KIL-KOR REGULON OF PROMISCUOUS PLASMID-RK2 - STRUCTURE, PRODUCTS, ANDREGULATION OF 2 OPERONS THAT CONSTITUTE THE KILE LOCUS

The kil-kor regulon of IncP plasmid RK2 is a complex regulatory networ k that includes genes for replication and conjugal transfer, as well a s for several potentially host-lethal proteins encoded by the kil4, ki lB, and kilC loci. While kilB is known to be involved in conjugal tran sfer, the functions of kil4 and kilC are unknown. The coregulation of kilA and kilC with replication and transfer genes indicates a possible role in the maintenance or broad host range of RK2. In this work, we found that a fourth kil locus, designated kilE, is located in the kb 2 .4 to 4.5 region of RK2 and is regulated as part of the kil-kor regulo n. The cloned kilE locus cannot be maintained in Escherichia coli host cells, unless korA or korC is also present in trans to control its ex pression. The nucleotide sequence of the kilE region revealed two pote ntial multicistronic operons. The kleA operon consists of two genes, k leA and kleB, predicted to encode polypeptide products with molecular masses of 8.7 and 7.6 kDa, respectively. The kleC operon contains four genes, kleC, kleD, kleE, and kleF, with predicted products of 9.2, 8. 0, 12.2, and 11.3 kDa, respectively. To identify the polypeptide produ cts, each gene was cloned downstream of the phage T7 phi10 promoter an d expressed in vivo in the presence of T7 RNA polymerase. A polypeptid e product of the expected size was observed for all six kle genes. In addition, kleF expressed a second polypeptide of 6 kDa that most likel y results from the use of a predicted internal translational start sit e. The kleA and kleC genes are each preceded by sequences resembling s trong sigma70 promoters. Primer extension analysis revealed that the p utative kleA and kleC promoters are functional in E. coli and that tra nscription is initiated at the expected nucleotides. The abundance of transcripts initiated in vivo from both the kleA and kleC promoters wa s reduced in cells containing korA or korC. When korA and korC were pr esent together, they appeared to act synergistically in reducing the l evel of transcripts from both promoters. The kleA and kleC promoter re gions are highly homologous and contain two palindromic sequences (A a nd C) that are the predicted targets for KorA and KorC proteins. DNA b inding studies showed that protein extracts from korA-containing E. co li cells specifically retarded the electrophoretic mobility of DNA fra gments containing palindrome A. Extracts from korC-containing cells al tered the mobility of DNA fragments containing palindrome C. These res ults show that KorA and KorC both act as repressors of the kleA and kl eC promoters. In the absence of korA and korC, expression of the clone d kleA operon was lethal to E. coli cells, whereas the cloned kleC ope ron gave rise to slowly growing, unhealthy colonies. Both phenotypes d epended on at least one structural gene in each operon, suggesting tha t the operons encode genes whose products interact with critical host functions required for normal growth and viability. Thus, the kilA, ki lC, and kilE loci of RK2 constitute a cluster of at least 10 genes tha t are coregulated with the plasmid replication initiator and the conju gal transfer system. Their potential toxicity to the host cell indicat es that RK2 is able to establish a variety of intimate plasmid-host in teractions that may be important to its survival in nature.