| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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