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coside to cultures growing in glucose-minimal medium, and the upshifts ... Second, we used a temperature-dependent copy mutant of ... t Presnt address: Department of Genetics, Stanford Uni- .... is difficult to achieve with the E. coli chromo- some. However, for plasmid Ri at ... somal DNA as well as in the number of copies.
Vol. 141, No. 1

JOURNAL OF BACTROLOGY, Jan. 1980, p. 106-110 0021-9193/80/01-0106/05$02.00/0

Control of Plasmid Ri Replication: Kinetics of Replication in Shifts Between Different Copy Number Levels PETTER GUSTAFSSONt AND KURT NORDSTROM Department of Molecular Biology, University of Odense, Dk-5230 Odense M, Denmark

Plasmid Rl replication was studied in shifts between two steady states of copy number. The copy number was varied in two ways. First, we utilized the fact that it decreases with increasing growth rate. To minimize the metabolic effects of changes in the growth rate, the downshifts were obtained by adding a-methylglucoside to cultures growing in glucose-minimal medium, and the upshifts were obtained by adding glucose to cultures growing in the presence of glucose plus a-methylglucoside. Second, we used a temperature-dependent copy mutant of plasmid Rl (pKN301). Plasmid pPK301 shows a threefold higher copy number at 40 than at 300C. In both types of shift, plasmid replication immediately adjusted to the postshift differential rate. The copy number asymptotically adjusted to the new steady state. Hence, the system that controls plasmid Rl replication sets the frequency of replication without measuring the actual copy number. It has been suggested that plasmid R1 replication is under negative control by an Rl-mediated repressor protein. Among the replication control models that involve negative control, the Pritchard inhibitor dilution model, the Sompayrac-Maal0e autorepressor model, and the plasmid Xdv system all predict gene dose-independent copy number control.

Replication of bacterial chromosomes and plasmids is carefully regulated; in an exponentially growing population of bacteria the plaid population grows with the same generation time as that of the bacteria, giving a defined average number of copies per cell (4, 9). For many plasmids this number is low, 2 to 5 per cell (4, 9). Nevertheless, the system is very stable, and plasmidless cells are rare. Replication is generally assumed to be controlled at the level of initiation (29). The initiation mass (cell mass per origin) of the Escherichia coli chromosome is the same at all growth rates (at least above one generation per h), suggesting that initiation mass (or rather origin concentration) is the parameter used to control replication (7, 27, 29). This is reasonable, since it implies the measurement of a concentration. It is also reasonable that other replicons than the chromosome should use the same type of control measure. A number of plasmids (Rl, F'lac, P1, R6K) show different initiation masses under different conditions of growth (2, 5, 8, 26). This enables a test of the hypothesis that the control system measures origin concentration; such a test should be possible by shifting a plasmid-containing bacterial population between different steady states. We report that (at least for plasmid R1) the control system does not titrate the concentration of origins, but only

defines the frequency of replication. In a shift between two steady states the plasmid copy number is not actively adjusted but slowly approaches the postshift value. MATERIALS AND METHODS

t Presnt address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305. 106

Bacteria and plasmids. The bacterial strain used throughout this work was E. coli K-12 strain EC1005 (met nal relA) (10). The plasmids used were Rldrd19 (19) and pKN301 (11). The latter is a temperaturedependent copy mutant of plasmid Rldrd-19. Both plasmids carry the genes aadA', aphA', bla', cat+, 8Ur, and tra' (for symbols, see Novick et al. [25]). Media and growth conditions. The media used were morpholinepropanesulfonic acid medium (22) supplemented with glucose (0.4%, wt/vol), Casamino Acids (1.5%, wt/vol), thiamine (1 yg/ml), and adenosine (250 ,Lg/ml) and minimal medium M9 (1) supplemented with adenosine (250 pg/ml), glucose, and amethyiglucoside (aMG) as indicated below. TESbuffer contained 50 mM Tris-hydrochloride, 5 mM EDTA, 50 mM NaCl, and 3 mM sodium azide (pH 8.0). The bacteria were grown as shaken cultures in thermostated water baths. Growth of the cultures was monitored by following the increase in either optical density or protein content. Optical density was measured in a Klett-Summerson colorimeter equipped with filter W66. A recording of 100 Klett units corresponds to a cell density of 4 x 10i cells per ml. Materials. All chemicals were of analytical grade and were obtained from E. Merck AG (Darmstadt, West Germany) or Sigma Chemical Co. (St. Louis, Mo.). Casamino Acids were obtained from Difco Lab-

