Abstract
BackgroundCorynebacterium glutamicum is a well-known producer of various l-amino acids in industry. During the fermenting process, C. glutamicum unavoidably encounters oxidative stress due to a specific reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfide bond-based regulatory devices to control a specific set of antioxidant genes. However, nothing is known about the mixed disulfide between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sensor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroperoxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specific Ohr was a core detoxification system.ResultsIn this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expression. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not H2O2, hypochlorous acid, and diamide; this outcome was substantiated by the fact that the ohsR-deficient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifically activated by CHP. Mutational analysis of the two cysteines (Cys125 and Cys261) showed that Cys125 was primarily responsible for the activation of DNA binding. The oxidation of Cys125 produced a sulfenic acid (C125-SOH) that subsequently reacted with MSH to generate S-mycothiolation that was required to activate the ohr expression. Therefore, OhsR regulated the ohr expression using an S-mycothiolation mechanism in vivo.ConclusionThis is the first report demonstrating that the regulatory OhsR specifically sensed OHPs stress and responded to it by activating a specific ohr gene under its control using an S-mycothiolated mechanism.
Highlights
Corynebacterium glutamicum is a well-known producer of various l-amino acids in industry
OhsR was required for the resistance to organic peroxides To obtain homologs of the B. subtilis OhrR in the C. glutamicum proteome, a direct subsequent BLAST search was performed
No hits for the OhrR homologs in C. glutamicum were found, while one candidate that was annotated as a potential regulator due to the presence of the “winged helix” type of helix-turn-helix (HTH) motif encoded by the gene of C. glutamicum ATCC 13032 was found to be located adjacently downstream of the ohr gene
Summary
Corynebacterium glutamicum is a well-known producer of various l-amino acids in industry. During normal cellular functions or under unfavorable external environmental stimuli, bacteria unavoidably produce significant levels of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide radical (O2·−), and organic peroxides (OHPs) [1]. The OHPs detoxification enzymes alkyl hydroperoxide reductase subunit CF (AhpCF) and organic hydroperoxide resistance (Ohr) are the two core detoxification systems in bacteria [6, 7]. They can reduce OHPs into their corresponding alcohols. The low molecular weight thiols have a role in OHPs detoxification in vivo, confirmed by the increased sensitivity to cumene hydroperoxide (CHP), menadione (MD), and tert-butyl hydroperoxide (t-BHP) in MSHdeficient Mycobacterium smegmatis, M. tuberculosis and Corynebacterium glutamicum mutant strains [8, 9, 11]
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