The role of methionine sulfoxide reductase in Helicobacter pylori
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Msr catalyzes the reduction of oxidized methionine residues and is an important antioxidant enzyme in many bacterial pathogens as well as in humans. An H. pylori msr mutant could not reduce oxidized methionine substrates resulting in an oxidative stress sensitive phenotype. Under oxidative stress conditions the mutant had higher levels of oxidized proteins than the wild-type. An msr strain could not colonize the mouse stomach for the longer time periods tested (2, 3 and 4 weeks). A complemented msr strain had stress resistance properties and host colonization ability similar to the wild-type, associating the earlier phenotype to msr inactivation. The gene was also up-regulated in cells under peroxide stress or iron starvation conditions. Using a co-immunoprecipitation (CIP) approach I identified catalase, site specific recombinase, GroEL, and the known Msr reductant thioredoxin-1 as putative Msr interacting proteins in H. pylori. Under oxidative stress conditions the msr strain showed approximately 50 % of the wild-type catalase specific activity, indicating an Msr-dependent repair of oxidized catalase. Pure Msr only reduced the R-epimer of methionine sulfoxide, with a Km(app.) of 4.1 mM for the substrate. MutS is a part of the DNA mismatch repair system in E. coli. An H. pylori mutS strain was sensitive to oxidative stress and had reduced colonization ability. Under stress conditions the mutant contained a greater amount of 8-OG in its DNA than did the wild-type. MutS bound to the Holliday junction DNA and to that containing the 8-oxo-guanine nucleotide with higher efficiency than MutS binding to DNA mismatch bases; this indicates an additional role for MutS as an enzyme involved in repairing oxidatively damaged DNA. The [Fe-S] cluster synthesis proteins NifS/NifU are essential in H. pylori. The nifS/nifU operon was maximally expressed when the bacterium is in high oxygen or in iron supplemented conditions. Such expression was not observed in an isogenic fur mutant, suggesting a Fur dependent activation of the gene. Unlike for regulations of other genes, only iron-loaded Fur recognized the nifS/nifU promoter region. Similarly, the region recognized by Fur was farther upstream than the conventional Fur binding regions associated with the promoters of other Fur regulated genes.