Multiple allosteric effectors control the affinity of DasR for its target sites

Abstract

a b s t r a c tThe global transcriptional regulator DasR connects N-acetylglucosamine (GlcNAc) utilization to the onset of morphological and chemical differentiation in the model actinomycete Streptomyces coelicolor. Previous work revealed that glucosamine-6-phosphate (GlcN-6P) acts as an allosteric effector which disables binding by DasR to its operator sites (called dre, for DasR responsive element) and allows derepression of DasR-controlled/GlcNAc-dependent genes. To unveil the mechanism by which DasR controls S. coelicolor development, we performed a series of electromobility shift assays with histidinetagged DasR protein, which suggested that N-acetylglucosamine-6-phosphate (GlcNAc-6P) could also inhibit the formation of DasR-dre complexes and perhaps even more efficiently than GlcN-6P. The possibility that GlcNAc-6P is indeed an efficient allosteric effector of DasR was further confirmed by the high and constitutive activity of the DasR-repressed nagKA promoter in the nagA mutant, which lacks GlcNAc-6P deaminase activity and therefore accumulates GlcNAc-6P. In addition, we also observed that high concentrations of organic or inorganic phosphate enhanced binding of DasR to its recognition site, suggesting that the metabolic status of the cell could determine the selectivity of DasR in vivo, and hence its effect on the expression of its regulon.

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…An example of metabolic control is presented by the pleiotropic nutrient sensory regulator DasR, which is essential for development and pleiotropically represses antibiotic production (see below). DNA binding by DasR is controlled by the binding of GlcNAc-related metabolites as ligands (186,225). An overview of key developmental events and regulatory networks in streptomycetes is presented in Fig.…”

The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci

“…E). However, the relative high concentration of NlpR used to achieve a complete binding in EMSA assays suggests the need of a yet unknown ligand or effector that could increase its affinity for the operator regions, as has been reported for other pleiotropic regulators (Tenconi et al ., ). The potential ligand, which may include / or metabolites from the synthesis of pyrrole‐ or lipid‐derivatives, may bind to the domain located in the N‐terminal region of the protein.…”

Prodiginine Production inStreptomyces coelicolorCorrelates Temporally and Spatially to Programmed Cell Death

“…After sequence confirmation, the fragment was retrieved through EcoRI and BamHI restriction digest, gel purified, and cloned into an EcoRI/BamHI-linearized plJ2587 (62) upstream of redD resulting in plasmid pELT003. The complementation construct, as well as the empty plJ2587 plasmid, were introduced into the redD mutant through intergeneric conjugation as described previously (63). All thiostreptone resistant colonies transformed with pELT003 (gene tsr in plJ2587) presented the intracellular red pigmentation and red fluorescence associated with PdG production confirming the complementation of the redD mutant phenotype.…”