Crystal structure of a putative HTH-type transcriptional regulator yxaF from Bacillus subtilis.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Proteins Structure Function and Bioinformatics (Impact Factor: 3.34). 07/2006; 63(4):1087-91. DOI: 10.1002/prot.20924
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    ABSTRACT: DNA microarray analysis revealed that transcription of the Bacillus subtilis yetM gene encoding a putative flavin adenine dinucleotide-dependent monooxygenase was triggered by certain flavonoids during culture and was derepressed by disruption of the yetL gene in the opposite orientation situated immediately upstream of yetM, which encodes a putative MarR family transcriptional regulator. In vitro analyses, including DNase I footprinting and gel retardation analysis, indicated that YetL binds specifically to corresponding single sites in the divergent yetL and yetM promoter regions, with higher affinity to the yetM region; the former region overlaps the Shine-Dalgarno sequence of yetL, and the latter region contains a perfect 18-bp palindromic sequence (TAGTTAGGCGCCTAACTA). In vitro gel retardation and in vivo lacZ fusion analyses indicated that some flavonoids (kaempferol, apigenin, and luteolin) effectively inhibit YetL binding to the yetM cis sequence, but quercetin, galangin, and chrysin do not inhibit this binding, implying that the 4-hydroxyl group on the B-ring of the flavone structure is indispensable for this inhibition and that the coexistence of the 3-hydroxyl groups on the B- and C-rings does not allow antagonism of YetL.
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    ABSTRACT: Bacillus subtilis collectively inhabits the rhizosphere, where it contributes to the promotion of plant growth, although it does not have a direct symbiotic relationship to plants as observed in the case of rhizobia between leguminous plants. As rhizobia sense the flavonoids released from their host roots through the NodD transcriptional factor, which triggers transcription of the nod genes involved in the symbiotic processes, we supposed that B. subtilis utilizes certain flavonoids as signaling molecules to perceive and adapt to the rhizospheric environment that it is in. Our approaches to identify the flavonoid-responsive transcriptional regulatory system from B. subtilis resulted in the findings that three transcriptional factors (LmrA/QdoR, YetL, and Fur) are responsive to flavonoids, with the modes of action being different from each other. We also revealed a unique regulatory system by two transcriptional factors, YcnK and CsoR, for copper homeostasis in B. subtilis. In this review, we summarize the molecular mechanisms of these regulatory systems with the relevant information and discuss their physiological significances in the mutually beneficial interaction between B. subtilis and plants, considering the possibility of their application for plant cultivation.
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    ABSTRACT: Bacillus subtilis LmrA and QdoR (formerly YxaF) are paralogous transcriptional regulators that repress their regulon comprising the lmrAB operon, the qdoR gene, and the qdoI-yxaH operon, by binding to the LmrA/QdoR boxes located in the promoter regions. Detachment of them followed by derepression of the target genes is induced by certain flavonoids. To identify the residues critical to the ligand response in QdoR, we selected eight residues based on structural information, produced eight single-mutated QdoRs in which each residue was replaced with alanine, and evaluated their capacities for DNA binding and the flavonoid response by gel retardation analysis. The three mutants, carrying the alanine substitution at Phe87, Trp131, or Phe135, showed features distinctly different from those of the wild type and from each other. We further examined the in vivo function of the mutant with alanine substitution at Trp131 by reporter assay. This largely supported the corresponding in vitro results. The in vitro and in vivo data suggest that Phe87, Trp131, and Phe135, forming a hydrophobic cluster in QdoR, play crucial roles in the DNA binding, flavonoid accommodation, and/or conformational change triggered by ligand binding.
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