Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.The ability of organisms to rearrange nucleic acid secondary structure is crucial for cellular function and is catalyzed by a diverse range of proteins or protein complexes that facilitate nucleic acid annealing and unwinding. Two protein families, nucleic acid-binding proteins and helicases, catalyze these reactions. RNA-binding proteins are structurally unrelated to helicases (1, 2) and rearrange RNA secondary structure through chaperone-mediated annealing or unwinding in ATPindependent reactions (3, 4). Helicases have been classified into five major groups based on characteristic amino acid motifs with the two largest families, superfamilies 1 and 2, composed of RNA and DNA helicases (5). The other helicase families include proteins possessing fewer conserved motifs and having different substrate specificities.Biochemically, helicases function as ATP-driven molecular motors, catalyzing NTP-dependent nucleic acid duplex destabilization or strand displacement (6, 7). Although a number of RNA helicases possess RNA unwinding activity in vitro, only three have been reported to exhibit intrinsic RNA annealing activity, the highly related yeast nuclear DEAD-box RNA helicases, p68 and p72 (8), and the nucleolar DExD-box protein, RNA helicase II/Gu (9, 10). Although these helicases unwind dsRNA, 1 the RNA substrates on which they catalyze RNA annealing differ with p68/72 capable of annealing complementary ssRNA...

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.Streptomyces spp. are unique among prokaryotic organisms because their life cycle involves filamentous, vegetative mycelia and a series of complex morphological changes resulting in the formation of aerial hyphae and chains of unigenomic spores.In Streptomyces coelicolor, the genetically best-characterized streptomycete, a number of genes have been characterized that function in the global regulation of morphological differentiation. Such genes are referred to as bld (bald) genes, since mutants lack the characteristic fuzzy coating of aerial hyphae seen in the wild-type strain. Interestingly, some of the bld mutants that have been isolated are also defective in the production of secondary metabolites, such as antibiotics, suggesting that the corresponding bld genes are involved in the regulation of both differentiation processes.To date, 14 S. coelicolor bld mutants have been isolated and eight of the bld genes have been cloned and characterized. The bld gene products are diverse in nature (7,16,28,33,35,36,43,53) and include a putative anti-anti-sigma factor, BldG (11) that is the subject of this study. Characterization of the bldG locus (11) revealed both a bldG-complementing ge...

The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.

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...