Master of Science (MS)
Being ubiquitous in nature, bacteria are often faced by environmental stresses. The predominant form of stress being the ‘oxidative’ one, prevalent during host invasion. As one of the host’s protective mechanisms, bacteria are bombarded by various kinds of organic and inorganic oxidants. Several bacterial antioxidant systems have been studied and characterized to be potent enough to scavenge such host-derived reactive oxygen species, aiding in bacterial survival within the host. Broadly, the antioxidant scavengers are categorized into those that are specific for inorganic or organic oxidants. Although a lot has been studied about the former, the latter still remains uncharacterized in several organisms. Burkholderia thailandensis, is one such soil dwelling bacterium, continually under oxidative stress by virtue of organic exudates released from plants in addition to other biotic compounds. In addition to sharing about 80% genetic homology with its pathogenic homologues such as B. psuedomallei and B. mallei, the oxidative environment (rich in organic oxidants) it thrives in makes it an interesting model organism to study the kind of oxidant sensor-responder proteins it possesses and their mechanism of action. With an emphasis on studying oxidant sensing proteins belonging to the MarR family, OhrR (organic hydroperoxide reductase regulator) protein from B. thailandensis was characterized using in vitro assays in this study. The gene was cloned from B. thailandensis genomic DNA and protein expressed in E. coli. The protein was determined to belong to the MarR family of proteins and to exist as a homodimer. It formed a series of reversible, oligomeric species on being treated with hydrogen peroxide, cumene hydroperoxide and tert-butyl hydroperoxide as was observed from SDS-PAGE. The reduced form of the protein was observed to be relatively thermostable (Tm of 63.5 °C), with an appreciable thermal instability observed in OhrR treated with higher concentration of oxidants (5 mM). DNA binding assays revealed specificity of B. thailandensis for the promoter region of ohr, although the DNA length to which bound seemed to be making a difference in the stability of protein-DNA complexes formed and detected using EMSA. Since OhrR is predicted to control production of organic hydroperoxide reductase, which is important for detoxifying organic hydroperoxides, the initial characterization and oxidant response of B. thailandensis OhrR, determined using in vitro assays, thus are indicative of its probable role as a crucial protein among the B. thailandensis protein machinery, helping the bacterium survive such drastic oxidative conditions.
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Pande, Anuja, "Oxidative stress based response of a transcriptional regulator, OhrR, from Burkholderia thailandensis" (2013). LSU Master's Theses. 2895.