title: Molecular Insights into Hydrogen Peroxide-sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses.
abstract: Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn2+content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn2+and H2O2sensors in mediating H2O2resistance by an oral streptococcus. H2O2disrupted So-MntR binding to Mn2+transportermntABCpromoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in H2O2-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished H2O2-induced disulfide-linked dimers and weakened H2O2damage on So-MntR binding, indicating that H2O2inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to H2O2oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon H2O2pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in H2O2-pulsed cells, confirming Cys-11 and Cys-156 contributed to H2O2-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression ofmntABCand cellular Mn2+decreased, but H2O2susceptibility increased. In the C123S mutant, increasedmntABCexpression, cellular Mn2+content, and manganese-mediated H2O2survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.
authors.affiliation: From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China.|School of Life Sciences, University of Chinese Academy of Sciences, No.19A Yuquan Road, Shijingshan District, Beijing 100049, China, and.
authors.forename: Zhaoyuan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China.|School of Life Sciences, University of Chinese Academy of Sciences, No.19A Yuquan Road, Shijingshan District, Beijing 100049, China, and.
authors.forename: Xinhui
authors.initials: X
authors.lastname: Wang
authors.affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.affiliation: From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China.|School of Life Sciences, University of Chinese Academy of Sciences, No.19A Yuquan Road, Shijingshan District, Beijing 100049, China, and.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Hu
authors.affiliation: From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China, tonghuichun@im.ac.cn.|School of Life Sciences, University of Chinese Academy of Sciences, No.19A Yuquan Road, Shijingshan District, Beijing 100049, China, and.
authors.forename: Huichun
authors.initials: H
authors.lastname: Tong
authors.affiliation: From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China, dongxz@im.ac.cn.|School of Life Sciences, University of Chinese Academy of Sciences, No.19A Yuquan Road, Shijingshan District, Beijing 100049, China, and.
authors.forename: Xiuzhu
authors.initials: X
authors.lastname: Dong
mesh_terms: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000502:physiology*; D019281:Dimerization; D006706:Homeostasis / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress / Q000187:drug effects; D012097:Repressor Proteins / Q000502:physiology*; D013291:Streptococcus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C433792:MntR protein, bacteria; D012097:Repressor Proteins; D008345:Manganese; D006861:Hydrogen Peroxide
keywords: Streptococcus; disulfide; hydrogen peroxide; oxidative stress; redox regulation