Separate and coordinate transcriptional control mechanisms link expression of the potentially lethal KilB spread locus to the upstream transmission operon on Streptomyces plasmid pIJ101

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Efficient conjugation of the high copy plasmid pIJ101 among members of the bacterial genus Streptomyces depends on a single plasmid gene (tra) for initial inter-mycelial transfer, and involves three additional pIJ101 functions (spdA, spdB, and kilB), which may promote intra-mycelial spread of the plasmid upon its entrance into the recipient. The genes tra, spdA, and spdB are co-transcribed as part of an operon, whose expression is negatively controlled by the pIJ101 repressor KorA. Downstream of this transmission operon and in the same orientation, the kilB spread gene possesses its own promoter, which is recognized by the pIJ101 KorB repressor protein; binding of KorB appears to prevent the lethal overexpression of the KilB protein, which otherwise shows a temporally increasing pattern of production or accumulation during the streptomycete life cycle. To define better the mechanism(s) controlling the concentration of the potentially toxic KilB protein in cells, a variety of transcriptional analyses involving the kilB promoter and kilB-specific mRNA were performed. These studies demonstrated that transcription originating from the kilB promoter on pIJ101 is dramatically reduced by KorB binding under non-mating conditions; more significantly, however, as judged by evidence of readthrough transcription across the kilB promoter region and polarity effects of upstream insertion and deletion mutations, kilB was found to be expressed also as part of the transmission operon with optimal KilB production being necessarily tied to such co-transcription. Our data indicate that the genes tra, spdA, spdB, and kilB comprise an unusual operon in which separate tight control of the distal gene (kilB) by the KorB repressor is superimposed on coordinate regulation of full operon transcription by KorA. Moreover, our results imply that potential interactions between elongating RNA polymerase molecules synthesizing transmission operon transcripts and KorB repressor bound to the intercistronic kilB promoter region are important for modulating kilB expression.

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Journal of molecular biology

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