Genes encoding the pKM101 conjugal mating pore are negatively regulated by the plasmid-encoded KorA and KorB proteins

Abstract

The IncN plasmid pKM101 contains a group of 11 genes thought to be required for the synthesis of its conjugal pilus and mating pore. Within this region are two genes, kilA and kilB, either of which is conditionally lethal to the cell. kilA was previously shown to be allelic with traL, and we now show that kilB is allelic with traE. In the same region, genetic studies previously defined two loci, korA and korB (kor for kill override), which together prevent lethality mediated by kilA and kilB. We now identify the genes that encode KorA and KorB functions. To determine whether KorA and KorB proteins influence tra gene transcription, we constructed beta-galactosidase fusions to three promoters in this region and measured their expression in the presence of KorA, KorB, and both proteins. KorA and KorB together repressed transcription of all three promoters, while neither protein alone affected transcription. We identified all three transcriptional start sites by primer extension analysis. Two putative binding sites for these proteins, designated kor boxes, contain 26 identical nucleotides in a 29-nucleotide region. The electrophoretic mobilities (of DNA fragments containing kor boxes were retarded by cell extracts containing both KorA and KorB but were not retarded by extracts containing just KorA or just KorB. DNase I footprinting analysis of one of these promoters demonstrates that KorA and/or KorB binds to a region containing a kor box.