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 expr ession. The functions of the other operons, including those of the fou r coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined. Here, we present the first evidence that a kil d eterminant is involved in IncP plasmid maintenance. Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kil C and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aer uginosa but had little effect on plasmid maintenance in Escherichia co li. Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes. The resulting plasm id showed significant loss of stability in P. aeruginosa only. The def ect could be complemented by reintroduction of kilE at a different pos ition on the plasmid. The instability of the RK2lac Delta kilE mutant did not result from a reduction in average plasmid copy number, reduce d expression of kilC, decreased conjugative transfer, or loss of the k orE regulator. We found that both the par and kilE loci are required f or full stability of RK2lac in P. aeruginosa and that the par and kilE functions act independently. These results demonstrate a critical rol e for the kilE locus in the stable inheritance of RK2 in P. aeruginosa .