INTRODUCTION

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The studies on the bacteriophage  DNA early (, or circle-to-circle) replication in Escherichia coli have been greatly stimulated by the availability of the phage -derived plasmids, called originally dv (for a review, see reference 17). The  plasmids may be produced from  phage DNA by cutting out and circularization of the  replication region o_Rp_R-cro-cII-O-P. The origin of replication, ori, is situated in the middle of the O gene, and the p_R promoter-initiated transcription plays a dual role: it is required for the synthesis of  replication proteins, O and P, as well as for the transcriptional activation of ori (for reviews, see references 14 and 30). The binding of Cro repressor to the o_R operator represents an autoregulatory loop, which is assumed to play an important role in the control of  plasmid replication (17). The cII protein, so important for the lysis-or-lysogeny decision in the early development of phage , is dispensable for  plasmid replication. The  O protein, the initiator of  DNA replication, is rapidly degraded in vivo by the E. coli ClpP/ClpX protease (3, 8, 46), but it becomes protected from proteolysis in the pathway of the  replication complex (RC) assembly (21, 34, 42). This event occurs after interaction of the P-DnaB helicase complex with the O initiator bound to ori at the step of the O-P-DnaB preprimosome formation (34). The next step, the release of DnaB helicase from P-mediated inhibition, is performed by DnaK, DnaJ, and GrpE proteins, the molecular chaperones of the Hsp70 system (2, 6, 48). The action of DnaB helicase loaded between transiently separated (due to transcriptional activation of ori) complementary strands of  DNA permits the binding of at least DnaG primase and the DNA polymerase III holoenzyme, leading to the assembly of a functional  RC (1, 32, 43-45, 48).

The studies on the in vitro-reconstituted  plasmid DNA replication led to an attractive model suggesting that the  RC functional in elongation resembles that of its host, E. coli: both complexes would be devoid of the respective initiator proteins, O or DnaA, and the respective DnaB helicase inhibitors, P or DnaC. Moreover, it is tacitly assumed that in both systems,  and the E. coli chromosome, the RC would be completely disassembled after one round of replication. By implication, the next round of replication should depend on the binding of the initiator, O or DnaA, to ori or oriC, respectively (14). However, the present in vitro system of  plasmid DNA replication may not exactly reflect the conditions occurring in the cell and is not suitable for studying more than one replication round.

We developed two systems for blocking the de novo assembly of the  RC. In amino acid-starved E. coli relA cells, there was no synthesis of the rapidly destroyed O initiator (33, 35, 36) and in E. coli wild-type (wt) cells growing in a complete medium, the Pts1 mutation (in the presence of the dnaA⁺ allele) blocked RC assembly at 43°C (33, 36, 44). Density shift and other experiments demonstrated that in both systems  plasmid DNA replication does occur and occurs linearly: only one of two plasmid daughter copies was able to initiate the next round of replication (28, 33, 36, 40). This replication could occur due to RCs assembled before the onset of amino acid starvation or temperature upshift, respectively. It was dependent on O, as well as on DnaK, DnaJ, and GrpE chaperone functions (33, 35, 36, 40, 41). We concluded that the assembled RC, containing O and probably also P, is not disassembled after termination of a round of replication (30, 40). This multiprotein complex (or at least the part that protects O from proteolysis) is inherited by one of two  plasmid daughter copies at each round of replication (29, 36). In amino acid-starved E. coli relA cells, the "old" RC-driven replication ceases after several rounds (39), but in E. coli wt cells growing in a complete medium, this replication does not seem to be time restricted (36). The above studies, together with the observation that neither the absence of nor the presence of excess O-digesting ClpP/ClpX protease affects  plasmid or phage replication (27), rule out the model of O binding to ori as the crucial event in the control of initiation frequency.

