K-092. Characterization of the Putative Anti-Sigma Factor Antagonist BldG in Streptomyces coelicolor

A. Parashar, B. K. Leskiw;
Univ. of Alberta, Edmonton, AB, CANADA.

Background: Streptomyces are renowned for their complex life cycle and production of various secondary metabolites including antibiotics. In Streptomyces coelicolor, several bld genes, including bldG, regulate morphological differentiation and antibiotic production. The bldG locus codes for BldG and Orf3, which show sequence homology to Bacillus anti-anti-sigma and anti-sigma factors, respectively. The Bacillus proteins regulate sigma factors by phosphorylation-dependent protein interactions. We hypothesize that BldG and Orf3 function as a regulatory pair to control target sigma factor(s) in S. coelicolor. Our goal is to identify this interaction and elucidate the role of BldG and Orf3. Methods: Chemical crosslinking was done on S. coelicolor cell extracts with a variety of protein crosslinkers, and complexes were purified by immunoaffinity chromatography using BldG antibodies. Protein interactions were analyzed using a Bacterial-two-hybrid (BTH) system based on interaction-mediated reconstitution of B. pertussis adenylate cyclase enzyme activity in E. coli. The role of BldG phosphorylation was investigated by analyzing interactions of Orf3 with two different mutant BldG proteins that mimic the phosphorylated and the nonphosphorylatable forms. Results: Comparative western analysis of various crosslinked extracts identified BldG-protein complexes of molecular weights corresponding to the size of the BldG dimer and the BldG-Orf3 complex. Purification of BldG and Orf3 from E. coli indicated that they form homodimers. BTH analyses confirmed interaction between BldG-Orf3, BldG-BldG, and Orf3-Orf3. The nonphosphorylatable form of BldG interacts with Orf3, while the phosphorylated form is unable to interact. Conclusion: Based on these results, we propose a model for BldG function: BldG and Orf3 interact as dimers, and most likely play antagonistic roles. The phosphorylation state of BldG affects interaction with Orf3, thus regulating BldG activity. This is the first experimental evidence of interaction, and the first indication that these proteins function as a pair to regulate morphological differentiation and antibiotic production in S. coelicolor.