Dataset Information

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.

SUBMITTER: Yao LL

PROVIDER: S-EPMC4702613 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

Yao Li-Li LL Ye Bang-Ce BC

Applied and environmental microbiology 20151030 1

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. Interesting ...[more]

PMID: 26519391

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Project description:Starch-degrading enzymes hydrolyze starch- and starch-derived oligosaccharides to yield glucose. We investigated the transcriptional regulation of genes encoding starch-degrading enzymes in the industrial actinobacterium Saccharopolyspora erythraea We observed that most genes encoding amylolytic enzymes (one α-amylase, one glucoamylase, and four α-glucosidases) were regulated by GlnR and PhoP, which are global regulators of nitrogen and phosphate metabolism, respectively. Electrophoretic mobility shift assays and reverse transcription-PCR (RT-PCR) analyses demonstrated that GlnR and PhoP directly interact with their promoter regions and collaboratively or competitively activate their transcription. Deletion of glnR caused poor growth on starch, maltodextrin, and maltose, whereas overexpression of glnR and phoP increased the total activity of α-glucosidase, resulting in enhanced carbohydrate utilization. Additionally, transcript levels of amylolytic genes and total glucosidase activity were induced in response to nitrogen and phosphate limitation. Furthermore, regulatory effects of GlnR and PhoP on starch-degrading enzymes were conserved in Streptomyces coelicolor A3(2). These results demonstrate that GlnR and PhoP are involved in polysaccharide degradation by mediating the interplay among carbon, nitrogen, and phosphate metabolism in response to cellular nutritional states. Our study reveals a novel regulatory mechanism underlying carbohydrate metabolism, and suggests new possibilities for designing genetic engineering approaches to improve the rate of utilization of starch in actinobacteria.IMPORTANCE The development of efficient strategies for utilization of biomass-derived sugars, such as starch and cellulose, remains a major technical challenge due to the weak activity of associated enzymes. Here, we found that GlnR and PhoP directly regulate the transcription of genes encoding amylolytic enzymes and present insights into the regulatory mechanisms of degradation and utilization of starch in actinobacteria. Two nutrient-sensing regulators may play important roles in creating a direct association between nitrogen/phosphate metabolisms and carbohydrate utilization, as well as modulate the C:N:P balance in response to cellular nutritional states. These findings highlight the interesting possibilities for designing genetic engineering approaches and optimizing the fermentation process to improve the utilization efficiency of sugars in actinobacteria via overexpression of the glnR and phoP genes and nutrient signal stimulation.

Project description:Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest ("metabolic switch") preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO(2) enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of actinomycetes.

Dataset Information

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

Publications

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

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