Genetic analysis of the vancomycin resistance Enterococcus highly conjugative pMG1-1 ike plasmids.

pHT质粒pHTalpha （65.9 kbp）、pHTbeta （63.7 kbp）和pHTgamma （66.5 kbp）是高度偶联的不依赖信息素的pmg1样质粒，携带编码万古霉素抗性的tn1546i样转座子。pHTbeta是原型质粒，pHTalpha和pHTgamma质粒分别是插入到pHTbeta中的is232样元件（2.2 kbp）和II族内含子（2.8 kbp）的衍生物。测定了pHTbeta质粒的完整核苷酸序列，除tn1546样插入（10,851 bp）外，发现全长52,890 bp。共鉴定出61个具有相同转录本取向的开放阅读框（orf）。同源性研究显示，22个pHTbeta （pHT）质粒orf与pX02质粒（96.2 kbp）上的orf相似，pX02质粒是炭疽芽孢杆菌形成荚膜所必需的毒力质粒；然而，大多数orf的功能仍然未知。大多数其他orf没有显示出任何显著的同源性。为了研究pHT质粒的高效转移机制，我们获得了174个独特的pHT β转座子Tn917-lac插入突变体。在174个衍生物中，92个衍生物的传递频率降低或丧失，74个衍生物的传递频率和LacZ表达正常。8个衍生物转移正常，无LacZ表达。174个衍生物中的插入片段在pHTbeta上被定位到124个不同的位点。导致转移频率改变的Tn917-Jac插入映射到三个独立的区域I， II和III，这些区域被Tn917-1ac插入不影响转移的片段分开。在pHT质粒中未发现与先前报道的oriT序列同源的区域。选择具有动员载体质粒pAM401能力的oriT进行克隆。oriT区位于ORF31和ORF32之间的非编码区（192 bp），包含3个直接重复序列和2个反向重复序列。0RF34编码一个506个氨基酸的蛋白，位于oriT区下游，包含移动质粒DNA松弛酶/缺口酶的3个保守基序（I至III）。0RF34的tne17 -lac插入突变体或框架内motif III缺失的0RF34突变体的转移能力完全丧失。ORF34的oriT区序列和推导出的松弛酶/镍酶蛋白序列分别与其他共轭质粒的oriT和松弛酶/镍酶没有显著的相似性。推测的0RF34松弛酶/缺口酶蛋白可归类为MOB（MG）家族的新成员。少

The pHT plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) are highly conjugative pheromone-independent pMG1-like plasmids that carry Tn1546i-like transposons encoding vancomycin resistance. pHTbeta is the prototype plasmid, and the pHTalpha and pHTgamma plasmids are derivatives of the insertion into pHTbeta of an IS232-like (2.2 kbp) element and a group II intron (2.8 kbp), respectively. The complete nucleotide sequence of the pHTbeta 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 pHTbeta (pHT) plasmid ORFs showed similarities to the ORFs identified on the pX02 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 signi … More ficant 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 pHTbeta 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 pHTbeta. The Tn917-Jac 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 Tn917-1ac 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. 0RF34, 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 Tne17-lac-insertion mutants of 0RF34 or a mutant of 0RF34 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 0RF34 could be classified as a new member of the MOB(MG) family. Less