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

Abstract

The single-component RcoM transcription factor couples an N-terminally bound heme cofactor with a C-terminal "LytTR" DNA-binding domain. Here the RcoM Bx -1 protein from Burkholderia xenovorans LB400 was heterologously expressed and then purified in a form with minimal bound CO (ϳ10%) 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 ϳ60-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 RcoM Bx -1 to a variety of DNA probes was demonstrated in vitro. We posit that for the RcoM Bx -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. Carbon monoxide (CO) is a prevalent toxin and yet is also a bacterial nutrient, a crucial intermediate in anaerobic central metabolism, and an enzyme cofactor (3,10,23,38). Bacteria sense and respond to CO by using two biochemically characterized nonhomologous transcription factors, CooA (CO oxidation activator) and RcoM (regulator of CO metabolism), that have been shown to regulate one of three metabolic processes in different organisms: (i) the expression of cox-encoded aerobic CO oxidation, (ii) the expression of coo-encoded anaerobic CO oxidation, or (iii) the expression of cowN, whose gene product protects nitrogenase function from CO inhibition (21,22,43). These single-component transcription factors link an N-terminal domain that enfolds a CO-binding heme cofactor with a C-terminal DNA-binding region, either the common helix-turn-helix domain (CooA) or the rarer "LytTR" domain (RcoM) (14).The LytTR DNA-binding domain, named for the prototypical LytT and LytR transcription factors (34), typically occurs as a DNA-binding response regulator component of two-component signal transduction systems. These systems often regulate virulence functions, including the synthesis of exopolysaccharides, bacteriocins, toxins, excreted enzymes, and fimbriae (13, 34), and given their absence from eukaryotes, the interference of LytTR domain protein function is considered a useful therapeuti...

Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands

“…The observed DNA binding constant Kd of <2 nM is higher than what has been observed under the same assay conditions for the NO-dependent transcriptional regulator DNR (44 nM (18) ) and the anaerobic CO-dependent transcriptional activator CooA (values between 6 and 16 nM have been reported (18,37,38)). It is in agreement with a high functional affinity, similar to what has been observed for binding of RcoM-1 to the a + b + c region (14), and an efficient CO-specific recruitment of RcoM-2.…”

“…RcoM-1 sensor (14). As has been discussed (14), these findings and the notion that aerobic RcoM sensors should be sensitive to persistent CO levels in the low nanomolar range, where CO oxidation in Burkholderia occurs (36), concord. The observed slow ligand exchange in this range also avoids rapid fluctuations in the sensor activity.…”

“…This fraction is far smaller than in the isolated heme domain RcoMH-2 that is almost completely purified in the CO-bound form (12). For RcoM-1, differences in CO-binding in the purified protein have been related to differences in protein expression (14). We used the same expression system and E. coli growth conditions for our full-length and heme domain constructs.…”

“…RcoM (regulator of CO metabolism) proteins have been discovered a decade ago and are active in the aerobic bacterium Burkholderia xenovorans (11). They are less well characterized (in particular structural data are lacking), but display some surprising properties (11)(12)(13)(14)(15)(16). B. xenovorans expresses two RcoM proteins, RcoM-1 and RcoM-2, which are highly homologous (11).…”

Gas‐Sensing Transcriptional Regulators

“…In particular, RcoM‐1 exhibits a higher affinity toward CO than RcoM‐2 so that it can sense lower CO concentration. [ 46 ]…”