BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes inStreptomyces coelicolor

Abstract

The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.Streptomyces coelicolor A3(2), the well-studied model organism for processes of bacterial multicellular development and antibiotic production, possesses a large genome (8.67 Mbp) with a high degree of regulatory complexity (6). A large proportion of the coding sequence (12.3%) is predicted to encode the multitude of regulatory factors required to support a complex life cycle, involving the formation of sporulating aerial hyphae, that responds to the changing soil environment. Of particular note is the presence of 64 sigma factors, which are thought to play a critical role in the modulation of gene expression; this group is comprised of 4 housekeeping sigma factors, as well as 50 extracytoplasmic function sigma factors and 9 group 3 subfamily sigma factors (6, 23). The activity of alternative sigma factors is typically regulated by a number of mechanisms, including phosphorylation-dependent partner switching by antagonistic proteins. The best-studied examples of this regulatory mechanism, which is active against the group 3 sigma factors, are found in Bacillus subtilis, where partner switching controls the activity of both the sporulation-specific factor F and the general stress response factor B (1,17,18,44,50,57,59). In these systems, an anti-sigma factor protein (SpoIIAB and RsbW, respectively) sequesters the cognate sigma factor, preventing the expression of target genes. Upon sensing some activating signal, an anti-sigma factor antagonist (or anti-anti-sigma factor; SpoIIAA and RsbV, respectively) binds to the anti-sigma factor, mediating release of the active sigma factor to direct regulon transcription.Partner-switching systems are also thought to play a critical role in sigma factor regulation in S. coelicolor. The first characterized example in this organism is the RsbV-RsbA partnerswitching pair that controls the activity of the osmotic-stressresponsive factor B (36). Many genes encoding additional putative paralogues of these regulatory factors are...

The Anti-Anti-Sigma Factor BldG Is Involved in Activation of the Stress Response Sigma Factor σ H in Streptomyces coelicolor A3(2)

“…Interestingly, in contrast to ApgA, which formed a complex with BldG at an ϳ1:1 molar ratio, UshX and BldG have been found to form a complex at an ϳ1:2 molar ratio. The 1:1 molar ratio of the ApgA-BldG complex is consistent with the proposed heterotetrameric complex consisting of an ApgA dimer interacting with a BldG dimer (43). Assuming that BldG forms a dimer, it is likely that it may interact with one molecule of UshX.…”

“…BldG has also been found to interact with another antisigma factor homologue, ApgA (SCO3548) (43), as verified also by our results. Based on these results, BldG has several anti-sigma partners to regulate, and in addition to H , likely also other B homologues, as H does not interact with the other BldG-specific anti-sigma factor, ApgA (Fig.…”

“…2, confirmed that BldG and UshX physically interact with each other. Interestingly, based on the activity of ␤-galactosidase, this interaction seems to be stronger than the previously verified interaction between SigH and UshX (47) and even between BldG and ApgA (43). The interaction was comparable to that for the positive BACTH control leucine zipper interaction (20).…”

“…Downstream of bldG is the gene apgA (SCO3548) that encodes a homologue of B. subtilis anti-sigma factor RsbW, and these two genes are cotranscribed (7). BldG has been shown to interact with ApgA as a part of a regulatory system to control both antibiotic production and differentiation in S. coelicolor, suggesting that they form a partner-switching pair; however, no target sigma factor has been identified for this system (43).…”

Regulation of an alternative sigma factor σI by a partner switching mechanism with an anti-sigma factor PrsI and an anti-anti-sigma factor ArsI in Streptomyces coelicolor A3(2)

“…An anti-sigma factor RsbA (SCO0599) with an anti-anti-sigma factor RsbV (SCO7325) has been shown to be involved in osmotic stress activation of σ B (Lee et al, 2004a), an anti-sigma factor RsmA (SCO7313) regulates the activity of σ M (Gaskell et al, 2007), an anti-sigma factor RsfA has been found to interact with σ F and two homologues for antianti-sigma factors, SCO0781 and SCO0869 (Kim et al, 2008), and an anti-anti-sigma factor BldG (SCO3549) has been shown to be involved in osmotic stress activation of σ H , through sequestering its anti-sigma factor UshX/PrsH (SCO5244) (Sevcikova et al, 2010). The regulation seems to be more complex, as BldG has been shown to interact with an anti-sigma factor ApgA (Parashar et al, 2009;Sevcikova et al, 2010).…”