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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.

Rodríguez-García A, Sola-Landa A, Apel K, Santos-Beneit F, Martín JF - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006, León, Spain.

ABSTRACT
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.

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Analysis by EMSA of the promoters. Lane P, probe without protein; lanes 1–4, increasing concentrations of GST-PhoPDBD (0.125, 0.25, 0.5 and 1 µM, respectively). An excess (more than 1000-fold) of poly[d(I-C)] is included in every lane as internal control to avoid an unspecific binding of the protein to the DNA. Controls with competing excess of unlabelled probe are shown as Supplementary Data (Figure S1). The different shift bands are indicated by arrows. The assays were repeated three times.
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Figure 1: Analysis by EMSA of the promoters. Lane P, probe without protein; lanes 1–4, increasing concentrations of GST-PhoPDBD (0.125, 0.25, 0.5 and 1 µM, respectively). An excess (more than 1000-fold) of poly[d(I-C)] is included in every lane as internal control to avoid an unspecific binding of the protein to the DNA. Controls with competing excess of unlabelled probe are shown as Supplementary Data (Figure S1). The different shift bands are indicated by arrows. The assays were repeated three times.

Mentions: The glnR 5′-region was cloned by PCR as a fragment of 362 bp that also included 98 bp of the upstream coding sequence (CDS). Electrophoretic mobility shift assays (EMSA) of this fragment with the GST-PhoPDBD protein revealed the formation of three retarded bands (Figure 1). As reported previously (11), each retarded DNA–protein complex correspond to a number of protein monomers bound to the DNA fragment. The established model of the PhoP-binding site indicates that each PhoP monomer binds a direct repeat unit (DRu) of 11 nt. Two or three consecutive DRus form the core of the binding site. Once the core is occupied, further protein monomers can bind adjacent DRus, what account for the DNA–protein complexes of lower electrophoretic mobility (see the detailed analysis below).Figure 1.


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.

Rodríguez-García A, Sola-Landa A, Apel K, Santos-Beneit F, Martín JF - Nucleic Acids Res. (2009)

Analysis by EMSA of the promoters. Lane P, probe without protein; lanes 1–4, increasing concentrations of GST-PhoPDBD (0.125, 0.25, 0.5 and 1 µM, respectively). An excess (more than 1000-fold) of poly[d(I-C)] is included in every lane as internal control to avoid an unspecific binding of the protein to the DNA. Controls with competing excess of unlabelled probe are shown as Supplementary Data (Figure S1). The different shift bands are indicated by arrows. The assays were repeated three times.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691820&req=5

Figure 1: Analysis by EMSA of the promoters. Lane P, probe without protein; lanes 1–4, increasing concentrations of GST-PhoPDBD (0.125, 0.25, 0.5 and 1 µM, respectively). An excess (more than 1000-fold) of poly[d(I-C)] is included in every lane as internal control to avoid an unspecific binding of the protein to the DNA. Controls with competing excess of unlabelled probe are shown as Supplementary Data (Figure S1). The different shift bands are indicated by arrows. The assays were repeated three times.
Mentions: The glnR 5′-region was cloned by PCR as a fragment of 362 bp that also included 98 bp of the upstream coding sequence (CDS). Electrophoretic mobility shift assays (EMSA) of this fragment with the GST-PhoPDBD protein revealed the formation of three retarded bands (Figure 1). As reported previously (11), each retarded DNA–protein complex correspond to a number of protein monomers bound to the DNA fragment. The established model of the PhoP-binding site indicates that each PhoP monomer binds a direct repeat unit (DRu) of 11 nt. Two or three consecutive DRus form the core of the binding site. Once the core is occupied, further protein monomers can bind adjacent DRus, what account for the DNA–protein complexes of lower electrophoretic mobility (see the detailed analysis below).Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006, León, Spain.

ABSTRACT
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.

Show MeSH