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

万古霉素抗性肠球菌高度接合pMG1-1 ike质粒的遗传分析。

基本信息

批准号：
16017216
负责人：
IKE Yasuyoshi
金额：
$ 9.6万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

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

pHT质粒pHT alpha（65.9 kbp）、pHT beta（63.7 kbp）和pHT gamma（66.5 kbp）是高度接合性信息素非依赖性pMG 1样质粒，其携带编码万古霉素抗性的Tn 1546 i样转座子。pHTbeta是原型质粒，pHTalpha和pHTgamma质粒分别是在pHTbeta中插入IS 232样（2.2kbp）元件和II组内含子（2.8kbp）的衍生物。测定了pHTbeta质粒的完整核苷酸序列，除Tn 1546样插入（10，851 bp）外，发现其为52，890 bp。鉴定出61个具有相同转录本方向的开放阅读框架（ORF）。同源性检索显示，22个pHT beta（pHT）质粒ORF与pX 02质粒（96.2 kbp）上鉴定的ORF相似，pX 02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未显示任何显著的 ...更多信息与已经分析功能的报道基因的虚构同源性。为了研究pHT质粒的高效转移机制，获得了pHT β的具有174个独特插入的转座子Tn 917-lac插入突变体的突变。在174个衍生物中，92个显示转移频率降低或丧失，74个显示正常的转移频率和LacZ表达。8个衍生物显示正常的转移和无LacZ表达。174个衍生物中的124个位点被定位在pHTbeta上。导致转移频率改变的Tn 917-Jac插入被映射到三个独立的区域，称为I、II和III，它们被其中Tn 917 -1ac插入不影响转移的片段分开。在pHT质粒中没有与先前报道的oriT序列同源的区域。通过选择动员载体质粒pAM 401的能力克隆oriT。oriT区位于ORF 31和ORF 32之间的非编码区（192 bp），包含3个正向重复序列和2个反向重复序列。0 RF 34编码一个506个氨基酸的蛋白质，位于oriT区的下游，包含移动的质粒的DNA松弛酶/切口酶的三个保守基序（I至III）。0 RF 34的Tne 17-lac-insertion突变体或具有框内基序III缺失的0 RF 34的突变体的转移能力被完全消除。ORF 34的oriT区域和推导的松弛酶/切口酶蛋白的序列分别与其他接合质粒的oriT和松弛酶/切口酶没有显著的相似性。推测0 RF 34的松弛酶/切口酶蛋白可被归类为MOB（MG）家族的新成员。少