Burkholderia xenovorans RcoMBx-1 a Transcriptional Regulator System for Sensing Low and Persistent Levels of Carbon Monoxide

摘要

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal “LytTR” DNA-binding domain. Here the RcoMBx-1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (∼10%) and was found to stably bind this effector with a nanomolar affinity. DNase I protection assays demonstrated that the CO-associated form binds with a micromolar affinity to two ∼60-bp DNA regions, each comprised of a novel set of three direct-repeat binding sites spaced 21 bp apart on center. Binding to each region was independent, while binding to the triplet binding sites within a region was cooperative, depended upon spacing and sequence, and was marked by phased DNase I hyperactivity and protection patterns consistent with considerable changes in the DNA conformation of the nucleoprotein complex. Each protected binding site spanned a conserved motif (5′-TTnnnG-3′) that was present, in triplicate, in putative RcoM-binding regions of more than a dozen organisms. In vivo screens confirmed the functional importance of the conserved “TTnnnG” motif residues and their triplet arrangement and were also used to determine an improved binding motif [5′-CnnC(C/A)(G/A)TTCAnG-3′] that more closely corresponds to canonical LytTR domain/DNA-binding sites. A low-affinity but CO-dependent binding of RcoMBx-1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoMBx-1 protein, the high CO affinity combined with multiple low-affinity DNA-binding events constitutes a transcriptional “accumulating switch” that senses low but persistent CO levels.