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

从黑链霉菌SN22中分离到一个11kbp的多拷贝质粒pSN22。PSN22是自传递的(接合)，在变铅青链霉菌中保持稳定，并在广泛的链霉菌菌株中形成麻点。突变分析表明，pSN22的一个片段包含5个与质粒转移和麻点形成有关的基因。TRAb是质粒转移所必需的。TRAA是麻点形成所必需的，但不是质粒转移所必需的。SPDA或SPDB突变使麻点变小。第五个基因Trag可以与其他基因一起缺失，得到不可传递的载体，但只在Trag内插入/缺失的载体变得不可见。体外启动子探测实验证明，一条550 bp的Bglll-SmaI DNA片段在两个方向都具有启动子活性；非杂交发现TraR和TraA-TRAB-SPDB操纵子的相应差异转录片段分别为1和5.2kb。TRAR基因产物抑制自身转录，也抑制traAtraB-SPDB操纵子的转录。含有功能性TRAb基因的质粒在TRAR不存在的情况下不能“存活”，无论是顺式还是反式，这可能是因为TRAb的非调控表达对宿主是致命的。含有TraA功能基因但不含TraR基因的质粒转化效率低，抑制了宿主细胞的生长；pSN22的转移促进了非可传递质粒的共转移，促进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细胞质的混合。

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.