STRUCTURE, EXPRESSION, AND REGULATION OF THE KILC OPERON OF PROMISCUOUS INCP-ALPHA PLASMIDS

The kil-kor regulon was first identified on the broad-host-range IncP alpha plasmid RK2 by the presence of multiple kil loci (kilA, kilB, ki lC, and recently kilE) that are lethal to Escherichia coli host cells in the absence of regulation by kor functions in various combinations. Whereas the kilB operon is required for mating-pair formation during conjugation, the functions encoded by the other kil loci are not known . They are not essential for replication or conjugal transfer, but the ir coregulation with replication and transfer genes indicates that the y are likely to be important for RK2. In this report, we describe mole cular and genetic studies on kilC. We determined the nucleotide sequen ce of the kilC region, which is located between the origin of vegetati ve replication (oriV) and transposon Tn1 on RK2. Primer extension anal ysis identified the transcriptional start site and showed that a seque nce corresponding to a strong sigma(70) promoter is functional. The ab undance of RNA initiated from the kilC promoter is reduced in the pres ence of korA and korC, as predicted from genetic analysis of kilC regu lation. The first gene of the kilC operon (klcA) is sufficient to expr ess the host-lethal phenotype of the kilC determinant in the absence o f korA and korC. By comparing RK2 to the related IncP alpha plasmids p UZ8 and R995, we determined that the Tn1 transposon in RK2 interrupts a gene (klcB) immediately downstream of klcA. Thus, the kilC determina nt is normally part of an autoregulated operon of three genes: klcA, k lcB, and korC. klcA is predicted to encode a 15,856-Da polypeptide tha t is related to the ArdB antirestriction protein of the IncN plasmid p KM101, suggesting a role for klcA in the broad host ranges of IncP alp ha plasmids. The predicted product of the uninterrupted klcB gene is a polypeptide of 51,133 Da that contains a segment with significant sim ilarity to the RK2 regulatory proteins KorA and TrbA. Located 145 bp u pstream of the kilC promoter is a 10th copy of the 17-bp oriV iteron s equence in inverted orientation relative to that of the other nine ite rons of oriV. Iteron 10 is identical to the ''orphan'' iteron 1, and b oth have identical 6-bp flanking sequences that make them likely to be strong binding sites for the TrfA replication initiator protein. The locations and relative orientation of orphan iterons 10 and 1 raise th e possibility that these iterons promote the formation of a DNA loop v ia protein-protein interactions by bound TrfA and lead us to propose t hat they demarcate the functional origin of replication. This analysis of the kilC region and our previous studies on the other kil loci of RK2 have revealed that the region between oriV and the korABF operon i n wild-type IncP alpha plasmids is saturated by the kilC, kilE, and ki lA loci arranged in four kor-regulated operons encoding a total of 12 genes.