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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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DNase I footprints of the GST-PhoPDBD protein bound to the promoter regions of glnR (A, B), glnA (C, D), glnII (E, F) and amtB (G, H). In each panel, the upper electrophoregram (black line) is the control reaction. The protected nucleotide sequence is boxed; partially protected nucleotides (*), and hypersensitive sites (arrows) are also indicated. Sequencing reactions are not included except in panel A. Coordinates are from the translation start codon.
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Figure 2: DNase I footprints of the GST-PhoPDBD protein bound to the promoter regions of glnR (A, B), glnA (C, D), glnII (E, F) and amtB (G, H). In each panel, the upper electrophoregram (black line) is the control reaction. The protected nucleotide sequence is boxed; partially protected nucleotides (*), and hypersensitive sites (arrows) are also indicated. Sequencing reactions are not included except in panel A. Coordinates are from the translation start codon.

Mentions: To locate the PhoP-binding site, we carried out DNase I footprinting experiments. Electrophoretic separation of digestion products was facilitated by using a smaller fragment of 257 bp that comprised only the glnR promoter sequence. The GST-PhoPDBD protein at a concentration of 2 μM protected from DNase I digestion a stretch of 33 nt located at positions −139 to −107 in the coding strand (all coordinates are referred to the translation start site; Figure 2A). This stretch comprised a DRu with sequence matching the first seven PHO box consensus bases (GTTCACC). In addition, some upstream and downstream nucleotides were protected to a lesser extent by protein binding. As previously observed in other PhoP footprintings (11), DNase I hypersensitive sites appeared next to the binding site at its 3′-end. The complementary strand showed protection from −112 to −140 nt, partial protections up to position −103, and hypersensitive sites at positions −144 and −145 (Figure 2B).Figure 2.


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)

DNase I footprints of the GST-PhoPDBD protein bound to the promoter regions of glnR (A, B), glnA (C, D), glnII (E, F) and amtB (G, H). In each panel, the upper electrophoregram (black line) is the control reaction. The protected nucleotide sequence is boxed; partially protected nucleotides (*), and hypersensitive sites (arrows) are also indicated. Sequencing reactions are not included except in panel A. Coordinates are from the translation start codon.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: DNase I footprints of the GST-PhoPDBD protein bound to the promoter regions of glnR (A, B), glnA (C, D), glnII (E, F) and amtB (G, H). In each panel, the upper electrophoregram (black line) is the control reaction. The protected nucleotide sequence is boxed; partially protected nucleotides (*), and hypersensitive sites (arrows) are also indicated. Sequencing reactions are not included except in panel A. Coordinates are from the translation start codon.
Mentions: To locate the PhoP-binding site, we carried out DNase I footprinting experiments. Electrophoretic separation of digestion products was facilitated by using a smaller fragment of 257 bp that comprised only the glnR promoter sequence. The GST-PhoPDBD protein at a concentration of 2 μM protected from DNase I digestion a stretch of 33 nt located at positions −139 to −107 in the coding strand (all coordinates are referred to the translation start site; Figure 2A). This stretch comprised a DRu with sequence matching the first seven PHO box consensus bases (GTTCACC). In addition, some upstream and downstream nucleotides were protected to a lesser extent by protein binding. As previously observed in other PhoP footprintings (11), DNase I hypersensitive sites appeared next to the binding site at its 3′-end. The complementary strand showed protection from −112 to −140 nt, partial protections up to position −103, and hypersensitive sites at positions −144 and −145 (Figure 2B).Figure 2.

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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