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Journal of Bacteriology, November 2005, p. 7727-7737, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7727-7737.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of Transfer-Related Regions of the Vancomycin Resistance Enterococcus Conjugative Plasmid pHTß: Identification of oriT and a Putative Relaxase Gene

Department of Bacteriology and Bacterial Infection Control, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan,¹ Laboratory of Bacterial Drug Resistance, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan²

Received 13 June 2005/ Accepted 26 August 2005

The pHT plasmids pHT (65.9 kbp), pHTß (63.7 kbp), and pHT (66.5 kbp) are highly conjugative pheromone-independent pMG1-like plasmids that carry Tn1546-like transposons encoding vancomycin resistance. pHTß is the prototype plasmid, and the pHT and pHT plasmids are derivatives of the insertion into pHTß of an IS232-like (2.2 kbp) element and a group II intron (2.8 kbp), respectively. The complete nucleotide sequence of the pHTß plasmid was determined and, with the exception of the Tn1546-like insertion (10,851 bp), was found to be 52,890 bp. Sixty-one open reading frames (ORFs) having the same transcript orientation were identified. A homology search revealed that 22 of the pHTß (pHT) plasmid ORFs showed similarities to the ORFs identified on the pXO2 plasmid (96.2 kbp), which is the virulence plasmid essential for capsule formation by Bacillus anthracis; however, the functions of most of the ORFs remain unknown. Most other ORFs did not show any significant homology to reported genes for which functions have been analyzed. To investigate the highly efficient transfer mechanism of the pHT plasmid, mutations with 174 unique insertions of transposon Tn917-lac insertion mutants of pHTß were obtained. Of the 174 derivatives, 92 showed decrease or loss in transfer frequency, and 74 showed normal transfer frequency and LacZ expression. Eight derivatives showed normal transfer and no LacZ expression. Inserts within the 174 derivatives were mapped to 124 different sites on pHTß. The Tn917-lac insertions which resulted in altered transfer frequency mapped to three separate regions designated I, II, and III, which were separated by segments in which insertions of Tn917-lac did not affect transfer. There was no region homologous to the previously reported oriT sequences in the pHT plasmid. The oriT was cloned by selection for the ability to mobilize the vector plasmid pAM401. The oriT region resided in a noncoding region (192 bp) between ORF31 and ORF32 and contained three direct repeat sequences and two inverted repeat sequences. ORF34, encoding a 506-amino-acid protein which was located downstream of the oriT region, contains the three conserved motifs (I to III) of the DNA relaxase/nickase of mobile plasmids. The transfer abilities of the Tn917-lac-insertion mutants of ORF34 or a mutant of ORF34 with an in-frame motif III deletion were completely abolished. The sequence of the oriT region and the deduced relaxase/nickase protein of ORF34 showed no significant similarity to the oriT and relaxase/nickase of other conjugative plasmids, respectively. The putative relaxase/nickase protein of ORF34 could be classified as a new member of the MOB_MG family.

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