Molecular genetic analysis of pheromone-independent highly transferable drug resistance plasmid of Enterococcus.

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1) DNA sequence of whole pMG1 plasmid was determined.2) By computer analysis of the sequence, putative ORFs were determined. Probes for each OFR was designed to examine ils expression during conjugation by Northern hybridization and it was suggested that tra genes made some operons.3) Insertion mutant of ORF20 abolished the expression of the biggest operon and reduced the expression of the operon downstream. From this observation, ORF20 was suggested to be a positive regulator of tra genes. The presence of a negative regulator was also suggested.4) All ORFs, which were examined and seemed to be involved in conjugation, were repressed during conjugation except that ORF2 (traA), had been reported to be upregulated early period of conjugation.5) The role of recipient cell in conjugation was examined by the mating with killed recipient. The expression of ORF42 was downregulated during conjugation, but was not changed in the mating with killed recipient. This observation indicated that recipient had to be alive to initiate conjugation, and interaction between donor and recipient was required.6) The mutants which could not be good recipients were isolated by Tn916 mutagenesis. They could not downregulate the ORF42 expression during conjugation. Mutation was mapped and DNA sequence of the gene was determined. This gene had significant homology with a positive regulator of transcription.