In amino acid-starved E. coli relA cells, the Cro-mediated regulation did not work, probably due to titration out of Cro by the growing number of o_R operator sequences (40, 41). In E. coli wt cells growing in complete medium, the replication of crotsPts1 plasmid after a temperature upshift was studied; hence, Cro regulation also did not work in this system (36). However, when the same procedure was applied to E. coli wt harboring cro⁺Pts1, plasmid replication was blocked and RC disassembly (judged from proteolysis of the otherwise stable O) was observed (36). The disassembly of RC also occurred for E. coli wt harboring cro⁺P⁺. Examination of this phenomenon revealed that for RC disassembly observed in cro⁺ plasmid-harboring wt cells, heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system, is indispensable (37). This was the first observation that the Hsp60 chaperone proteins, known to mediate deaggregation of denatured protein aggregates, may also disassemble in vivo a highly organized protein structure (dispensable for cell survival) under stress caused by temperature upshift.

Here we present our study on the disassembly of RC, which emphasizes the role of DNA gyrase-mediated negative resupercoiling of  plasmid DNA, which counteracts DNA relaxation that occurs immediately after the temperature upshift (20). It seems that the negative resupercoiling of  plasmid DNA results in dissociation of RC that now becomes disassembled with the help of GroEL and GroES chaperone proteins. This finding allowed us to extend our studies on the crots plasmid and to formulate a hypothesis concerning heat shock resistance of RC when Cro repressor function is absent.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, phages, and gene fusions. Most of the experiments (all except the gene fusion experiments) were performed in an E. coli MG1655 genetic background (12). Particular mutations were transferred by P1 transduction by the method of Silhavy et al. (25). The mutant strains used were BM270 (groEL44 linked to Tn10), obtained from C. Georgopoulos and M. Zylicz (see also reference 37); HI515 (nalA26), described by Ikeda et al. (11) and provided by K. Sekimizu and Y. Ogata; and CAG354 [rpoD800(Ts) linked to Tn10], described by Liebke et al. (16) and obtained from C. Gross. To achieve expression of the groE operon from plasmid pGELS2 (see below), it was necessary to remove a tetA gene activity from a tetracycline-resistant strain (like the groEL44 mutant). This was performed by the method of Bochner et al. (5). Plasmid pGELS1 was constructed by cloning the groE operon from pOF39 (7) into the pCattTrE18 vector (constructed in the laboratory of W. Szybalski [University of Wisconsin] by M. Koob). Plasmid pGELS2 is a derivative of pGELS1 lacking the groE promoter region; thus, transcription of groEL and groES genes is exclusively dependent on the p_tetA promoter activity. Details of the construction of pGELS1 and pGELS2 are provided in Fig. 1. Plasmids derived from bacteriophage , pKB2 (wt), and pRLM4 (as pKB2 but crots) were described by Kur et al. (15) and Wold et al. (47), respectively. Experiments with gene fusions were performed with a lac strain (WAM106) described by Thomas and Glass (31). The single-copy p_L-lacZ fusion carried on cryptic cI857 prophage was provided by D. Court. The single-copy fusion of the ³²-dependent groE promoter with lacZ was described by Benvenisti et al. (4) and obtained from A. B. Oppenheim. Single-stranded DNA of phage M13mp18I (29) was used as a template for preparation of the labeled probe used in Northern blotting experiments. Phage clb2 (from our collection) was also used.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Construction of pGELS1 and pGELS2.

DNA techniques. All DNA manipulations (molecular cloning and preparation of labeled probes for Northern blotting) were carried by the methods of Sambrook et al. (24).

O protein decay. Decay of the O protein was measured as described previously (34, 37, 42). Briefly, bacteria harboring a  plasmid were pulse-labeled with [³⁵S]methionine and the label was chased by addition of excess of unlabeled L-methionine (time zero in the figures). The samples were withdrawn at the indicated times, the bacteria were lysed, and the cellular lysates were immunoprecipitated with anti-O serum. Then the isotope-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% polyacrylamide) and visualized by autoradiography. The bands of the O protein remaining after the different chase times were quantitated by densitometry.

