Distinct characteristics of OxyR2, a new OxyR‐type regulator, ensuring expression of Peroxiredoxin 2 detoxifying low levels of hydrogen peroxide in Vibrio vulnificus

Abstract

SummaryTwo peroxiredoxins, Prx1 and Prx2, were previously identified in Vibrio vulnificus. Besides OxyR1, a homologue of Escherichia coli OxyR (EcOxyR), OxyR2 that shares low homology with EcOxyR was first identified in V. vulnificus. OxyR2 activated prx2 during aerobic growth, while OxyR1 activated prx1 only when exposed to exogenous H2O2. OxyR2 was oxidized to form a reversible C206 to C215 disulphide bond by sensing low levels of H2O2, which were insufficient to oxidize OxyR1, and only the oxidized OxyR2 activated prx2. OxyR25CA, in which all cysteine residues except for C206 and C215 were replaced with alanines, and its mutants, OxyR25CA-C206S and OxyR25CA-C215S, were constructed. OxyR25CA and OxyR25CA-C215S directly bound to a specific binding sequence centred at −56.5 from the prx2 transcription start site, albeit with different binding affinities. The binding sequence consisted of four ATCGnt elements spaced by a helical turn and aligned in the twofold dyad symmetry, suggesting that OxyR2 binds DNA as a tetramer. OxyR25CA-C206S also directly bound to DNA comprising more extended sequences, indicating that oxidized and reduced OxyR2 adopt different conformational states, leading to altered DNA contacts. The oxyR2 mutation reduced cytotoxicity and growth during infection, indicating that OxyR2 is essential for the pathogenesis of V. vulnificus.

Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

“…Ϫ, grown anaerobically; ϩ, grown aerobically. (22,31,48,49). This study demonstrated for the first time that IscR, rather than OxyR, activates the expression of a Prx (Prx3) (Figs.…”

“…Anerobic conditions were obtained by using an anaerobic chamber with an atmosphere of 90% N 2 , 5% CO 2 , and 5% H 2 (Coy Laboratory Products, Grass Lake, MI). For anaerobic culture, the medium was preincubated to remove resolved O 2 in the anaerobic chamber, which was verified by adding 0.00001% (w/v) resazurin salt (Sigma) to the medium as described previously (31).…”

OxyR2 Modulates OxyR1 Activity and Vibrio cholerae Oxidative Stress Response

“…5C and D). In Vibrio vulnificus, there are two OxyR homologs, which control different peroxiredoxin genes (25). V. vulnificus OxyR2 senses H 2 O 2 with high sensitivity (26).…”

Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

“…Studies of these two homologues within D. radiodurans have revealed each individually to be necessary for resistance to oxidative stress but also to function synergistically in this regard, as a mutant lacking both oxyR homologues exhibits increased sensitivity to oxidative stress (64,66). In V. vulnificus, it was found that each homologue of OxyR functioned in sensing changes in oxidation; however, each had distinct gene targets and different sensing thresholds, with one homologue responding to very low H 2 O 2 in comparison to its counterpart (65). Perhaps, in L. pneumophila, oxyR1 also interacts with the OxyR Lp regulon, adding another layer of regulatory complexity.…”

“…However, the L. pneumophila Philadelphia genome, available on the LegioList database (http://genolist.pasteur.fr/LegioList), contains a second OxyR homologue designated oxyR1 (lpg0173). The only other organisms, to our knowledge, that contain two OxyR homologues are the Gram-positive bacterium Deinococcus radiodurans (64) and the Gram-negative pathogen Vibrio vulnificus (65). Studies of these two homologues within D. radiodurans have revealed each individually to be necessary for resistance to oxidative stress but also to function synergistically in this regard, as a mutant lacking both oxyR homologues exhibits increased sensitivity to oxidative stress (64,66).…”