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.     

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.     

Plasmid transfer in Streptomyces lividans: identification of a kil-kor system associated with the transfer region of pIJ101.

The 8.9-kilobase Streptomyces plasmid pIJ101 is self-transmissible at high frequency into recipient strains. By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal pIJ101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB. KilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever. Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively. These results suggest that kilA, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes.     

Identification of pKM101-encoded loci specifying potentially lethal gene products.

Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype).     

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.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli.

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.     

Plasmid RP4 host-lethal function kilA and its repressor korA sequences are part of the cryptic tellurite resistance transposon Tn521

The normally silent 4.5 kb tellurite resistance transposon Tn521 of RP4 has been shown to carry sequences from both the flanking kilA and korA loci of this broad host range plasmid. The major portion of both of these sequences were used as probes to examine DNA homology in Southern transfer hybridization experiments with plasmid recipients of Tn521 chosen from varying incompatibility groups. In the case of every recipients molecule analyzed using either probe, DNA homology was observed.     

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.     

Genetic and physiological control of host cell lysis by bacteriophage lambda.

The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.