Changes in plasmid DNA linking number. Changes in  plasmid DNA linking number after a temperature shift were monitored by the method of Ogata et al. (20). Briefly, samples of the bacterial culture were withdrawn at the indicated times and chilled immediately by being transferred into probes containing equal volume of ice. Plasmid DNA was isolated by the alkali lysis method (24) and separated by agarose gel electrophoresis (1% agarose) in TBE buffer (24) containing 15 µg of chloroquine per ml (electrophoresis was performed at 2 V per cm of gel length). The changes in the average linking number of plasmid DNA () were calculated by the method of Keller (13).

Analysis of the level of p_R-t_R2 transcripts. Estimation of the amounts of the p_R-t_R2 transcripts relative to the level of rRNA (internal RNA control) and to the level of  plasmid (template DNA) was performed as described previously (19, 29). Briefly, samples containing the same bacterial mass (estimated by measurement of the optical density at 575 nm of the culture) were withdrawn at the indicated times. Each sample was divided in two parts. RNA was isolated from the first part (see reference 29 for a detailed description of the procedure), and plasmid DNA was isolated by the alkali lysis method (24) from the second part. The relative level of rRNA was estimated densitometrically on the basis of analysis of ethidium bromide-stained agarose gels. The p_R-t_R2 transcripts were detected by Northern blotting and quantitated by densitometry (the template for labeling of the probe designed to detect these transcripts was single-stranded DNA isolated from phage M13mp18I). The amount of  plasmid DNA was estimated on the basis of densitometric analysis of DNA bands after agarose gel electrophoresis and staining with ethidium bromide, by the method of Herman et al. (9). The relative values presented in the figure were calculated by the method of Obuchowski et al. (19) by dividing the relative intensity of bands on a particular blot by the relative intensity of ethidium bromide-stained rRNA bands and then by dividing by the relative amount of  plasmid. A value of 1 corresponds to the relative value calculated for the sample taken from the culture of bacteria harboring wild-type  plasmid (pKB2) at 30°C (time zero), and the other values are expressed relative to this value.

Estimation of the relative amount of  plasmid. The relative amount of  plasmid DNA per bacterial mass was estimated as described by Herman et al. (9) (see above). A value of 1 corresponds to the relative value calculated for the sample taken from the culture of bacteria harboring wild-type  plasmid (pKB2) at 30°C (time zero).

Measurement of -galactosidase activity. The activity of -galactosidase was measured by the method of Miller (18).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Fine-tuned expression of the groE operon. We assumed that RC disassembly may be due not only to an increased cellular concentration of GroEL/S chaperone proteins but also to some other concomitant process(es) (to be discovered), both induced by heat shock. To verify this hypothesis, it was important to raise the cellular concentration of GroEL/S proteins while keeping the temperature constant. This aim has been achieved by construction of a plasmid, pGELS2 (Fig. 1), containing the groE operon under the exclusive control of the promoter p_tetA, responsible for the expression of tetracycline resistance in the Tn10 transpozon. The pGELS2 plasmid contains the tetR gene coding for the tetracycline repressor that blocks the p_tetA-initiated transcription in the absence of the antibiotic. Autoclaved chlortetracycline, aCT, which has lost its antibiotic functions, inactivates TetR repressor (5, 23, 26) causing transcription of the groE operon and efficient synthesis of GroEL and GroES proteins (Fig. 2). The effect of increasing concentration of aCT on the level of GroEL/S in the pGELS2-harboring E. coli groEL44 cells was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its densitometric evaluation led to the conclusion that the level of GroEL at an aCT concentration of 0.05 µg/ml was equivalent to its level in plasmidless cells under the conditions used in our experiments before the temperature shift from 30 to 43°C; aCT at 2.0 µg/ml resulted in a three- to fourfold-higher level of GroEL (data not shown). The aCT-mediated derepression of the groE operon resulted in the production of functional GroEL/S chaperone proteins, as revealed by their activity in phage  development (Table 1). It is worth noting that these proteins have been discovered due to their involvement in phage  morphogenesis (for a review, see reference 22). At concentrations as low as 0.01 µg/ml, aCT caused derepression of the p_tetA promoter, as revealed by the expression of the groE operon from pGELS2 (Table 1); at high aCT concentrations (e.g., 25 µg/ml), the system seems to be useful for the production of a high concentration of the desired protein in bacteria (Fig. 2, lane 9).

