The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa.

Eight coordinately regulated operons constitute the kor regulon of the IncP alpha plasmid RK2. Three operons specify functions required for replication initiation, conjugative transfer, and control of gene expression. The functions of the other operons, including those of the four coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined. Here, we present the first evidence that a kil determinant is involved in IncP plasmid maintenance. Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kilC and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aeruginosa but had little effect on plasmid maintenance in Escherichia coli. Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes. The resulting plasmid showed significant loss of stability in P. aeruginosa only. The defect could be complemented by reintroduction of kilE at a different position on the plasmid. The instability of the RK2lac delta kilE mutant did not result from a reduction in average plasmid copy number, reduced expression of kilC, decreased conjugative transfer, or loss of the korE regulator. We found that both the par and kilE loci are required for full stability of RK2lac in P. aeruginosa and that the par and kilE functions act independently. These results demonstrate a critical role for the kilE locus in the stable inheritance of RK2 in P. aeruginosa.     

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.     

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 kilA operon of promiscuous plasmid RK2: the use of a transducing phage (λpklaA-1) to determine the effects of the lethal klaA gene on Escherichia coli cells

The kil-kor regulon of promiscuous plasmid RK2 includes the replication initiator gene trfA and several potentially host-lethal kil loci (kilA, kilB, kilC, kilE), whose functions may be involved in plasmid maintenance or broad host range. The kilA locus consists of a single operon of three genes (klaA, klaB, klaC), each of which is lethal when expressed from the klaA promoter in the absence of repressors encoded by korA and korB. In this study, we examined the effects of the unregulated klaA gene on the host cell. Bacteriophage lambda was used to construct a transducing phage (lambda pklaA-1) that allows efficient introduction of the klaA gene into Escherichia coli. Cells lacking korA and korB (to allow uncontrolled expression of klaA) and expressing lambda repressor (to prevent phage lytic growth) are killed by lambda pklaA-1. Cell death is dependent on the klaA structural gene, independent of the SOS system of the host, and is prevented by the presence of korA and korB. lambda pklaA-1 was used to synchronously infect cells lacking korA and korB to determine the effects of klaA on the cells over time. The earliest effects, visible at two hours post-infection, are inhibition of growth of the culture, formation of elongated cells, and striking changes in the appearance of the outer membrane. After four to five hours, the viability of the culture declined sharply and macromolecular synthesis ceased. The distinct class of early events is consistent with the hypothesis that the KlaA polypeptide interacts with a specific target in the host cell.     

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.     

Conditionally lethal genes in the N pilus region of plasmid pCU1.

Plasmid genes or regions that are conditionally lethal to Escherichia coli have been called kil and those lethal to Klebsiella but not to E. coli have been called kik. Both classes of genes are found in or close to the N pilus region of the plasmid pCU1 and the closely related plasmid pKM101. Here we describe two new and overlapping lethal genes that are located between kikA and traA of the plasmid pCU1 and display host specificity. KilC is lethal in E. coli and Klebsiella while kikC is lethal only in Klebsiella. The previously identified korA gene is sufficient to override the lethality of kilC in trans or in cis but is insufficient to override kikC. kilC expression in E. coli leads to cell death accompanied by an increase in average cell length without affecting septum formation.     

A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids.

The kor regulon of broad host-range, incompatibility group P (IncP) plasmids uses the KorA, KorB, and KorC repressors to regulate expression of genes for replication, conjugation, segregation, and host range. One operon, kilC, encodes the KorC repressor and two genes of unknown function (klcA and klcB). The predicted sequences of the 51.1 kDa KlcB protein, the 11.3 kDa KorA repressor, and another small (13.5 kDa) regulatory protein, TrbA, show a highly related 35 amino acid residue segment (V-L-P domain). We found that induction of the klcB gene is toxic to Escherichia coli host cells harboring an IncP plasmid. We confirmed a model in which the V-L-P domain of KlcB interacts directly with the V-L-P domain of KorA to derepress KorA-regulated operons, thereby allowing expression of toxic genes. First, a lacZ reporter fused to the kleA promoter, which is regulated by KorA and KorC, revealed that klcB induction specifically releases KorA-repression but has no effect on KorC repression. Second, induced expression of the V-L-P domains from KorA or KlcB is sufficient to release KorA-repression at the kleA promoter. Third, purified GST-KlcB fusion protein interacts specifically with His-tagged KorA. Fourth, fusion of the V-L-P domains of KorA and TrbA and full-length KlcB polypeptide to the DNA-binding domain of bacteriophage lambda repressor leads to the formation of functional, dimeric repressors, and mutations that alter conserved residues of the V-L-P domain adversely affect dimerization. Fifth, crosslinking experiments demonstrated that the V-L-P domain of KorA is able to dimerize in solution and form heterodimers in mixtures with full-length KorA polypeptide. These findings show that the V-L-P domain is a protein-protein interaction module that is likely to be responsible for dimerization of KorA and TrbA, and important for KlcB dimerization. We speculate on the possible significance of KlcB-KorA heterodimers in IncP plasmid maintenance.     

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.     

Genetic, functional and sequence analysis of the xylR and xylS regulatory genes of the TOL plasmid pWW0

Mutant derivatives of a plasmid, pCF20, which carries the XhoI-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the xylR and xylS regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the XhoI-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the xylR gene. No clear candidate for the xylS polypeptide was observed. The nucleotide sequence of the xylS region, the intergenic region and part of the xylR region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated xylS appears capable of encoding a polypeptide of approximately 37 kDa.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.