated stalked cell (at early G2 period) has two targets against gammaFIG. 8. SDS-polyacri ylamide slab gel electropho- ray inactivation (7). resis of pulse-chased cellular protems. A swarmer ACKNOWLEDGMENT cell culture was pulse-chased as described in the legend of Fig. 7. At intervals, 1 ml of the chased This work was supported by a grant from the Scientific culture was withdrawn for slab gel electrophoresis. Research Fund of the Ministry of Education, Science and Labeled proteins (14 #l each) were separated on 7% Culture of Japan. gel. The numbers at the top represent the time after LITERATURE CrTED chase (minutes).
(33.d
92K 85K
64K
also suggested in Escherichia coli (8, 10). The existence of a novel control of the ,B subunit synthesis might be expected from the observation in Fig. 6A that the absolute rate of f8 subunit synthesis increased after the addition of rifampin which inhibits RNA synthesis by binding to the ,B subunit (5). Streptolydigin, which also binds to the ,8 subunit (5), did not have a similar effect. The differential effect of rifampin and streptolydigin treatment on ft subunit synthesis was also reported in E. coli (11). The degradation rate of most cellular major
1. Barnsley, P. G. H., and B. H. Sells. 1977. Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins of Esche-
richia coli. Mol. Gen. Genet. 153:121-127. L. K., and 1. Shapiro. 1973. Deoxyribonucleic acid-dependent ribonucleic acid polymerase of Caulo-
2. Bendis,
bacter crescentus. J. Bacteriol. 115:848-857. 3. Cheung, K. K., and A. Newton. 1977. Patterns of protein synthesis during the development in Caulobacter crescentus. Dev. Biol. 56:417-425. 4. Degnen, S. T., and A. Newton. 1972. Chromosome replication during development in Caulobacter crescentus. J. Mol. Biol. 64:671-680. 5. Heil, A., and W. Zilling. 1970. Reconstitution of bacterial DNA-dependent RNA polymerase from isolated subunits as a tool for elucidation of the role of the subunits
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PROTEIN SYNTHESIS IN CAULOBACTER CELL CYCLE
in transcription. FEBS Lett. 11:165-168. 6. Iba, H., A. Fukuda, and Y. Okada. 1977. Chromosome replication in Caulobacter crescentus growing in a nutrient broth. J. Bacteriol. 129:1192-1197. 7. Iba, H., A. Fukuda, and Y. Okada. 1977. Gamma-ray sensitivity during synchronous cell differentiation in Caulobacter crescentus. J. Bacteriol. 131:369-371. 8. Iwakura, Y., K. Ito, and A. Ishihama. 1974. Biosynthesis of RNA polymerase in Escherichia coli. 1. Control of RNA polymerase content at various growth rates. Mol. Gen. Genet. 133:1-23. 9. Laemmli, U. K. '1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 10. Matzura, H., B. S. Hansen, and J. Zeuthen. 1973. Biosynthesis of the fi and ff subunits of RNA polymerase in Escherichia coli. J. Mol. Biol. 74:9-20. 11. Nakamura, Y., and,T. Yura. 1976. Effects of rifampicin on synthesis and functional activity of DNA-dependent RNA polymerase in Escherichia coli. Mol. Gen. Genet.
655
145:227-237. 12. Newton, A. 1972. Role of transcription in the temporal control of development in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 69:447-451. 13. Ohta, N., M. Sanders, and A. Newton. 1977. Characterization of unstable poly (A)-RNA in Caulobacter crescentus. Biochim. Biophys. Acta 366:149-158. 14. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 15. Shapiro, L. 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377-407. 16. Siddhikol, C., J. W. Erbstoeszer, and B. Weisblunu 1969. Mode of action of streptolydigin. J. Bacteriol.
99:151-155. 17. Studier, F. W. 1973. Analysis on bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79:237-248. 18. Wehrli, W., and M. Staehelin. 1971. Actions of the rifamycins. Bacteriol. Rev. 35:290-309.