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Overproduction of GroEL and GroES proteins from plasmids pOF39, pGELS1, and pGELS2. Bacteria were grown in Luria-Bertani medium at 30°C. When indicated, the culture was transferred to 43°C or treated with aCT (final concentration, 25 µg/ml) and incubated further for 60 min. Cell lysates were separated on 12.5% polyacrylamide gels, and protein bands were visualized by staining with Coomassie brilliant blue. Lanes: M, molecular mass standards (from top to bottom, 94, 67, 43, 30, 20.1, and 14.4 kDa), 1, MG1655; 2, MG1655/pOF39; 3, MG1655/pOF39 after a shift to 43°C; 4, MG1655/pCattTrE18; 5, MG1655/pCattTrE18 treated with aCT; 6, MG1655/pGELS1; 7, MG1655/pGELS1 treated with aCT; 8, MG1655/pGELS2; 9, MG1655/pGELS2 treated with aCT. The positions of GroEL and GroES are indicated.

View this table: [in this window] [in a new window]   TABLE 1.   Suppression of the effect of the groEL44 mutation on phage  growth at 30°C and bacterial growth at 43°C by expression of the groE operon from plasmid pGELS2

Role of an increased GroEL/S chaperonin concentration. It was then possible to perform heat shock experiments with  plasmid-harboring and pGELS2 plasmid-harboring groEL44 cells that could produce a functional GroEL protein exclusively due to aCT-mediated derepression of the groE operon of pGELS2. The aCT was added at the time of the temperature upshift. In the pulse-chase experiments in Fig. 3 (also see Fig. 4, 5, 7, and 9), the decay of O protein occurring immediately after the start of isotope chasing at 30°C (time zero) represents ClpP/ClpX-mediated proteolysis of an excess of synthesized free O (27, 42). The stable fraction of O corresponds to O protected from this protease by other components of the ori-bound O-P-DnaB preprimosome or RC (34, 37). There was no RC disassembly in the absence of aCT (Fig. 3A) or at an aCT concentration of 0.05 µg/ml (Fig. 3B), which should result in a level of functional GroEL equivalent to that present in wt cells before the heat shock (see above). However, when aCT was added to 2.0 µg/ml (see above), RC disassembly was observed (Fig. 3C). The same result was obtained at an aCT concentration of 25 µg/ml (Fig 3D). The above results support our previous finding that one of the requirements of heat shock-provoked RC disassembly is the induction of the groE operon (37). We were then ready to ask whether an increase of the concentration of GroEL/S chaperone proteins alone is sufficient to cause RC disassembly. As shown in Fig. 4, the answer was negative.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Decay of the O protein at 30°C (triangles) and after a shift to 43°C (circles) in the groE44 mutant harboring cro+ (pKB2) and pGELS2 plasmids, untreated (A) or treated with aCT at 0.05 µg/ml (B), 2 µg/ml (C), or 25 µg/ml (D) at the time of the temperature shift (arrow).

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Decay of the O protein at 30°C in the groE44 mutant harboring cro+ (pKB2) and pGELS2 plasmids, untreated (triangles) or treated with aCT at 25 µg/ml (circles) at the time indicated by the arrow.

