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

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1)Transcription units were determined by northern blot analysis and RT-PCR. They were ORF13-15,ORF16-19,ORF20-43 and ORF44-49. The biggest one, ORF20-43, was transcribed from strong promoter in front of ORF20 and ORF20 product supplied in trans by cloned ORF20 gene increased the activity of LacZ fusion of ORF20 promoter, indicating that ORF20 was a positive regulator for this transcript.2)The insertion upstream of ORF20 resulted in the overexpression of ORF20-43, indicating the presence of a negative regulator for ORF20-43 expression. The fragment, which complemented the effect of the insertion, was mutagenized by in vitro transposon mutagenesis to identify the negative regulator. The negative regulator was located between the end of ORF13 and the start of ORF15, but it needed the transcript from ORF13 promoter, because ORF13 alone did not complement and the insertion in ORF15 did not complement either.3)OriT region was determined. It was located in the region of 180bp just in front of ORF44. The oriT plasmid carrying the 180bp region was mobilized by pMG1 and transferred at the frequency of 10^<-4> per donor cell in 3 h broth mating.4)Cell surface proteins of conjugation-defective recipient mutant and parent were extracted and analyzed by 2D SDS-PAGE. No difference was observed between the protein profile of the mutant and that of parent, so far.