Molecular gentic analysis of pheromone-independent highly transferable plasmid of Enterococci

1.选用粪肠球菌耐药质粒pMG1(Gm^r，Km^r)，研究tra基因在接合过程中的调控。为了揭示trA基因调控系统的存在，我们搜索了在接合过程中转录受到调控的质粒基因。一个名为71ORF2的基因转录本在20分钟的匹配中被发现增加。通过基因阻断实验，发现71个ORF2基因与交配聚集体的形成或稳定有关。71ORF2基因在部分反式突变株中呈结构性表达，提示71ORF2基因处于负调控状态。在tra13101突变株中，710RF2基因得到了结构性表达，对trA 13101突变进行了定位和测序，发现75ORF4基因调控71ORF2基因的表达。接合反应使75ORF4基因的转录本减少，而71ORF2基因的转录本增加。从75ORF4基因在反式突变株中的表达模式来看，75ORF4基因似乎受到多种方式的调控。

1. Enterocuccus faecium drug resistance plasmid pMG1 (Gm^r, Km^r) was chosen to study the regulation of tra genes in conjugation. To reveal the presence of the regularotry system of tra genes, we searched the plasmid gene, of which transcription was regulated during conjugation. One transcript of the gene named 71ORF2 was found to increase in 20 min mating.2. By gene disruption experiment, 71 ORF2 gene was found to be responsible for the formation or stabilization of mating aggregates.3. 71ORF2 gene was expressed constitutively in some tra mutants, implying that 71ORF2 gene is negatively regulated.4. In tra13101 mutant strain 710RF2 gene was expressed constitutively.tra 13101 mutation was mapped and DNA sequence around it was determined.75ORF4 gene was found to regulate the expression of 71ORF2 gene.5. Transcript of 75ORF4 gene was found to decrease in response to conjugation, while that of 71ORF2 gene incresed.6. From the patterns of 75ORF4 gene expression in tra mutant strains, 75ORF4 gene seems to be regulated by more than one way.