Understanding the regulatory effects of a biofilm regulator (BifR) and the implication of its interactions in Burkholderia thailandensis

Biofilm formation plays a key role in the pathogenesis of chronic infections. In pathogenic Burkholderia species, biofilm formation is a severe complication, and it makes bacteria resistant to host defenses and antibiotics. BifR (Biofilm regulator) is a recently described redox-sensitive member of the multiple antibiotic resistance regulator (MarR) protein family that has been found to repress biofilm formation in Burkholderia thailandensis. BifR appears to regulate the expression of other proteins critical to biofilm formation, but its overall functions are poorly understood. BifR is encoded in the same operon with an efflux pump (emrB) and divergently oriented to a gene (ecsC) that codes for a protease (LasA) that has been shown to be implicated in virulence by its contribution to elastin degradation in Pseudomonas aeruginosa. Previously, BifR was shown to repress the expression of emrB-bifRand ecsCand to repress the expression of these genes further under oxidizing conditions. However, the substrate of the efflux pump (EmrB) is unknown. To determine the substrate of EmrB, we carried out antibiotic sensitivity tests on the Wild Type (WT), emrB-bifRmutant and bifRmutant strains. Following a 24-hour incubation at 37 o C, our results showed that the bifR mutant was resistant to novobiocin at concentrations of 50 ug/ml, 75 ug/ml and slightly resistant at 100 ug/ml. The bifRstrain was also more resistant to ampicillin at concentrations of 50 ug/ml, 75 ug/ml and 100 ug/ml. Similar results were obtained for the related β-lactam, penicillin G. This indicates that novobiocin and β-lactam antibiotics are substrates for EmrB. We also conducted onion tissue maceration and tested for the survival of Caenorhabditis elegans after infection with the three strains. Our results showed that after 72 hours of incubation of the onions at 30 o C, the bifRmutant strain was more virulent compared to the WT.Meanwhile, with a 70 % survival of the C. elegansafter 12 hours of incubation at room temperature, the emrB-bifRmutant strain was more virulent. This study reveals that BifR plays a role in virulence and that the role may be host specific.