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 exclusi on phenotype, is located within a cluster of tra genes that are though t to encode a mating bridge. We have determined the DNA sequence of th e region containing eex, including the points of insertion of two flan king tra=Tn5 insertions. Sequence analysis of this interval strongly s uggests that it contains only one gene. This gene is transcribed in a clockwise direction on the circular map of pKM101 and appears to be tr anslationally coupled to neighboring tra genes. A Tn5 insertion that c auses 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 moeities. eex was amplified by polym erase chain reaction, subcloned, and fused to the Escherichia coli lac promoter. The resulting plasmid was sufficient to mediate entry exclu sion. The degree of exclusion was greatly enhanced by overexpression o f this gene. (C) 1994 Academic Press, Inc.