Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces coelicolor M145

GlnR is the global regulator of nitrogen assimilation in Streptomyces coelicolor M145 and other actinobacteria. Two-dimensional polyacrylamide gel electrophoresis analyses were performed to identify new GlnR target genes by proteomic comparison of wild-type S. coelicolor M145 and a ΔglnR mutant. Fifty proteins were found to be differentially regulated between S. coelicolor M145 and the ΔglnR mutant. These spots were identified by nanoHPLC–ESI-MS/MS and classified according to their cellular role. Most of the identified proteins are involved in amino acid biosynthesis and in carbon metabolism, demonstrating that the role of GlnR is not restricted to nitrogen metabolism. Thus, GlnR is supposed to play an important role in the global metabolic control of S. coelicolor M145.     

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.     

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Atypical OmpR/PhoB Subfamily Response Regulator GlnR of Actinomycetes Functions as a Homodimer,Stabilized by the Unphosphorylated Conserved Asp-focused Charge Interactions

The OmpR/PhoB subfamily protein GlnR of actinomycetes is an orphan response regulator that globally coordinates the expression of genes related to nitrogen metabolism. Biochemical and genetic analyses reveal that the functional GlnR from Amycolatopsis mediterranei is unphosphorylated at the potential phosphorylation Asp⁵⁰ residue in the N-terminal receiver domain. The crystal structure of this receiver domain demonstrates that it forms a homodimer through the α4-β5-α5 dimer interface highly similar to the phosphorylated typical response regulator, whereas the so-called “phosphorylation pocket” is not conserved, with its space being occupied by an Arg⁵² from the β3-α3 loop. Both in vitro and in vivo experiments confirm that GlnR forms a functional homodimer via its receiver domain and suggest that the charge interactions of Asp⁵⁰ with the highly conserved Arg⁵² and Thr⁹ in the receiver domain may be crucial in maintaining the proper conformation for homodimerization, as also supported by molecular dynamics simulations of the wild type GlnR versus the deficient mutant GlnR(D50A). This model is backed by the distinct phenotypes of the total deficient GlnR(R52A/T9A) double mutant versus the single mutants of GlnR (i.e. D50N, D50E, R52A and T9A), which have only minor effects upon both dimerization and physiological function of GlnR in vivo, albeit their DNA binding ability is weakened compared with that of the wild type. By integrating the supportive data of GlnRs from the model Streptomyces coelicolor and the pathogenic Mycobacterium tuberculosis, we conclude that the actinomycete GlnR is atypical with respect to its unphosphorylated conserved Asp residue being involved in the critical Arg/Asp/Thr charge interactions, which is essential for maintaining the biologically active homodimer conformation.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.     

A complex role of <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis> GlnR in nitrogen metabolism and related antibiotics production

Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain’s secondary metabolism, resemblance to the phenotypes of a unique S. coelicolor glnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.