Reciprocal Regulation of GlnR and PhoP in Response to Nitrogen and Phosphate Limitations in Saccharopolyspora erythraea

Abstract

Nitrogen and phosphate source sensing, uptake, and assimilation are essential for the growth and development of microorganisms. In this study, we demonstrated that SACE_6965 encodes the phosphate regulator PhoP, which controls the transcription of genes involved in phosphate metabolism in the erythromycin-producing Saccharopolyspora erythraea. We found that PhoP and the nitrogen regulator GlnR both regulate the transcription of glnR as well as other nitrogen metabolism-related genes. Interestingly, both GlnR-and PhoP-binding sites were identified in the phoP promoter region. Unlike the nonreciprocal regulation of GlnR and PhoP observed in Streptomyces coelicolor and Streptomyces lividans, GlnR negatively controls the transcription of the phoP gene in S. erythraea. This suggests that GlnR directly affects phosphate metabolism and demonstrates that the cross talk between GlnR and PhoP is reciprocal. Although GlnR and PhoP sites in the glnR and phoP promoter regions are located in close proximity to one another (separated by only 2 to 4 bp), the binding of both regulators to their respective region was independent and noninterfering. These results indicate that two regulators could separately bind to their respective binding sites and control nitrogen and phosphate metabolism in response to environmental changes. The reciprocal cross talk observed between GlnR and PhoP serves as a foundation for understanding the regulation of complex primary and secondary metabolism in antibiotic-producing actinomycetes.

GlnR and PhoP Directly Regulate the Transcription of Genes Encoding Starch-Degrading, Amylolytic Enzymes in Saccharopolyspora erythraea

“…In many actinobacterial species, nitrogen metabolism and phosphate metabolism are, respectively, mediated by the regulator GlnR and the two-component system PhoR-PhoP in response to nitrogen and phosphate availability (28,29,34,35). We investigated growth, starch consumption, and ␣-glucosidase activity of S. erythraea under the conditions of different nitrogen and phosphate availabilities.…”

“…The sequences of glnR and phoP were amplified from S. erythraea NRRL23338 genomic DNA by PCR using the primers shown in Table 2. The overexpression and purification of GlnR and PhoP in E. coli were performed as described previously (35,38). The purified proteins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined using the Bradford reagent.…”

“…Overexpression mutants were generated by protoplast transformation with pIB139 (Table 1) by inserting the 681 bp of the phoP open reading frame (SACE_6965) or 711 bp of glnR (SACE_7101) between NdeI and EcoRV (35,38), to obtain the phoP (pIB139-phoP) or glnR (pIB139-glnR) overexpression plasmid (Table 1). These plasmids were integrated into the S. erythraea genome by phiC31.…”

“…The MEME/MAST tools (http://meme -suite.org/) and the PREDetector software program were used to identify GlnR and PhoP binding sites in the upstream region of glucosidase-encoding genes in S. erythraea NRRL23338. The training set of sequences used to generate weight matrices and prediction methods has previously been described (34,35,41).…”

“…However, GlnR has no direct effect on phosphate metabolism (33). Interestingly, we have previously shown that S. erythraea possesses an extensive cross talk mechanism between PhoP and GlnR that is involved in the global coordination of nitrogen and phosphate metabolism (34,35). Recently, we found that GlnR controlled the transport of nonphosphotransferase system carbon sources by directly activating the transcription of most carbohydrate ABC transporter loci in response to nitrogen starvation in actinobacteria (36).…”