Role of DNA gyrase-mediated negative supercoiling. What else, besides induction of the groE operon, is changing after heat shock that is required for  plasmid RC disassembly? We turned our attention to changes in supercoiling of plasmid DNA, known to occur after a temperature shift from 30 to 50°C (20), and started to study the effect of DNA gyrase inhibitors on RC disassembly under our standard experimental conditions. The disassembly of RC (Fig. 5A) was blocked by each of two DNA gyrase inhibitors, nalidixic acid (Fig. 5B) and coumermycin (Fig. 5C). The specificity of action of nalidixic acid was checked in an experiment performed with a nalidixic acid-resistant mutant, nalA26 (Fig. 5D). Gyrase may cause DNA relaxation or negative supercoiling; both processes are inhibited by nalidixic acid, in contrast to coumermycin, which specifically inhibits the ATP-dependent negative supercoiling of DNA (for a review, see reference 14).

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Decay of the O protein at 30°C (triangles) and after a shift to 43°C (circles) in the wild-type strain (A to C) and the nalA26 mutant (D) harboring cro+ (pKB2) plasmid, untreated (A), or treated with 100 µg of nalidixic acid per ml (B and D) or 50 µg of coumermycin per ml (C) at the time of the temperature shift (arrow).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Changes in the average linking number () of cro+ (pKB2) plasmid DNA in the wild-type strain at 30°C (triangles) and after a shift to 43°C (squares). The values are estimated relative to the linking number observed for the pKB2 plasmid at time zero in bacteria growing at 30°C. The temperature shift is indicated by an arrow.

crots plasmid: heat shock resistance of RC requires p_R-initiated transcription. The results presented in previous sections with bacteria harboring the cro⁺ plasmid led us to propose a model assuming that DNA gyrase-mediated  DNA negative resupercoiling (occurring after initial plasmid relaxation caused by a temperature upshift) results in dissociation of RC from  DNA. We suggest that RC disassembly helped by GroEL/S chaperone proteins then occurs and that O is no longer protected and becomes hydrolyzed by ClpP/ClpX protease. It was now possible to ask why the temperature upshift does not cause RC disassembly in crots plasmid-harboring cells. The Cro repressor blocks the p_R-initiated transcription required for initiation of  plasmid replication as well as for Cro synthesis (30). According to the generally accepted model (17), the transient derepression event that should occur due to dilution of Cro in the growing cell volume should produce a wave of transcription leading to a round of replication. Synthesis of Cro would restore the initial, silent state of  plasmid. In contrast to the cro⁺-harboring cells, the p_R-initiated transcription should be constantly derepressed in crots plasmid-harboring bacteria at 43°C. Therefore, an obvious question was whether unhindered transcription from p_R is indeed required for RC stability.

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Decay of the O protein at 30°C (triangles) and after a shift to 43°C (circles) in the wild-type strain harboring crots (pRLM4) plasmid, untreated (A) or treated with 25 µg of rifampin per ml (B) or 25 µg of rifampin per ml and 50 µg of coumermycin per ml (C) at the time of the temperature shift (arrow).

View larger version (19K): [in this window] [in a new window]   FIG. 8.   Relative level of the GroEL protein in the wt strain after a shift from 30 to 43°C (arrow). Bacteria were untreated (triangles) or treated with 25 µg of rifampin per ml (squares) at the time of the temperature shift.

View larger version (21K): [in this window] [in a new window]   FIG. 9.   Decay of the O protein at 30°C (triangles) and after a shift to 43°C (circles) in the rpoD800(Ts) mutant harboring cro+ (pKB2) (A) or crots (pRLM4) (B). The temperature shift is indicated by the arrows.

crots plasmid: kinetics of transcription and replication. In crots plasmid-harboring cells, the plasmid was stably maintained after the temperature upshift (36) and the release from Cro repression did not lead to uncontrolled  DNA replication, in contrast to the situation that would be expected on the basis of previously proposed model (17), in which the Cro repressor autoregulatory loop plays a crucial role in the regulation of replication and maintenance of  plasmids in E. coli (see below). Therefore, transcription of the O-P region, leading to the synthesis of  replication proteins and to activation of ori situated in the middle of the O gene, should be controlled by a Cro-independent system. It was interesting to check how rapidly this control is established after the temperature upshift. A convenient measure of the transcription of the O-P region is the level of the p_R-t_R2 transcript; the t_R2 terminator is situated downstream of the P gene. The level of the p_R-t_R2 transcripts, calculated per plasmid copy, increased up to 10 min after the temperature upshift (Fig. 10). Since this level results from the synthesis and decay of the transcript, one may assume that its synthesis attained its peak even earlier. The rapid decrease in the level of the p_R-t_R2 transcripts and its stabilization observed later (Fig. 10) reveal an important element of a Cro-independent control system.

