Complete nucleotide sequencing and analysis of the 65-kb highly conjugativeEnterococcus faeciumplasmid pMG1: identification of the transfer-related region and the minimum region required for replication

Abstract

pMG1 (65.1 kb) is a highly conjugative, pheromone-independent Enterococcus faecium plasmid that carries a Tn4001-like transposon encoding gentamicin resistance. The complete nucleotide sequence (65 029 bp) of the pMG1 plasmid was determined and 73 ORFs lying in the same transcription orientation were identified. Sixty-one of the 73 ORFs showed a high degree of similarity (90-100% identity at the amino acid level) to the ORFs of the pHTbeta plasmids. Like the pHTbeta plasmid, 22 of the pMG1 plasmid ORFs showed homology with ORFs present on the pXO2 plasmid (96.2 kb), which is the virulence plasmid essential for capsular formation by Bacillus anthracis. Analysis of tra mutants created by Tn917 insertion and Northern analysis of transcripts indicated that ORFs 15-49, lying in the 31.7 kb region between 13.6 and 45.3 kb on the plasmid map, were related to transfer. This region was designated as the Tra I region of pMG1. A 5.9-kb HindIII fragment that replicates autonomously in Enterococcus faecalis was cloned and analysis of this fragment by deletion and in vitro insertion mutations showed that ORF10 (rep) and the inverted repeat sequence in the noncoding region between ORF8 and ORF9 were necessary for pMG1 replication.