VOL. 141, 1980

COPY NUMBER SHIFTS WITH PLASMID Ri

oratories (Detroit, Mich.). Radioactive chemicals were purchased from The Radiochemical Centre (Amersham, Buckinghamshire, England). Lysozyme and RNase A were obtained from Sigma. To destroy DNase activity, the solution of RNase was heated at 90°C for 10 min before use. Analytical methods. Protein was measured according to Lowry et al. (15). DNA analysis was performed by harvesting cells by centrifugation at 0°C and washing them twice with ice-cold TES-buffer. The washed cells were treated with lysozyme (1 mg/ml) and RNase (500 tig/ml) for 15 min at 37°C. The cells were lysed by the addition of sodium dodecyl sulfate (0.1%, wt/vol). The lysates were analyzed by CsClethidium bromide density gradient centrifugation or by alkaline sucrose gradient centrifugation (24). In the latter case the lysates were sheared before centrifugation as described before (24). Shift experiments. In the aMG shifts, strain EC1005 carrying plasmid Rldrd-19 was grown exponentially in minimal medium M9 containing adenosine (250 ug/ml) at 37°C with glucose (0.2%, wt/vol) (culture B) or with glucose (0.2%, wt/vol) and aMG (2% wt/vol) (culture A). In both cases the media also contained [14C]thymidine (specific activity, '61 mCi/ mmol; 0.25 ,uCi/ml). At a cell density of about 108 cells per ml, glucose (1.8%, wt/vol) was added to culture A and aMG (2% wt/vol) was added to culture B. From each culture a sample was taken for determination of plasmid DNA. The two cultures were divided into aliquots that were incubated at 37°C. [3H]thymidine (specific activity, 18.5 Ci/mmol; 5 yCi/ml) was added to the subcultures at different times. After a pulse of 10 (culture A) or 20 (culture B) min, cold thymidine (100 ,ug/ml) was added, and incubation was continued for another 5 min. The cells were harvested by the addition of an equal volume of cold TES-buffer. The bacteria were collected by centrifugation at 0°C, washed, and lysed, after which the DNA was analyzed by ethidium bromide-cesium chloride density gradient centrifugation (24). Protein was determined according to Lowry et al. (15). The temperature shifts were performed essentially in the same way as the aMG shifts. Strain EC1005 carrying plasmid pKN301 was grown in morpholinepropanesulfonic acid medium and pulse-labeled with [3H]thymidine for 10 (30 -- 40°C) and 20 (40 -* 30°C) min. The DNA was analyzed on alkaline sucrose gradients.

RESULTS Shifts between different plasmid copy numbers. For the E. coli chromosome, it has been suggested that the origin concentration (origins/mass) is the parameter used to control replication (7, 27, 29). It is reasonable that this hypothesis should apply for plasmid copy number control. A test of this hypothesis should be possible if the steady-state concentration of the replicon could be varied. Due to the constant initiation mass at all growth rates (7, 27, 29), this is difficult to achieve with the E. coli chromosome. However, for plasmid Ri at least two

107

possibilities for shifts between different copy numbers exist. One is based on the fact that the plasmid Rl content decreases with increasing growth rate; there is a decrease in the ratio of covalently closed circular Rl DNA to chromosomal DNA as well as in the number of copies of plasmid Rl per cell (8). The other type of shift utilizes the temperature-dependent copy mutant (11), plasmid pKN301. This plasmid has a copy number close to that of the wild type at temperatures below 350C and shows a fourfold higher value at temperatures above 360C. The theoretical behavior of plasmid replication in shifts between two steady-state levels of copy number, assuming a titration of copy number (concentration of origins), would be the following: (i) in a shift from a low to a high copy number there should be a burst of plasmid synthesis lasting until the proper postshift value has been obtained; (ii) in a shift from a high to a low copy number there should be a complete cessation of plasmid replication until the plasmid has been diluted to the postshift concentration. In the following the plasmid copy number is expressed as covalently closed circular DNA per chromosomal DNA. Since the ratio of chromosomal DNA to protein is nearly constant, the ratio of covalently closed circular DNA to chromosomal DNA will also be a measure of plasmid concentration. Copy number shifts by changes in growth rate. Growth rate can be varied by using different carbon and nitrogen sources (16). However, there are great differences in metabolism and catabolism in these cases. Therefore, shifts from a low to a high growth rate are not immediate, and in shifts from a high to a low growth rate there is a very long period without any growth (16). Therefore, such shifts are not really suitable for a proper test of the hypothesis that there is a mechanism that titrates the copy number. It would be ideal if it were possible to shift growth rate without changing the biosynthetic and catabolic pattern of the cells. One way to approach this ideal is to use aMG and glucose-minimal medium. aMG is a competitive inhibitor of the uptake of glucose, but it cannot be catabolized by the cells (14). It is, therefore, possible to affect the growth rate by varying the ratio of aMG to glucose (12). Both in upshifts and in downshifts there is an immediate change from the preshift to the postshift growth rate (12). Kessler and Rickenberg (14) have shown that the growth rate can be changed over a threefold range without changing the composition of the cells. Strain EC1005 carrying plasmid Rldrd-19 was grown at 370C in minimal medium M9. The