View larger version (20K): [in this window] [in a new window]   FIG. 10.   Relative level of the pR-tR2 transcripts normalized to the level of rRNA (internal RNA control) and to the amount of plasmid (i.e., template) DNA in the wt strain harboring cro+ (pKB2) (triangles) or crots (pRLM4) (squares) plasmid after a shift from 30 to 43°C (arrow).

View larger version (19K): [in this window] [in a new window]   FIG. 11.   Relative level of plasmid DNA per bacterial mass in the wt strain harboring cro+ (pKB2) (triangles) or crots (pRLM4) (squares) plasmid after a shift from 30 to 43°C (arrow).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The results presented for the cro⁺ plasmid demonstrate that heat shock-provoked disassembly of the O initiator-containing structure, RC, requires both DNA gyrase-mediated negative supercoiling of plasmid DNA and induction of the groE operon. We have no data concerning the temporal sequence of action of DNA gyrase and GroEL/S chaperone proteins. However, the most probable scenario is that at first the RC dissociates from  DNA due to gyrase-mediated negative resupercoiling, which occurs after plasmid relaxation caused by a temperature upshift (Fig. 12A). Then disassembly of the free RC, helped by the GroEL/S proteins, may occur, exposing O to the action of ClpP/ClpX protease. We assume that, this chain of events cannot start earlier than 10 min after the temperature shift from 30 to 43°C, since the negative resupercoiling of the heat shock-relaxed plasmid DNA begins at this time (Fig. 6).

View larger version (23K): [in this window] [in a new window]   FIG. 12.   Proposed mechanism of heat shock-provoked disassembly of the  replication complex in bacteria harboring cro+ plasmid (A) and of heat shock resistance of the  replication complex in bacteria harboring crots plasmid (B). In the case of a cro-null plasmid, the  replication complex should also interact with other DNA-bound proteins before temperature upshift. See the text for details.

An obvious and unanswered question is how the GroE chaperone system distinguishes between the DNA-bound and free states of RC, acting exclusively in the latter case on this viral protein structure. The exact composition of the structure which protects the O protein from proteolysis, referred to as RC, is not yet known, but it was demonstrated that this structure must contain at least O, P, and DnaB proteins (34). Therefore, perhaps the crucial role in the process of distinguishing among the different states of RC by the GroE system is played by the surface of RC formed by sequential binding of RC components (O, P, DnaB, etc.) to ori and their mutual interactions, which change due to the action of DnaK, DnaJ, and GrpE chaperone proteins. After dissociation of RC from  DNA, this part of the RC surface may appear conformationally unnatural (possibly with exposed hydrophobic groups) and become prone to GroEL/GroES attack.

