Doctor of Philosophy (PhD)
Multiple antibiotic resistance regulator (MarR) family is the most ubiquitous category of transcriptional regulators that exists among different bacteria and archaea. MarR family transcriptional regulators have been studied for their involvement in various biological processes, such as environmental chemical response, pathogenicity, and environmental stress responses. This work elucidates the role of MarR homologs MftR (major facilitator transcriptional regulator) and BifR (biofilm regulator) in the soil bacterium Burkholderia thailandensis. Burkholderia thailandensis-encoded MarR homolog MftR is divergently oriented to a gene that encodes the efflux pump MftP (major facilitator transport protein). MftR binds two cognate sites (each site consist of 9 bp imperfect inverted repeats) in the mftR-mftP intergenic region with equivalent affinity. For each site, urate attenuates DNA binding by MftR with equivalent sensitivity. MftR shows two-step unfolding transition (dimerization and DNA binding region) and urate binding to MftR and variants (mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter (now known as Agrobacterium fabrum) PecS) results in one step thermal unfolding transition. Further, data suggest the binding of urate in the cleft between the dimer interface and the DNA-binding lobes. DNA binding by MftR is attenuated by urate. MftR binds DNA with lower affinity at 37 °C. Collectively, this study suggests that MftR upregulates the genes under its control by responding to urate and by thermal upshift. Secondary metabolites are often produced during host invasion by a pathogen and function as virulence factors to survive in host. In normal condition biosynthetic gene cluster that produces drug or drug like molecules remains inactive for unknown reason. The signal required to activate these biosynthetic gene clusters is hard to identify. Global gene expression data of mftR strains suggests that MftR is a master regulator, which represses the various biosynthetic gene clusters required for the production of antimicrobial bactobolins, the iron siderophore malleobactin, and the virulence factor malleilactone among others. Along with that this study also identifies urate as a physiologically relevant inducer of biosynthetic gene clusters responsible for producing virulence factors. Burkholderia thailandensis also encodes a redox-sensitive MarR homolog, BifR that represses biofilm formation. Binding of BifR at two sites between the intergenic region of ecsC and emrB-bifR represses the expression of ecsC (putative LasA protease) and emrB-bifR. Oxidized BifR also binds to the intergenic region with nM affinity. However, oxidizing conditions further represses the expression. Oxidized BifR forms dimer-of-dimers. BifR also represses an operon that is required for the enzymatic synthesis of phenazine antibiotic. Phenazine acts as an alternative respiratory electron acceptor. Biofilm formation generates oxygen-limiting environment. This study suggests that BifR regulates LasA protease and expression of genes, which are involved in biofilm formation. Overall, my study identifies novel properties of MarR homologs in B. thailandensis, which suggest the role of MarR homologs in awakening of cryptic gene clusters that facilitate identification of novel pharmaceuticals and regulation of synthesis of alternate electron acceptor to survive in oxygen-limiting environment in biofilm.
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Gupta, Ashish, "The Role of Burkholderia thailandensis-Encoded MarR Homologs in Response to Host-Derived Signals" (2016). LSU Doctoral Dissertations. 4449.