Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2

In broad host-range plasmid RK2, several kil loci (kilA, kilB, kilC, kilE) and the replication initiator gene (trfA) are regulated by combination of kor determinants (korA, korB, korC, korE) in a regulatory network known as the kil-kor region. Although the kil determinants are not essential for replication, their coregulation with trfA suggests an involvement in plasmid maintenance or host-range. Plasmids carrying the cloned kilB region of RK2 cannot be maintained in the absence of korB owing to two phenotypically distinguishable, kor-regulated determinants: (1) kilB1 (kilD), which can be controlled by korA or korB, and (2) kilB2, which requires korB for control. In this study, we have determined the nature of the functions responsible for the kor-sensitive phenotypes of the kilB region. We found that insertion of transcription terminators within or downstream of the trfA operon allows plasmids carrying the kilB1 portion of the kilB region to be maintained in cells lacking korA or korB. In addition, mutants of the kilB1 region that can be maintained in the absence of korA and korB have alterations in the trfA promoter. These results show that the phenotype of the cloned kilB1 region in kor-deficient cells depends on trfA transcription but does not involve expression of any gene of the trfA operon. Therefore, the kilB1 determinant is not a structural gene. The phenotype results from entry of trfA-initiated transcription into adjacent sequences of the plasmid vector. The ability to block the kilB2 phenotype with transcriptional terminators allowed us to show conclusively that the kilB2 determinant is a host-lethal gene (klbA) whose regulation is dependent on korB. These findings have implications for the structure of the basic replicon of RK2.