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steady-state concentration of the plasmid was 2.0% (per chromosomal DNA) in glucose-minimal medium (0.2%, wt/vol) (growth rate, 1.04 generations per h) and 5.0% in the same medium to which 2.0% (wt/vol) aMG had been added (growth rate of 0.51 generations per h). The difference in copy number between these two conditions is 2.5-fold, which gives a reasonable resolution in the analysis of the transient plasmid replication during shifts. We have ignored that the ratio of chromosomal DNA to protein decreases by 5 to 10% when the growth rate increases from 0.5 to 1.0 generation per h (according to the Cooper-Hehmstetter theory [6]). A metabolic upshift was performed by changing the ratio between aMG and glucose from 2% aMG-0.2% glucose to 2% each. The metabolic downshift was performed by adding 2% aMG to a culture growing in the presence of 0.2% glucose. Both in the upshift and in the downshift there was an immediate change from the preshift to the postshift growth rate (Fig. 1). Plasmid DNA and protein were measured in the shift experiments described. The total amount of plasmid DNA in the population (expressed as relative units of DNA per milliliter of culture) has been plotted against the total amount of cell protein (micrograms per milliliter of culture) in the differential plot shown in Fig. 2. The results evidently show that the specific rate of plasmid replication immediately shifted to the value characteristic of the postshift conditions. To increase the resolution of the experiment at short times after the shifts, plasmid DNA was pulse-labeled with [3H]thymidine during short intervals after the shift. The result is presented by the histograms in Fig. 2, whichshow that there was no burst or shutoff of plasmid replication in the downshifts or upshifts, respectively. The plasmid continued to replicate with virtually the same initiation frequency (initiations per minute per mass unit) before, during, and after the shifts. The results described above were replotted to show the change in plasmid DNA to chromosomal DNA during one generation time after the shifts (Fig. 2, kinetic plot). There was only a slow adjustment of the plasmid concentration to the value characteristic of the postshift conditions. Copy number shifts with the temperature-dependent copy mutant pKN301. The copy mutant pKN301 shows a temperature-dependent change in copy number compared with the wild-type plasmid (11). The copy number shifts from the wild-type level to a four to five times higher level when the temperature is changed from 30 to 400C. Due to the increase in the amount of wild-type plasmid DNA per chromosomal DNA when decreasing the tempera-

J. BACTERIOL.

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FIG. 1. Growth of strain EC1005 carryingplasmid Rldrd-19 in minimal medium M9. (A) The bacteria were grown in the presence of 0.2% (wt/vol) glucose and 2.0% (wt/vol) aMG. At zero time the culture was supplemented with 1.8% (wt/vol) glucose. (B) The bacteria were grown in the presence of 0.2% (wt/vol) glucose. At zero time 2.0% (wt/vol) aMG was added.

ture, the copy number shift expressed as plasmid DNA per chromosomal DNA is only about

threefold. Populations of bacteria carrying plasmid pKN301 were grown exponentially at 30 and 400C and were shifted to 40 and 300C, respectively. Plasmid DNA and protein were measured during the shifts. The total amount of plasmid DNA in the population (expressed as relative units of DNA per milliliter of culture) has been plotted against the total amount of cell protein (micrograms per milliliter) in the differential plot shown in Fig. 3. Directly after the shifts, there was a change of the specific rate of plasmid replication from the preshift value to that characteristic ofthe postshift conditions. The specific rate of plasmid replication then remained constant, at least during the two doublings in protein during which plasmid DNA was measured. Also in these shifts, there was neither a burst of intensive plasmid replication nor a complete inhibition of plasmid synthesis. This is also evident from the pulse experiments also shown in Fig. 3 (kinetic plot). This means that in both temperature shifts, there was an asymptotic adjustment of the copy number.

