Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.
Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.
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对参与接合转移功能正向调节的粪肠球菌质粒 pCF10 区域进行遗传分析。
DOI：
10.1128/jb.177.8.2107-2117.1995
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发表时间：
1995
影响因子：
3.2
通讯作者：
Dunny,GM
中科院分区：
文献类型：
--
作者：
Chung,JW;Bensing,BA;Dunny,GM
prgB基因编码的表面蛋白Asc 10，介导细胞聚集，导致在粪肠球菌中的信息素诱导的四环素抗性质粒pCF 10的高频接合转移。先前对pCF 10的12-kb片段进行的Tn 5插入诱变和测序分析表明，激活prgB的表达需要位于prgB上游3 - 6 kb的含有prgX、-Q、-R、-S和-T的区域。互补研究表明，正调控区以方向依赖性方式顺式发挥功能（J. W. Chung和G. M. Dunny，Proc. Natl. Acad. Sci. USA 89：9020-9024，1992）。为了确定每个基因参与prgB的激活，进行调节区的Tn 5插入诱变和核酸外切酶III缺失分析。结果表明，prgB的表达需要prgQ和-S，而不需要prgX、-R和-T。这些突变体的蛋白质印迹（免疫印迹）分析表明，prgQ也是必不可少的表达prgA（编码表面排斥蛋白Sec 10），这是位于prgB和阳性对照区之间。互补分析表明，顺式作用调控元件位于prgQ区域，并且位于prgQ 3'端非翻译区的pCF 10序列是阳性对照系统的重要组成部分。对pCF 10基因中各种Tn 5插入的分析表明，该转座子的转录阅读在E.外读转录本可能起始于Tn 5的末端或连接序列。
The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.