The wild-type strain harboring this plasmid exhibited the wild-ty

The wild-type strain harboring this plasmid exhibited the wild-type phenotype; it formed aerial mycelium (Fig. 1a) and produced normal levels of streptomycin (data not shown), thereby

indicating that bldG suppresses the inhibitory activity of rshA. Originally, bldG was identified by Leskiw and colleagues to be an essential regulator for the initiation of aerial mycelium formation and antibiotic production in S. coelicolor A3(2) (Bignell et al., 2000, 2003). The amino acid sequence similarity strongly suggests Tacrolimus cell line that the BldG product is an anti-sigma factor antagonist. The bldG gene and a downstream cds for a putative anti-sigma factor (SGR3306 in S. griseus) comprise an operon. This operon, located at a locus different from the rshA-sigH operon, does not contain any cds for sigma factor (Fig. 1b). The gene organization at the bldG locus is highly conserved in the genome of Streptomyces and related bacteria. To observe

the interaction between RshA and BldG, we carried out a two-hybrid analysis using an E. coli host–vector system. The measurement of β-galactosidase activity, which enabled the evaluation of interaction activity, showed that the activity of the transformants harboring the rshA-containing bait and bldG-containing target plasmid (63.6 × 10−5; ΔA410 min−1 μg−1) was considerably higher than that of the control strains harboring an empty bait or target plasmid INNO-406 (8.3–15.1 × 10−5; ΔA410 min−1 μg−1). The interaction activity between RshA and BldG was higher than that between RshA and σH-family sigma factors described previously (23.4–47.0 × 10−5ΔA410 min−1 μg−1) (Takano et al., 2003). To verify the interaction, we performed an in vitro pull-down assay. As shown in Fig. 2, during glutathione column chromatography for the mixture

of GST-RshA and BldG-6xHis recombinants, both proteins were collected in the same fraction (lane 5), indicating that the latter protein was bound to the former. The binding complex of the two proteins was also observed in a native PAGE analysis (Fig. S1). To study the role of bldG in S. griseus, we generated GNAT2 a knockout mutant by the standard homologous recombination technique. The bldG mutant was unable to form aerial mycelium and produce streptomycin (Fig. 1c), indicating that BldG plays an essential role in the developmental control of S. griseus. The bald phenotype of this mutant was restored to the wild type by introducing an integration plasmid carrying an intact bldG cassette (data not shown). Transcriptional analysis using a low-resolution S1 protection assay revealed that the activities of σH-dependent promoters were downregulated in the bldG mutant (Fig. 3a). Among the three promoters preceding the rshA-sigH operon (PH1, PH2, and PH3), the activity of PH1, the σH-dependent promoter (Takano et al., 2007), was considerably reduced by bldG knockout.

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