COPY NUMBER SHIFTS WITH PLASMID Rl

VOL. 141, 1980

Kinetic plot

Differential plot

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FIG. 2. Plasmid replication in shifts between two copy number levels. E. coli strain EC10K)5 carrying was grown as described in the text and in Fig. 1. Symbols: 0, metabolic upshift; 0, metabolic downshift. The differential plot shows the total concentration ofplasmid DNA (relative units per milliliter) in the culture as a function of the total concentration of protein in the culture. The kinetic plot contains histograms of the results of the pulse experiments and curves for the ratio of covalently closed circular (CCC) DNA to chromosomal (chrom) DNA. The rate of plasmid synthesis is expressed as dRl/dt, where dRI is the amount ofplasmid synthesized during tine dt. Kinetic plot Differential plot

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FIG. 3. Replication of the temperature-dependent copy mutant plasmid pKN301 in shifts between two temperatures. Symbols: 0, 40 -* 30°C; 0, 30 -* 40°C. The experiment was performed as described in the text. The differentialplot shows the total concentration ofplasmid DNA (relative units per milliliter) in the culture as a function of the total concentration of protein in the culture. The kinetic plot contains histograms of the results of the pulse experiments and curves for the ratio of covalently closed circular (CCC) DNA to chromosomal (chrom) DNA. The rate ofplasmid synthesis is expressed as dRl/dt, where dRI is the amount ofplasmid synthesized during time dt.

DISCUSSION The results presented above argue against the idea that plasmid Rl replication is controlled by direct titration of the concentration of Rl origins (7, 27, 29). The data rather suggest that plasmid replication is controlled by a system that determines the frequency of replication without measuring the actual copy number (lOa). Similar results have been obtained for the related plas-

mid NR1 in Proteus mirabilis in shifts from stationary cultures (where the copy number is elevated compared with the copy number in a

logarithmically growing culture) to exponential growth (13). The control genes (at least some of them) are present on the plasmid itself (20, 21, 23, 24), and, according to the data presented above, the replication frequency is the same irrespective of the actual copy number. This means that there is no

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gene dosage effect monitored by the control system. The gene dose independence means that in a simple positive control system, activator cannot be formed constitutively from each activator gene, and in a simple negative control system, repressor cannot be formed constitutively from each repressor gene. There is genetic evidence that suggests that plasmid Rl replication is under negative control by an Rl-mediated protein (11). Several models involving repressors have been proposed in the literature to explain control of DNA replication. The main models seem to be the autorepressor model proposed by Sompayrac and Maal0e (31), the autorepressor system of plasmid Xdv (3, 17, 18), and the Pritchard inhibitor dilution model (28, 29). All these models predict gene dose independence of the type observed in Fig. 2 and 3.

von Meyenburg. 1975. Simple downshift and resulting lack of correlation between ppGpp pool size and ribonucleic acid accumulation. J. Bacteriol. 122:585-591. 13. Kasamatsu, H., and R. Rownd. 1970. Replication of Rfactors in Proteus mirabiis. replication under relaxed

control. J. Mol. Biol. 51:473-489. 14. Kesler, D. P., and H. V. Rickenberg. 1963. The competitive inhibition of a-methylglucoside uptake in Esch-

15. 16.

17. 18.

19. 20.

