1887

Abstract

The microaerophilic food-borne pathogen is exposed to highly variable oxygen concentrations during its life cycle and employs a variety of protection mechanisms to resist oxidative stress. However, not all of the enzymes that mediate such protection have yet been identified. Two genes in strain NCTC 11168, and , are predicted to encode unrelated methionine sulphoxide reductases, which may repair oxidized methionine residues in proteins and thus contribute to oxidative stress defence. Cj0637 and Cj1112 were overexpressed, purified and shown by a coupled thioredoxin–thioredoxin reductase–NADPH assay to catalyse the stereospecific reduction of the and diastereoisomers, respectively, of the model compound methyl tolyl sulphoxide. Cj0637 is thus identified as MsrA and Cj1112 as MsrB. The contribution of these enzymes to oxidative and nitrosative stress resistance in was assessed by phenotypic analysis of a set of isogenic and insertion mutants. As RT-PCR data suggested a polar effect on in the mutant, an merodiploid complementation strain was also constructed. The strain was severely growth inhibited under standard microaerobic conditions, whereas the and strains grew normally. Agar plate disc diffusion assays showed that all mutants displayed increased sensitivity to hydrogen peroxide, organic peroxide, superoxide, and nitrosative and disulphide stress, but quantitative cell viability assays showed that the double mutant was markedly more sensitive to both oxidative and nitrosative stress. All of the stress-sensitivity phenotypes observed for the mutant were restored to wild-type in the merodiploid. It is concluded that MsrA and MsrB make a significant contribution to the protection of against oxidative and nitrosative stress.

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2008-08-01
2024-04-25
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