The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriT pIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg 2؉ or Mn 2؉ and is highest at temperatures of between 42 and 45°C.pIP501 is a 30.2-kb Inc18 broad-host-range conjugative plasmid that was originally isolated from Streptococcus agalactiae. It confers resistance to macrolide, lincosamide, and streptogramin B antibiotics and to chloramphenicol. In addition to self-transfer to a wide variety of gram-positive genera, pIP501 is able to mobilize non-self-transmissible plasmids such as pMV158, the prototype of family 2 of rolling-circle-replicating plasmids (6). Krah and Macrina (16) postulated that the determinants conferring pIP501-mediated conjugation are encoded by two separate regions, a so-called region A and another region, region B, which has not been further characterized. Wang and Macrina (25) reported the nucleotide sequence of the complete 8,121-bp region A. Analysis of this DNA sequence revealed six contiguous open reading frames (orf1 to orf6) and an oriT site with high sequence identity to oriT regions of plasmids from gram-negative bacteria (Fig. 1). The oriT of pIP501 belongs to the RSF1010 family (27). An alignment of the nick regions of the RSF1010 family (modified from reference 27), including two more plasmids of grampositive origin, i.e., pGO1 from Staphylococcus aureus (5) and a new member, pRE25 from Enterococcus faecalis (GenBank accession no. X92945), is presented in Fig. 1. pRE25 is a 50.237-kb conjugative multiresistance plasmid whose putative tra region shows high overall identity with the tra region of pIP501 (B. Kurenbach and E. Grohmann, unpublished data).The precise nick site within oriT pIP501 was mapped by primer extension analysis on relaxed (open circular) pVA1857 DNA containing the entire tra region (26). To only one of the genes encoded by the pIP501 tra region A was a putative function ascribed. Wang and Macrina (26) demonstrated nicking of the oriT pIP501 site with plasmid pVA1860 carrying only oriT pIP501 and the traA gene, which gave rise to amounts of open circular plasmid DNA similar to those with pVA1857 containing the entire tra region.The six proteins encoded by tra region A of pIP501 show high overall similarity (between 24 and 36% identity over the whole amino acid sequence) with the gene products of the tra regions of the staphylococcal plasmids pGO1 (5) and pSK41 (3) and of the lactococcal conjugative plasmid pMRC01 (7). The TraA prote...
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