ACKNOWLEDGMENT This work was supported by the Danish Medical Research Council (projects 8000 and 10149). LITERATURE CMD 1. Adams, ML H. 1959. Bacteriophages, p. 446. Interscience Publisers, Inc., New York. 2. Arai, T., and R. Clowes. 1975. Replication of stringent and relaxed plasmids, p. 141-165. In D. Schlessinger (ed.), Microbiology-1974. American Society for Micro. biology, Washington D.C. 3. Berg, D. E. 1974. Genes of phage A essential for Adv plasnda. Virology 62:224-233. 4. Clowes, R. C. 1972. Molecular structure of bacterial plasmida Bacteriol. Rev. 36:361-406. 5. Collins, J., and R. H. Pritchard. 1973. Relationship between chromosome replication and F'lac episome replication in Escherichia coli. J. Mol. Biol. 78:143155. 6. Cooper, S., and C. E. Helmstetter. 1968. Chromosome replication and the division cycle of Escherichia coli B/r. J. Mol. Biol. 31:519-540. 7. Donachie, W. D. 1968. Relationship between cell size and time of initiation of DNA replication. Nature (London) 219:1077-1079. 8. Engberg, B., and K. Nordstr6m. 1975. Replication of the R-factor Rl in Escherichia coli K-12 at different growth rates. J. Bacteriol. 123:179-186. 9. Falkow, S. 1975. Infectious multiple drug resistance. Pion Ltd., London. 10. Grinsted, J., J. R. Saunders, L C. Ingram, R. B. Sykes, and ML H. Richmond. 1972. Properties of an R factor which originated in Pseudomonas aeruginosa 1822. J. Bacteriol. 110:529-537. lOa.Gustafeson, P., H. Dreisig, S. Molin, K. Nordstrom, and B. E. Uhlin. 1979. DNA replication control: studies of plasmid Rl. Cold Spring Harbor Symp. Quant. Biol. 43:419-425. 11. Gustafsson, P., and K. Nordstrom. 1978. Temperaturedependent and amber copy mutants of plasmid Rldrd19 in Escherichia coli. Plasmid 1:134-144. 12. Hansen, M. T., M. L Pato, S. Molin, N. P. FilB, and K.

21.

22. 23.

24.

25.

26. 27. 28.

29. 30.

31.

erichia coli. Biochem. Biophys. Res. Commun. 10:482487. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis, p. 97-124. Benjamin Co., Inc., New York. Matsubara, K. 1976. Genetic structure and regulation of a replicon of plasmid Adv. J. Mol. Biol. 102:427-439. Matsubara, K., and Y. Takeda. 1975. Role of the tof gene in the production and perpetuation of the Xdv plasmid. Mol. Gen. Genet. 142:225-230. Meynell, E., and N. Datta. 1967. Mutant drug-reitant factors of high isbility. Nature (London) 214: 8854887. Molin, S., and K. Nordstr6m. 1979. Control of plasmid RI replication: functions involved in replication, copy number control, and switch-off of replication. J. Bacteriol. 141:111-120. MolHn, S., P. Stougaard, B. E. Ublin, P. Gustafsson, and K. Nordstr6m 1979. Clustering of genes involved in replication, incompatibility, and stable maintenance of the resistance plasmid Rldrd-19. J. Bacteriol. 138: 70-79. Neidhardt, F. C., P. L Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119: 736-747. Nordstr6m, K., B. Engberg, P. Gustson, S. Moln, and B. E. Uhlin. 1977. Copy mutants of the plasmid RI as a tool in studies of control of plasmid replication, p. 299-332. In J. Drews and G. Hogenauer (eds.), Topics in infectious diseases, vol. 2. R-factors: their properties and posible control. Springer Verlag, Vienna. Nordstr6m, K., L. C. Ingram, and A. Lundback. 1972. Mutations in R factors in Escherichia coli causing an increased number of R-factor copies per chromosome. J. Bacteriol. 110:562-69. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtis. H, N. Datta, and S. Falkow. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. Prentki, P., M. Chandler, and L Caro. 1977. Replication of the prophage P1 during the cell cycle of Escherichia coli. Mol. Gen. Genet. 162:71-76. Pritchard, R. H. 1968. Control of DNA synthesis in bacteria. Heredity 23:472. Pritchard, R. H 1969. Control of replication of genetic material in bacteria, p. 65-74. In G. E. W. Wolstenholme and M. O'Connor (ed.), Bacterial episomes and plas. mids. A Ciba Foundation Symposium. J. & A. Churchill Ltd., London. Pritchard, R ILH. 1974. On the growth and form of a bacterial cell. Philos. Trans. R. Soc. London 262:303336. Pritchard, RI H., P. T. Barth, and J. Collins. 1969. Control of DNA synthesis in bacteria. Symp. Soc. Gen. Microbiol. 19:263-297. Sompayrac, L., and 0. Maale. 1973. Autorepressor model for control of DNA replication. Nature (London) New Biol. 241:133-135.