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In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.

The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication., (Copyright 1997 Academic Press Limited.)

Replication of mini-F plasmids requires the initiator protein RepE, which binds specifically to four iterons within the origin (ori2), as well as some host factors that are involved in chromosomal DNA replication. To understand the role of host factors and RepE in the early steps of mini-F DNA replication, we examined the effects of RepE and the Escherichia coli proteins DnaA and HU on the localized melting of ori2 DNA in a purified in vitro system. We found that the binding of RepE to an iteron causes a 50 degrees bend at or around the site of binding. RepE and HU exhibited synergistic effects on the localized melting within the ori2 region, as detected by sensitivity to the single-strand specific P1 endonuclease. This opening of duplex DNA occurred around the 13mer of ori2, whose sequence closely resembles the set of 13mers found in the chromosomal origin oriC. Further addition of DnaA to the reaction mixture increased the efficiency of melting and appeared to extend melting to the adjacent AT-rich region. Moreover, DNA melting with appreciably higher efficiencies was observed with mutant forms of RepE that were previously shown to be hyperactive both in DNA binding in vitro and in initiator activity in vivo. We propose that the binding of RepE to four iterons of ori2 causes bending at the sites of RepE binding and, with the assistance of HU, induces a localized melting in the 13mer region. The addition of DnaA extends melting to the AT-rich region, which could then serve as the entry site for the DnaB-DnaA complex, much as has been documented for oriC dependent replication.

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.

A subset of Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, is required for mini-F plasmid replication, presumably at the step of functioning of the RepE initiator protein. We have isolated and characterized mini-F plasmid mutants that acquired the ability to replicate in the Escherichia coli dnaJ259. The mutant plasmids were found to replicate in any of dnaJ, dnaK, and grpE mutant hosts tested. In each case, the majority of the mutant plasmids carried a unique amino acid alteration in a localized region of repE coding sequence and showed an increased copy number, whereas the minority contained a common single base change (C to T) in the promoter/operator region and produced an increased amount of RepE. All RepE proteins with altered residues (between 92 and 134) exhibited increased initiator activities (hyperactive), and many showed reduced repressor activities as well, indicating that this region is important for the both major functions of RepE protein. These results together with evidence reported elsewhere indicate that the subset of heat shock proteins serves to activate RepE protein prior to or during its binding to the replication origin and that the mutant RepE proteins are active even in their absence. We also found that a C-terminal lesion (repE602) reduces the initiator activity particularly of some hyperactive mutant RepE proteins but does not affect the repressor activity. This finding suggests a functional interaction between the central and C-terminal regions of RepE in carrying out the initiator function.

Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32. We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.

A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30 degrees C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.

Mutants of Escherichia coli K-12 defective in replication of F-like plasmids at a high temperature (42 degrees C) were found among threonine-independent (Thr+) revertants of a threonine-requiring F' stain after localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Transduction experiments with phage P1 permitted us to divide these mutations into two classes with respect to man location; some mutations were located between thr and ara at about 0.8 min, very close to maf-1 reported previously (Wada et al., J. Mol. Biol. 108:25-41, 1976 and the others probably were located between leu and azi at about 1.8 min. The former class of mutants designated mafA exhibited the same plasmid specificity as maf-1; replication of plasmids F and ColVB trp, but not R386 or R222, were affected at a high temperature. By contrast, the latter mutants designated mafB were defective in replication of nay of these plasmids at a high temperature. When a culture of mafA mutants carrying an F' plasmid was transferred from 30 to 42 degrees C, the plasmid replication as determined by incorporation of [3H]thymidine into covalently closed circular F DNA was markedly inhibited. Under certain conditions, the temperature shift-up caused severe growth inhibition of the mutant cells. Examination of merodiploids (mafA/FmafA+) for plasmid maintenance suggested that the two mafA mutations tested (mafA23 and mafA36) were both dominant, at least partially, over the wild-type mafA+ allele. These properties of the mafA mutants, manifested at the restrictive temperature, are similar to those previously reported for the maf-1 mutant. Taken together with other evidence it is likely that these mutations affect either the same gene (mafA) or a set of closely linked genes, playing a specific role in autonomous plasmid replication in E. coli.

The Escherichia coli htpR (= hin, rpoH) mutants are defective in the induction of heat-shock proteins due to a deficiency in sigma 32 and are unable to grow at high temperature. We found that these mutants are also defective in supporting replication of certain plasmids including F and mini-F. When a htpR mutation is introduced into an F' strain, the F' plasmid is effectively excluded. Similarly, when an F' or mini-F plasmid is introduced into htpR mutant cells, transconjugant or transformant clones are obtained at low frequencies and the plasmid is rapidly lost upon subsequent growth in a non-selective medium. In htpR amber mutants carrying a temperature-sensitive suppressor, mini-F replication occurs normally at 30 degrees C, but is inhibited upon transfer to 40 degrees C where the suppressor tRNA is inactivated. A temperature-resistant "pseudo-revertant" of the htpR6 (amber) mutant, that exhibits apparently normal induction of the major heat-shock proteins in the absence of functional sigma 32, fails to support mini-F replication at 40 degrees C, suggesting that inhibition of mini-F replication is not a secondary consequence of the defective induction of the major heat-shock proteins. It is proposed that the function of the sigma 32 protein is directly required for F plasmid replication.

Replication of F (including mini-F) and some related plasmids is known to be specifically inhibited in mafA mutants of Escherichia coli K-12. We have now isolated and characterized mini-F mutants that can overcome the replication inhibition. Such plasmids, designated pom (permissive on maf), were obtained spontaneously or after mutagenesis with hydroxylamine or by transposon (Tn3) insertion. In addition to their ability to replicate in mafA mutant bacteria, the pom mutant plasmids exhibit an increased copy number and resistance to "curing" by acridine dye in the mafA+ host. In agreement with these results, Tn3-induced pom mutants were found to carry Tn3 inserted at the incC region of mini-F DNA, known to be involved in incompatibility, control of copy number, and sensitivity to acridine dye. Furthermore, three of the seven mini-F plasmids tested that carry Tn3 within the tandem repeat sequences of the incC region (previously isolated by other workers) exhibit all the phenotypes of pom plasmids, the ability to replicate in the mafA strain, and high copy number and acridine resistance in the mafA+ strain. The rest of the plasmids that contain Tn3 just outside the tandem repeats remain wild type in all these properties. These results strongly suggest that the putative mafA gene product of host bacteria controls mini-F replication through interaction with the incC region.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.