Primer extension with RNA from an RNase III null mutant of Streptomyces coelicolor M145 and a primer complementary to the polynucleotide phosphorylase gene revealed two major extension products. Two different extension products were observed using RNA from either wild type M145 or the null mutant with a primer complementary to rpsO. Mapping of the 5'-ends of these extension products to the rpsO-pnp intergenic region indicated that all four putative transcription start sites were preceded by possible promoter sequences. These putative promoters were synthesized by the PCR and cloned into pIPP2, a xylE-based streptomycete promoter probe vector. Transfer of the pIPP2 derivatives to S. coelicolor and catechol dioxygenase assays demonstrated that all four cloned fragments had promoter activity in vivo. The activities of the four promoters changed over the course of growth of S. coelicolor and studies in three sigma factor mutant strains demonstrated that three of the promoters were σ(B) dependent. Northern blotting studies showed that the levels of the rpsO-pnp transcripts remained relatively constant over the course of growth of S. coelicolor M145, but that on a molar basis, the levels of the readthrough and pnp transcripts were considerably lower than those of rpsO. PNPase is a cold shock protein in S. coelicolor and the activity of the rpsO-pnp promoters increased during cold shock at 10°, resulting in a two-fold increase in PNPase activity, compared with the activity at 30°.
Keywords: APE; CATO(2)ase; Cold shock; PCR; PNPase; Polynucleotide phosphorylase; Promoter; SFM; SMM; Sigma factor; Streptomyces; Streptomyces minimal medium; acetone-phosphate-EDTA; catechol dioxygenase; polymerase chain reaction; polynucleotide phosphorylase; rpsO-pnp; soya flour-mannitol.
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