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We have shown that the bldD gene of Streptomyces coelicolor, while required for antibiotic production and morphological differentiation, is not essential for viability. We have also demonstrated that BldD forms a higher order complex both in solution and when bound to target DNA. Purified BldD exists in three forms in solution, as a tetramer, dimer and monomer, but only in the dimeric form when bound to its own promoter/operator.  相似文献   

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BldD is a central regulator of the developmental process in Streptomyces coelicolor. The 1.8 angstroms resolution structure of the DNA-binding domain of BldD (BldDN) reveals that BldDN forms a compact globular domain composed of four helices (alpha1-alpha4) containing a helix-turn-helix motif (alpha2-alpha3) resembling that of the DNA-binding domain of lambda repressor. The BldDN/DNA complex model led us to design a series of mutants, which revealed the important role of alpha3 and the 'turn' region between alpha2 and alpha3 for DNA recognition. Based on the fact that BldD occupies two operator sites of bldN and whiG and shows significant disparity in the affinity toward the two operator sites when they are disconnected, we propose a model of cooperative binding, which means that the binding of one BldD dimer to the high affinity site facilitates that of the second BldD dimer to the low affinity site. In addition, structural and mutational investigation reveals that the Tyr62Cys mutation, found in the first-identified bldD mutant, can destabilize BldD structure by disrupting the hydrophobic core.  相似文献   

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Lee CJ  Won HS  Kim JM  Lee BJ  Kang SO 《Proteins》2007,68(1):344-352
A homodimeric protein, BldD is a key regulator for developmental process of Streptomyces coelicolor and the bldD mutant exhibits severely pleiotropic defects in the antibiotic production and morphological differentiation of the bacterium. In the present work, we approached domain organization of BldD, to structurally and functionally characterize the protein as a DNA-binding protein. We first observed a proteolytic cleavage of BldD by the cytoplasmic extracts of S. coelicolor, which was highly dependent on the developmental stage of the bacterium. The resulting fragment of BldD was identified by mass spectrometry as the N-terminal domain resistant to the proteolysis. Recombinant proteins corresponding to the intact BldD, the N-terminal domain (residues 1-79) and the rest part (C-terminal domain; residues 80-167) were used for comparative analyses by several spectroscopic, thermodynamic, and biochemical experiments, respectively. The results of circular dichroism and nuclear magnetic resonance spectroscopies certified each of the two determined domains could be regarded as an individual folding unit possessing an independent thermodynamic cooperativity. Structural interaction between the two domains was little observed in the DNA-free and DNA-bound states. Strikingly, it was revealed by gel permeation chromatography, chemical crosslink, gel mobility shift, and NMR-monitored DNA-binding experiments, that only the N-terminal domain is responsible for the dimerization as well as DNA-binding of BldD. Detailed inspection of the present results suggests that BldD function in a unique and complicated mode to totally regulate the diverse developmental stages of S. coelicolor.  相似文献   

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The bacterium Streptomyces coelicolor produces two cell types during the course of its life cycle: the aerial hyphae, which metamorphose into spores, and the substrate hyphae, which synthesize antibiotics. We show that the genes ramC and ramR are required for the production of the aerial hyphae but are dispensable for vegetative growth and antibiotic synthesis. We find that ramC is expressed in the substrate hyphae and shut off in the aerial hyphae by the time visible signs of sporulation-associated septation are evident. Production of RamC requires the developmental regulators bldD, cprA and ramR, but not bldM or bldN, and we show that the RamR protein interacts directly with DNA in the ramC promoter region suggesting that it is, at least in part, responsible for regulating ramC expression.  相似文献   

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BldD‐(c‐di‐GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here, we report the identification and characterisation of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we show that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.  相似文献   

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