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.     

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.     

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.     

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.     

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.     

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.     

Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication

The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.     

Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria

A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA*. The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms. These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria.     

The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.     

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.     

Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2

Mini-RK2 plasmids pCT460 and pCT461 which contain the oriVRK2, trfA and trfB regions of RK2 in addition to tetracycline and kanamycin resistance determinants, have copy numbers of 17 and 35 copies per chromosome equivalent, respectively. The difference in copy number is due to a 56-bp deletion in oriVRK2 in pCT461. In Escherichia coli only pCT461 is markedly unstable in batch culture while both are unstable (although pCT461 is more so) in bacteria on stock plates. The instability of pCT461 in bacteria on stock plates is recA+ dependent and appears to involve loss of plasmid DNA from bacteria rather than selective cell death. After storage of recA+ bacteria carrying pCT461 for a few weeks the remaining antibiotic-resistant bacteria carry a mixture of plasmid DNA species including parental pCT461, transposable element insertion derivatives, and, by far the majority, deletion derivatives. It appears that one particular plasmid region, which includes the kilD gene (which inhibits plasmid maintenance in the absence of korD which, however, is present on pCT460 and pCT461), is responsible for this instability in a gene dosage-dependent way. Most of these deletion derivatives are dependent on pCT461-specified trfA gene (essential for replication) so that they do not displace pCT461 entirely. Their presence reduces the copy number of pCT461, thus reducing the instability, and is probably ultimately responsible for pCT461 survival on stock plates. In many bacteria the same process which gives rise to deletion derivatives may result in degradation of plasmid DNA extensive enough to cause loss of pCT461.     

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.