Molecular genetic analysis of pheromone-independent highly transferable drug resistance plasmid of Enterococcus.

1)通过北方印迹分析和RT-PCR确定转录单位。它们是ORF 13 -15、ORF 16 -19、ORF 20 -43和ORF 44 -49。其中最大的一个ORF 20 -43由位于ORF 20前的强启动子转录，克隆的ORF 20基因反式提供的ORF 20产物增加了ORF 20启动子的LacZ融合活性，表明ORF 20是该转录产物的正调控因子。2）ORF 20上游插入导致ORF 20 -43的过表达，表明存在ORF 20 -43表达的负调节因子。通过体外转座子诱变对补充插入效应的片段进行诱变以鉴定负调节子。负调控子位于ORF 13的末端和ORF 15的起始端之间，但需要ORF 13启动子的转录本，因为ORF 13本身不互补，ORF 15中的插入也不互补。它位于ORF 44前180 bp的区域。将含有180 bp区域的oriT质粒用pMG 1动员，并<-4>在3 h肉汤交配中以10 μ g/供体细胞的频率转移。4）提取接合缺陷的受体突变体和亲本的细胞表面蛋白，并通过2D SDS-PAGE分析。到目前为止，突变体的蛋白质谱与亲本没有差异。

1)Transcription units were determined by northern blot analysis and RT-PCR. They were ORF13-15,ORF16-19,ORF20-43 and ORF44-49. The biggest one, ORF20-43, was transcribed from strong promoter in front of ORF20 and ORF20 product supplied in trans by cloned ORF20 gene increased the activity of LacZ fusion of ORF20 promoter, indicating that ORF20 was a positive regulator for this transcript.2)The insertion upstream of ORF20 resulted in the overexpression of ORF20-43, indicating the presence of a negative regulator for ORF20-43 expression. The fragment, which complemented the effect of the insertion, was mutagenized by in vitro transposon mutagenesis to identify the negative regulator. The negative regulator was located between the end of ORF13 and the start of ORF15, but it needed the transcript from ORF13 promoter, because ORF13 alone did not complement and the insertion in ORF15 did not complement either.3)OriT region was determined. It was located in the region of 180bp just in front of ORF44. The oriT plasmid carrying the 180bp region was mobilized by pMG1 and transferred at the frequency of 10^<-4> per donor cell in 3 h broth mating.4)Cell surface proteins of conjugation-defective recipient mutant and parent were extracted and analyzed by 2D SDS-PAGE. No difference was observed between the protein profile of the mutant and that of parent, so far.