Identification of Aromatic Residues Critical to the DNA Binding and Ligand Response of theBacillus subtilisQdoR (YxaF) Repressor Antagonized by Flavonoids

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.

A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities

“…The ability of TetR repressors to bind different effectors with analogous structures is already known and was one of the factors leading us to perform this study (Cuthbertson and Nodwell, 2013). For example, the QdoR repressor, which has been implicated in the regulation of quercetin dioxygenase (QdoI) gene expression, can bind five other flavonoids (i.e., fisetin, tamarixetin, galangin, genistein, and coumestrol) in addition to quercetin (Hirooka and Fujita, 2011). Grkovic et al (2003) studied the QacA multidrug transporter, which confers resistance to cationic lipophilic antiseptics and disinfectants effective against Staphylococcus aureus .…”

Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion

“…We further examined the in vivo function of the mutant with alanine substitution at Trp131 by a reporter assay, which largely supported the corresponding in vitro results. 29) These in vitro and in vivo results 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 (Fig. 3).…”

“…A QdoR mutant capable of responding to The qdoI gene, one of the LmrA/QdoR regulon members, encodes quercetin 2,3-dioxygenase, which forms a dimer (two rotundate rectangles) 24) and catalyzes the C-ring cleavage of flavonols, as illustrated. 29) various flavonoids can be created by substituting the residues in the flavonoid-interacting domain probably including these three aromatic residues, and this mutant would be available as a novel biosensor to detect various flavonoids and related compounds in such a way as to introduce its gene into the B. subtilis strain carrying a reporter-fusion construct similar to that used in this study. 29) Siedler et al reported that a biosensor composed of QdoR and the qdoI promoter-gfp fusion functions in the Escherichia coli cells to detect flavonoids such as quercetin and kaempferol by the greenfluorescent signal, which is expected to be applied for isolation of the genes involved in the flavonoid biosynthetic pathways in plants.…”

“…29) (TAGTTAGGCGCCTAACTA). An in vitro gel retardation assay with the YetL protein and the DNA probes corresponding to the yetL and yetM promoter regions and an in vivo reporter assay of the B. subtilis strains carrying the yetL or yetM promoter-lacZ fusion indicated that the DNA binding of YetL is inhibited effectively by flavonoids such as kaempferol, apigenin, and luteolin, and its weaker interruption by flavonoids such as quercetin and fisetin appears to be different from the case of LmrA/QdoR (the responsive property of YetL to flavonoids with different structural features is discussed in our previous report 32) ).…”

Control of Gene Expression With Quercetin-Responsive Modular Circuits

“…QdoR represses the QdoI expression, binding to specific operators upstream of qdoI ( Hirooka et al, 2007 ). QdoR also interacts with an operator upstream of qdoR, repressing its own expression ( Hirooka et al, 2007 ; Hirooka and Fujita, 2011 ). Quercetin inhibits the binding of QdoR to DNA; thus, the transcription of qdoI and qdoR is induced ( Hirooka et al, 2007 ).…”