Entry exclusion of the IncN plasmid pKM101 is mediated by a single hydrophilic protein containing a lipid attachment motif.

The eex gene(s) of pKM101, which mediates that plasmid's entry exclusion phenotype, is located within a cluster of tra genes that are thought to encode a mating bridge. We have determined the DNA sequence of the region containing eex, including the points of insertion of two flanking tra::Tn5 insertions. Sequence analysis of this interval strongly suggests that it contains only one gene. This gene is transcribed in a clockwise direction on the circular map of pKM101 and appears to be translationally coupled to neighboring tra genes. A Tn5 insertion that causes a null eex mutation disrupts this ORF. Computer analysis of the predicted eex protein suggests that it may be exported and covalently modified by the addition of lipid moieties. eex was amplified by polymerase chain reaction, subcloned, and fused to the Escherichia coli lac promoter. The resulting plasmid was sufficient to mediate entry exclusion. The degree of exclusion was greatly enhanced by overexpression of this gene.