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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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Promoter activity in S. coelicolor M145 (squares, solid lines) and ΔphoP mutant (triangles, dashed lines) of glnR (A), glnA (B), glnII (C) and amtB (D) using the luxAB genes as reporter. Error bars correspond to the standard error of triplicated cultures in MG-3.2.
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Figure 4: Promoter activity in S. coelicolor M145 (squares, solid lines) and ΔphoP mutant (triangles, dashed lines) of glnR (A), glnA (B), glnII (C) and amtB (D) using the luxAB genes as reporter. Error bars correspond to the standard error of triplicated cultures in MG-3.2.

Mentions: Using these reporter constructions it was clearly observed that the four promoter regions were repressed by PhoP since reporter enzyme higher activities were observed in the ΔphoP mutant with respect to parental strain at all sampling times (Figure 4A–D). The reporter expression patterns, varied among the promoters assayed. Thus, compared to the other assayed promoters (of phosphate transporter genes pstS, pitH1 and pitH2, and of glycerophosphodiesterase genes glpQ1 and glpQ2) [(23,25), Santos-Beneit, unpublished] the promoter region of the glnR gene produced usual or low activities across the time course of the culture. Indeed, in the wild-type strain the luminescence signals were below the instrument detection limit at 70 h (Figure 4A). In contrast, the structural glnA, glnII and amtB genes appeared to have very strong promoters. Among these, glnIIp showed the highest activities in the wild-type strain. Besides, maximum activities of the different promoters were reached at distinct growth phases. In both strains glnRp activities increased until the transition phase (45 h, Figure 4A), and decreased thereafter. For the glnA and amtB promoter activities, the highest values were at the exponential growth phase in the wild-type (35 h, Figure 4B and D). The glnIIp showed high activities during the first growth phase and a maximum activity at 42 h (at the initial transition phase, Figure 4C). In all cases, the wild-type activities dropped rapidly after the transition phase resulting in low values in the stationary phase. In contrast, deletion of the phoP gene caused slower promoter activity drops, as seen in the INB201 plots (Figure 4A–D).Figure 4.


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)

Promoter activity in S. coelicolor M145 (squares, solid lines) and ΔphoP mutant (triangles, dashed lines) of glnR (A), glnA (B), glnII (C) and amtB (D) using the luxAB genes as reporter. Error bars correspond to the standard error of triplicated cultures in MG-3.2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Promoter activity in S. coelicolor M145 (squares, solid lines) and ΔphoP mutant (triangles, dashed lines) of glnR (A), glnA (B), glnII (C) and amtB (D) using the luxAB genes as reporter. Error bars correspond to the standard error of triplicated cultures in MG-3.2.
Mentions: Using these reporter constructions it was clearly observed that the four promoter regions were repressed by PhoP since reporter enzyme higher activities were observed in the ΔphoP mutant with respect to parental strain at all sampling times (Figure 4A–D). The reporter expression patterns, varied among the promoters assayed. Thus, compared to the other assayed promoters (of phosphate transporter genes pstS, pitH1 and pitH2, and of glycerophosphodiesterase genes glpQ1 and glpQ2) [(23,25), Santos-Beneit, unpublished] the promoter region of the glnR gene produced usual or low activities across the time course of the culture. Indeed, in the wild-type strain the luminescence signals were below the instrument detection limit at 70 h (Figure 4A). In contrast, the structural glnA, glnII and amtB genes appeared to have very strong promoters. Among these, glnIIp showed the highest activities in the wild-type strain. Besides, maximum activities of the different promoters were reached at distinct growth phases. In both strains glnRp activities increased until the transition phase (45 h, Figure 4A), and decreased thereafter. For the glnA and amtB promoter activities, the highest values were at the exponential growth phase in the wild-type (35 h, Figure 4B and D). The glnIIp showed high activities during the first growth phase and a maximum activity at 42 h (at the initial transition phase, Figure 4C). In all cases, the wild-type activities dropped rapidly after the transition phase resulting in low values in the stationary phase. In contrast, deletion of the phoP gene caused slower promoter activity drops, as seen in the INB201 plots (Figure 4A–D).Figure 4.

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