Three genes encoding citrate synthases in Saccharopolyspora erythraea are regulated by the global nutrient-sensing regulators GlnR, DasR, and CRP.

Saccharopolyspora erythraea has three citrate synthases encoded by gltA-2, citA, and citA4. Here, we characterized and identified the expression and regulatory properties of these synthases. Three pleiotropic global regulatory proteins of S. erythraea - CRP, GlnR, and DasR - are involved in carbon metabolism, nitrogen metabolism, and amino-sugar (chitin and GlcNAc) metabolism. Using electrophoretic mobility shift assays (EMSAs), we identified these regulators as proteins that bind directly to the promoter regions of all citrate synthase genes (gltA-2, citA, and citA4). Footprinting assays indicated the exact protect sequences of CRP, GlnR, and DasR on the promoter region of gltA-2, revealing binding competition between GlnR and DasR. Moreover, by comparing the transcription levels of citrate synthase genes between parental and glnR mutant or dasR mutant strains, or by comparing the transcription response of citrate synthases under various nutrient conditions, we found that GlnR and DasR negatively regulated citA and citA4 transcription but had no regulatory effects on the gltA-2 gene. Although no CRP mutant was available, the results indicated that CRP was a cAMP-binding receptor affecting gltA-2 transcription when the intracellular cAMP concentration increased. Thus, an overall model of CS regulation by C and/or N metabolism regulators and cAMP receptor protein was proposed.