The RNA polymerase omega factor RpoZ is regulated by PhoP and has an important role in antibiotic biosynthesis and morphological differentiation in Streptomyces coelicolor

The RNA polymerase (RNAP) omega factor (ω) forms a complex with the α₂ββ′ core of this enzyme in bacteria. We have characterized the rpoZ gene of Streptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of the rpoZ gene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZ strain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZ mutant with the wild-type rpoZ allele restored both phenotype and antibiotic production. Interestingly, the rpoZ gene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed that rpoZ promoter activity was increased in a ΔphoP background, it can be concluded that rpoZ is controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation in Streptomyces.     

New loci required for Streptomyces coelicolor morphological and physiological differentiation.

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.     

Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors.

Previous studies showed that Streptomyces coelicolor A3(2) has four genes (hrdA, hrdB, hrdC, and hrdD) that appear to encode RNA polymerase sigma factors very similar to the sigma 70 subunit of Escherichia coli and that hrdC and hrdD could be individually disrupted without causing obvious phenotypic defects. Here, hrdA was cloned and stable null hrdA and hrdD mutants were constructed by gene replacement. These two mutants and a previously constructed hrdC null mutant were used in crosses to generate hrdAC, hrdAD, hrdCD, and hrdACD strains. The inability to synthesize one, two, or all three of the nonessential hrd-encoded sigma factors had no obvious phenotypic consequences.     

A new RNA polymerase sigma factor, σF is required for the late stages of morphological differentiation in Streptomyces spp.

A gene ( sigF ) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial σ⁷⁰ family. Homologues were present in other Streptomyces spp., and that of the genetically well-studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 ( S. coelicolor ) and 295 ( S. aureofaciens ) amino acid residues, both showing greatest similarity to σ^B of Bacillus subtilis . However, while σ^B is involved in stationary-phase gene expression and in the general stress response in B. subtilis , σ^F affects morphological differentiation in Streptomyces , Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. σ^F is proposed to control the late stages of spore development in Streptomyces .     

Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.     

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ^F, suggesting that it is an antagonistic regulator of σ^F. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ^F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.