Identification and characterization of two entry exclusion genes of the promiscuous IncP plasmid R18

Two entry exclusion genes (designated eexA and eexB) from the promiscuous IncP alpha plasmid R18 have been isolated by molecular cloning. They are located between coordinates 26.6-27.4 kb and 27.4-27.6 kb, respectively and are transcribed clockwise on the conventional R18 map. The product of the eexA gene has an apparent molecular mass of 28 kDa and its N-terminus contains a putative signal sequence for protein export. A recombinant plasmid containing R18 eex genes exerted Eex activity towards another promiscuous IncP alpha plasmid, R702, about 50 times more strongly than plasmid R18 itself. Analysis of the DNA sequence revealed no similarity to the eex genes of the F plasmid of Escherichia coli. R18 eexA includes a potential korB binding site and is followed by a potential transcription terminator. A Tn7 insertion at coordinate 20.0 kb of R18 resulted in a host range mutant pM01185, which leads to loss of Eex activity and of conjugative transfer of the plasmid into some bacterial species.