Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.     

Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2

Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of RK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.     

Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA.

We previously reported that broad-host-range plasmid RK2 encodes multiple host-lethal kil determinants (kilA, kilB1, kilB2, and kilC) which are controlled by RK2-specified kor functions (korA, korB, and korC). Here we show that kil and kor determinants have significant effects on RK2 replication control. First, korA and korB inhibit the replication of certain RK2 derivatives, unless plasmid replication is made independent of the essential RK2 gene trfA. Second, kilB1 exerts a strong effect on this interaction. If the target plasmid is defective in kilB1, sensitivity to korA and korB is enhanced at least 100-fold. Thus, korA and korB act negatively on RK2 replication, whereas kilB1 acts in a positive manner to counteract this effect. A mutant RK2 derivative, resistant to korA and korB, was found to have fused a new promoter to trfA, indicating that the targets for korA and korB are at the 5' end of the trfA gene. We constructed a trfA-lacZ fusion and found that synthesis of beta-galactosidase is inhibited by korA and korB. Thus korA, korB, and kilB1 influence RK2 replication by regulating trfA expression. We conclude that the network of kil and kor determinants is part of a replication control system for RK2.     

Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2

We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.     

Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.

Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes. Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA. kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment. Transformation of E. coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions. If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes. KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans. These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.     

Comparison of 10 IncP plasmids: homology in the regions involved in plasmid replication.

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolated from several different countries. Two regions of RK2, the best-studied plasmid of this group, are required for vegetative DNA replication: the origin of replication (oriV) and the trfA region, which codes for a gene product necessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA regions as shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology with the RK2 trfA, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751, R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility but differed markedly from RK2 and the other six plasmids in the GC-rich region of the origin required for replication. These data indicate that the P-group plasmids can be divided into two subgroups: IncP alpha, which includes the RK2-like plasmids, and IncP beta which includes the R751-like plasmids.     

Narrow-host-range IncP plasmid pHH502-1 lacks a complete IncP replication system

Plasmid pHH502-1 shows incompatibility only towards members of the IncP group, but has a narrower host range than typical members of that group. In contrast to other IncP plasmids its replication was not affected by a high-copy-number plasmid carrying the replication origin (oriV) of IncP plasmid RK2. Southern blotting of pHH502-1 revealed homology to oriV, consistent with its incompatibility phenotype, but no homology to trfA, the essential replication gene of RK2. Thus it is probable that pHH502-1 does not possess a functional IncP replication system, accounting for its restricted host range. A restriction map of pHH502-1 was constructed and the mercury-resistance determinant was localized to Tn735, which also carries the trimethoprim-resistance determinant and is related to Tn21. The presence of a korB-like function on pHH502-1 was also demonstrated.     

Nucleotide sequence of korB, a replication control gene of broad host-range plasmid RK2

The korB gene is a major regulatory element in the replication and maintenance of broad host-range plasmid RK2. It negatively controls the replication gene trfA, the host-lethal determinants kilA and kilB, and the korA-korB operon. Here, we present the nucleotide sequence of an 1167 base-pair region that encodes korB. Using sequence data from korB mutants, we identified the korB structural gene. The predicted polypeptide product is negatively charged and has a molecular weight of 39,015, which is considerably less than that estimated by its electrophoretic mobility in SDS/polyacrylamide gels. Secondary-structure predictions of korB polypeptide revealed three closely spaced helix-turn-helix regions with significant homology to similar structures in known DNA-binding proteins. The korB gene, like all other sequenced RK2 genes, shows a strong preference for codons ending in a G or C residue. This is similar to codon usage by genes of Klebsiella and Pseudomonas, the original hosts for RK2 and some closely related plasmids. We also sequenced the site of transposon Tn76 insertion in the host-range mutant pRP761 and found it to be located immediately upstream from korB in the incC gene. Finally, we report the presence of sequences resembling a replication origin within the korB structural gene: a cluster of four 19 base-pair direct repeats and a nearby potential binding site for Escherichia coli dna A replication protein.     

Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA.

The trfA gene, encoding the essential replication initiation protein of the broad-host-range plasmid RK2, possesses an in-frame overlapping arrangement. This results in the production of TrfA proteins of 33 and 44 kDa, respectively. Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone. TrfA-44/98L is identical to wild-type TrfA-44 with the exception of a single conservative amino acid alteration from methionine to leucine at codon 98; this alteration removes the translational start codon for the TrfA-33 protein. Copy number and stability were virtually identical for plasmids containing both TrfA-44 and TrfA-33 proteins or TrfA-44/98L alone in Pseudomonas aeruginosa and Agrobacterium tumefaciens, two unrelated bacteria in which TrfA-33 is poorly functional. This, along with recent in vitro studies comparing TrfA-44, TrfA-33, and TrfA-44/98L, suggests that the functional activity of TrfA-44 is not significantly affected by the 98L mutation. Analysis of minimal RK2 derivatives in certain gram-negative bacterial hosts suggests a role of the overlapping arrangement of trfA in facilitating the broad host range of RK2. RK2 derivatives encoding TrfA-44/98L alone demonstrated decreased copy number and stability in Escherichia coli and Azotobacter vinelandii when compared with derivatives specifying both TrfA-44 and TrfA-33. A strategy employing the trfA-44/98L mutant gene and in vivo homologous recombination was used to eliminate the internal translational start codon of trfA in the intact RK2 plasmid. The mutant intact RK2 plasmid produced only TrfA-44/98L. A small reduction in copy number and beta-lactamase expression resulted in E. coli, suggesting that overlapping trfA genes also enhance the efficiency of replication of the intact RK2 plasmid.     

Control of the kilA gene of the broad-host-range plasmid RK2: involvement of korA, korB, and a new gene, korE.

Broad-host-range plasmid RK2 encodes several different kil genes which are potentially lethal to an Escherichia coli host. The kil genes and the essential RK2 replication gene trfA are regulated by the products of kor genes. We have shown previously that kilA can be controlled by a constitutively expressed korA gene. In this study, we have found that the wild-type, autoregulated korA gene is insufficient for control of kilA cloned on high-copy-number plasmids. One of two other genes must also be present with korA. One gene is korB, originally discovered by its ability to control the determinants in the kilB region and later found to affect expression of both trfA and korA. The other is a new gene, korE, which has been cloned from the 2.2' to 4.1' region located between korC and kilA. Studies with a kilA-cat fusion suggest that korA, korB, and korE all participate in the control of kilA gene expression.     

The trfA and trfB promoter regions of broad host range plasmid RK2 share common potential regulatory sequences

The positions of the trfA and trfB promoters of broad host range IncP plasmid RK2 (identical to RP1, RP4, R68 and R18 ) were identified by RNA polymerase protection studies, and the nucleotide sequences of the promoter regions determined. A mutation within the trfA promoter sequence is associated with loss of kilD activity. In addition a probable promoter region for the kilB locus was identified. The three promoter regions share common palindromic sequences which may serve as sites for the coordinate regulation of replication and kil functions.