Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. ‘Information theory’ quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.     

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.     

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

Two-component regulatory systems play a key role in the cell metabolism adaptation to changing nutritional and environmental conditions. The fidelity between the two cognate proteins of a two-component system is important since it determines whether a specific response regulator integrates the signals transmitted by different sensor kinases. Phosphate regulation in Streptomyces coelicolor is mostly mediated by the PhoR-PhoP two-component system. Previous studies elucidated the mechanisms that control phosphate regulation as well as the genes directly regulated by the response regulator PhoP (pho regulon) in this organism. However, the role of the histidine kinase PhoR in Streptomyces coelicolor had not been unveiled so far. In this work, we report the characterization of a non-polar ΔphoR deletion mutant in S. coelicolor that keeps its native promoter. Induction of the phoRP operon was dependent upon phosphorylation of PhoP, but the ΔphoR mutant expressed phoP at a basal level. RT-PCR and reporter luciferase assays demonstrated that PhoR plays a key role in the activation of the pho regulon in this organism. Our results point towards a strict cognate partner specificity in terms of the phosphorylation of PhoP by PhoR thus corroborating the tight interaction between the two-components of this system.     

Overlapping binding of PhoP and AfsR to the promoter region of glnR in Streptomyces coelicolor

Growth of soil bacteria is often limited by the availability of essential nutrients such as carbon, nitrogen and phosphate. The reaction to a specific nutrient starvation triggers interconnected responses to equilibrate the metabolism. It is known that PhoP (response regulator involved in phosphate control) specifically binds to several promoters of genes involved in nitrogen metabolism which are also regulated by GlnR (regulator involved in nitrogen control). In this article we report a novel cross-talk between GlnR and the SARP-like regulator, AfsR. AfsR binds to some PhoP-regulated promoters including those of afsS (a small regulatory protein of secondary metabolism), pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). We have characterized the regulation exerted upon the nitrogen regulator glnR gene by AfsR, using EMSA and DNase I footprinting assays as well as in vivo expression studies with ΔphoP, ΔafsR and ΔafsR-ΔphoP mutants. Both PhoP and AfsR proteins are able to bind to overlapping regions within the glnR promoter producing different effects. This work demonstrates a cross-talk of three different regulators of both primary and secondary metabolism.