Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary 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° 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.     

Inhibition of initiation of mini-F plasmid replication in temperature-sensitive mafA mutants of Escherichia coli K-12

When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [³H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.     

Purification and DNA binding of the D protein, a putative resolvase of the F-factor of Escherichia coli

Summary The D protein encoded by plasmid mini-F promotes resolution of plasmid cointegrates or dimers of the F-factor or mini-F. In addition, two rfsF sequences are essential for this site-specific, recA-independent recombination event. The D gene was cloned into an expression vector and the gene product was overproduced in Escherichia coli and purified to homogeneity. The sequence of the N-terminus of the D protein was determined, thus permitting identification of the correct translational start codon in the nucleotide sequence that results in a 29.6 kDa protein. The binding site for the purified D protein is located within the mini-F NcoIHpaI DNA fragment (192 bp). Binding seems to be affected by DNA methylation, since the protein did not bind to DNA isolated from a dam mutant of E. coli. The binding site, which is a region of approximately 28 bp and is located 160 by downstream of the rfsF site, was identified by DNase I footprinting using fluorescence labelled DNA.     

The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication

Summary At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression.     

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters

Summary A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasrnid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, -lactamase.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

IncD, a genetic locus in F responsible for incompatibility with several plasmids of the IncFI group

Summary Cloning of mini-F DNA segments has led to the identification and mapping of a locus, incD, involved in incompatibility reactions with many IncFI plasmids. The cloned incD locus expressed incompatibility with F, R386, and six other IncFI plasmids but not with ColV3-K30 or pHH507 which lack sequence homology with the incD region. A sequence of 360 bp (48.66–49.02 FKB) was found to be sufficient for expression of incD incompatibility. Multicopy vectors containing incD are compatible with each other, but can be displaced by mini-F plasmids deleted for incD. These results indicate that incD-mediated incompatibility reactions require the presence of replication genes to which incD is normally linked. The degree of incompatibility exercised by incD is moderate compared with that of other inc loci in F, suggesting that incD is involved in an aspect of plasmid maintennce, such as partition, different from the functions of the other inc loci.     

Replication Control and Switch-Off Function as Observed with a Mini-F Factor Plasmid

Mini-F is a fragment of the F plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. Its copy number is one to two per chromosome. This plasmid is joined to ColE1, whose copy number is 16 to 20. Under normal circumstances the composite plasmid replication exhibited ColE1 characteristics, maintaining a high copy number. However, when ColE1 replication was inhibited by deoxyribonucleic acid polymerase I inactivation, its replication exhibited mini-F characteristics, maintaining a low copy number. These observations are in complete agreement with those of Timmis et al. (Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560, 1974), who examined the behavior of a recombinant plasmid formed between pSC101 and ColE1. The transition from high to low copy number allowed us to examine the control system acting in cells carrying plasmids exhibiting intermediate copy numbers. The initiation of the mini-F replication system as represented by deoxyribonucleic acid synthesis of the composite plasmid was completely blocked when there were multiple copies of mini-F in a cell. It was not restored until the copy number was lowered to one to two, after which replication was first detected. ppF, a mini-F replicon packaged in a phage λ head behaved similarly: its replication was completely shut off when the resident mini-F genome copy number was high and was inhibited partially when the resident mini-F genome copy number was low. These experiments clearly demonstrate that there is a switch-off mechanism acting on deoxyribonucleic acid synthesis (initiation) in a cell carrying mini-F, and its intensity is related to the plasmid copy number. This result supports the “inhibitor dilution model” proposed by Pritchard et al. (Symp. Soc. Gen. Microbiol. 19:263-297, 1969). The nature of the hypothetical inhibitor is discussed.     

An amber replication mutant of F plasmid mapped in the minimal replication region

We have constructed a mini-F derivative (pKP1013) consisting of a 5.4 kilobase pairs (kb) segment (44.0 to 49.4 kb) of mini-F and fragments carrying the chloramphenicol and spectinomycin resistance genes that originated from the R plasmid NR1. The plasmid pKP1013 replicates autonomously in a manner indistinguishable from that of the parental mini-F. An amber mutant defective in replication has been isolated from pKP1013 by localized mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The virtual absence of incorporation of [3H]-thymidine into the plasmid DNA as well as the kinetics of appearance of plasmid-free segregants suggest that plasmid DNA synthesis is primarily affected under nonpermissive conditions. The amber mutation has been mapped within the 530 base pairs (bp) region that extends from 45.25 (XmaI) to 45.78 Kb (PstI) by extensive analysis of in vitro recombinants constructed from rep+ and rep- plasmids.     

Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini-replicon of pCU1 and the ori-2 of F

The dominant, polA1-independent replicon of pGSH500, repβ (1.8 kb), consists of a cis-acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of repβ resembles that of mini-pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini-F. In addition, an 80% identity over 200 bp was identified between the cis-acting βoriV region and the equivalent region of ori-2 (mini-F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini-F) in trans, supporting the hypothesis that repβ is a natural hybrid between a pCU1-like and F-like replicon.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

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.     

Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication.

When folded chromosomes are purified from plasmid-containing bacteria, a reproducible fraction of the host's covalently closed, circular (CCC) plasmid DNA copurifies with the chromosomes. From this copurification, we infer the existence of nonintegrative plasmid-chromosome (NPC) complexes. Previously, we noted that plasmids dependent on DNA polymerase III and with stringent control of replication complex to a greater extent than plasmids dependent on DNA polymerase I and with relaxed control of replication. We have examined this subject in more depth and find that: (i) The composite plasmids formed by in vitro recombination of a “stringent” with a “relaxed” replicon complex to chromosomes at the frequency of the component replicon which directs replication; (ii) all of the detectable replicative intermediates, but only 25% of the CCC forms, of plasmid ColE1 complex to chromosome; and (iii) when a mini-F plasmid is deleted for the DNA sequences which include the primary origin of replication, the complexing frequency decreases 30 to 40%. We conclude from these findings that NPC complexes either indirectly or directly relate to plasmid replication. Further, we find that the EcoRI kan⁺ fragments of pML31 and the ampicillin resistance transposon, Tn3, promote complexing of both ColE1 and mini-F plasmids to host chromosomes. The biological significance of this latter complexing is unknown. However, we conclude from these studies and from point (iii) that complexing is determined in part by unique plasmid sequences.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2

Summary A DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc ₂ incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc ₂ incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene

Summary The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42°C. We cloned the wild-type E. coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein. Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR-thrA-seg-2-leuB, consistent with the locus of the dnaK gene. Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution. These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene. The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30°C in contrast to plasmids pBR322, pACYC184 and pSC101, which did. The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30° and 42°C.