The knowledge accumulated in the study of heat shock-provoked disassembly of RC in cro⁺ plasmid-harboring cells allowed us to look for the cause of the heat shock resistance of RC in the case of the crots plasmid (36) (see above). This feature appears to be characteristic of cro plasmids as supported by an experiment with a recently constructed cro-null plasmid (10). In this case, the p_R-initiated transcription, unhindered by Cro repression, could occur all the time, i.e., before and after the temperature upshift. We found that the RC is heat shock resistant in cro-null plasmid-harboring bacteria (38). We postulate that dissociation of RC from  DNA during the negative resupercoiling does not occur, because RC is more tightly bound to DNA than in the case of the cro⁺ plasmid, possibly due to interaction with other DNA-bound proteins (Fig. 12B). One of the potential candidates for such proteins may be the host DnaA protein. This protein stimulates the activity of the p_R promoter (43). Several putative DnaA-binding sequences were found in the  replication region, and one of them is located near p_R (30). Therefore, one may assume a competition between Cro and DnaA in binding to the region of this promoter. If DnaA is able to interact with components of  RC, it might stabilize this structure more efficiently in the absence of Cro function. An alternative hypothesis is that the heat shock resistance of RC under cro mutant conditions (when p_R-initiated transcription is more efficient due to the lack of the Cro repressor) is based on the assumption of positive supercoiling in front of RNA polymerase, which might counteract the DNA gyrase-mediated negative resupercoiling. However, the observations that in crots plasmid-harboring cells the very intensive p_R-initiated transcription and  DNA replication caused by the temperature upshift terminates before the period of DNA gyrase-mediated negative resupercoiling (compare Figs. 6, 10, and 11) do not support this hypothesis. On the other hand, they suggest that the heat shock resistance of RC and the replication control specific for crots plasmid at 43°C are established within 10 min after the temperature upshift and before the process of negative resupercoiling of  plasmid DNA.

The construction of the cro-null plasmid, which is stably maintained in E. coli (10), and our observation that the copy number of the crots plasmid increases only a few times at 43°C relative to 30°C are in contrast to previously reported findings that Cro function is a key element in the regulation of  plasmid replication since it is necessary for the stable maintenance of such a plasmid (17). However, the older data came from experiments with  plasmids produced in vivo, called dv, which usually contained relatively large fragments of phage  genome, including the cI gene (coding for a strong repressor of p_R). Currently used  plasmids (including those used in this work) were constructed in vitro and do not contain this gene. These differences between the plasmids used previously and in this work may be responsible for the differences between the results obtained previously (17) and those presented in this paper.

As mentioned above, in crots plasmid-harboring cells, the heat shock resistance of RC should be established before the period of DNA gyrase-mediated negative supercoiling of plasmid DNA, which begins 10 min after the temperature shift from 30 to 43°C. In the same 10-min period, a Cro-independent control of  plasmid replication is established. If the Cro-mediated repression of the p_R promoter, and thus the Cro autoregulatory loop, were the crucial process in the control of  plasmid replication, as suggested previously (17), one should expect a rapid and continuous increase in the crots plasmid copy number after a temperature upshift. However, the release of the p_R promoter from Cro repression does not result in a runaway plasmid replication. On the contrary, after rapid doubling in copy number, the plasmid DNA replication proceeds in step with the growth of host cells (Fig. 11) providing a new argument in favor of the existence of a Cro-independent control of replication. We assume that the wave of the p_R-initiated and t_R2-terminated transcription occurring shortly after inactivation of the cro gene product, which activates ori, is responsible for the rapid doubling of the plasmid copy number (Fig. 10 and 11). However, this transcription soon falls off and proceeds further at a constant level (Fig. 10). This level may reflect the transcriptional activation of ori that should occur to ensure replication in the Cro-independent system of replication control, as described previously (36). The low level of the p_R-t_R2 transcripts in the absence of the functional Cro repressor may mean that the p_R-initiated transcription is inhibited, prematurely terminated, or paused on the way to ori and finally to t_R2. Since there is no reason to suspect that activation of the p_R promoter by DnaA protein is abolished under these conditions, the pausing of RNA polymerase seems to serve as the best working hypothesis concerning the model of the Cro-independent control. It is tempting to speculate that, the paused RNA polymerase and other DNA-bound proteins (for example DnaA, as discussed above) can interact with the ori-bound RC, forming a complex which may sequester ori during most of the cell cycle and can be responsible for the block of reinitiation of replication which is characteristic for cro mutant plasmids (36, 38). Although the molecular mechanism of the Cro-independent control awaits elucidation, the data presented here suggest that strong binding of RC to  DNA (proposed in this work on the basis of the heat shock resistance of RC) is one of the elements of this control.