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Functional Analysis of Transmission of a Streptomyces plasmid and Its Application for Streptomyces Genetics.

链霉菌质粒传播的功能分析及其在链霉菌遗传学中的应用。

基本信息

批准号：
02660118
负责人：
SEKI Tatsuji
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660118/
关键词：
Streptomyces Plasmid Conjugation Plasmid Transmission Gene Expression Pock Formation Mutation Tranformation 形貭転換

项目摘要

A 11 kbp multi-copy plasmid, pSN22, was isolated from Streptomyces nigrifaciens SN22. pSN22 is self-transmissible (conjugative) , maintained stably in S. lividans, and forms pocks in a wide range of Streptomyces strains. Mutational analyses showed that a fragment of pSN22 contained five genes involved in plasmid transfer and pock-formation. trab was essential for plasmid transfer. traa was required for pock-formation, but not for plasmid transfer. Mutation of spda or spdb decreased the pock size. The fifth gene, trag, could be deleted together with other genes to give non-transmissible plasmids, but plasmids with insertions/deletions only within trag became non-viable.In vitro promoter-probing experiments identified a 550 bp Bglll-SmaI DNA fragment with promoter activity in both orientations ; nonhem hybridization identified corresponding divergent transcripts of 1 and 5.2 kb for traR and the traA-traB-spdB operon, respectively. The traR gene product repressed its own transcription and also the transcription of the traAtraB-spdB operon. Plasmids containing a functional traB gene could not "survive" without traR being present in the same cell either in cis or in trans, presumably because unregulated expression of traB is lethal to the host. Plasmids with a functional traA gene but without traR gave a low transformation efficiency and inhibited the growth of host cells.The plasmid transfer of pSN22 promoted the co-transfer of non-transmissible plasmids and enhanced the chromosome recombination between the host and recipient strains, suggesting that plasmid transfer accompanied cytoplasmic mixing.

从产黑链霉菌SN 22中分离到一个11 kbp的多拷贝质粒pSN 22。pSN 22是一种自传的接合型基因，在S.变铅青，并在广泛的链霉菌菌株中形成麻点。突变分析表明，pSN 22的片段含有五个参与质粒转移和麻点形成的基因。TRAB是质粒转移所必需的。TRAA是痘形成所必需的，但不是质粒转移所必需的。spda或spdb的突变减少了痘的大小。第五个基因trag可以与其他基因一起缺失，得到非传递性质粒，但仅在trag内插入/缺失的质粒变得不能存活。nonhem杂交分别鉴定了traR和traA-traB-spdB操纵子的1和5.2kb的相应不同转录物。traR基因产物抑制其自身的转录以及traAtraB-spdB操纵子的转录。含有功能性traB基因的质粒不能在没有traR以顺式或反式存在于同一细胞中的情况下“存活”，这可能是因为traB的不受调节的表达对宿主是致命的。含有功能性traA基因而不含traR基因的质粒转化效率低，抑制宿主细胞生长，pSN 22质粒转移促进了非传递性质粒的共转移，增强了宿主和受体菌株之间的染色体重组，表明质粒转移伴随着胞质混合。