title: Catalase (KatA) plays a role in protection against anaerobic nitric oxide in Pseudomonas aeruginosa.
abstract: Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Shengchang
authors.initials: S
authors.lastname: Su
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Warunya
authors.initials: W
authors.lastname: Panmanee
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Wilson
authors.affiliation: Departments of Anesthesiology, Cell, Developmental and Integrative Biology, and Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Harry K
authors.initials: HK
authors.lastname: Mahtani
authors.affiliation: Departments of Anesthesiology, Cell, Developmental and Integrative Biology, and Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Vanderwielen
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Makris
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Melanie
authors.initials: M
authors.lastname: Rogers
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Cameron
authors.initials: C
authors.lastname: McDaniel
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: John D
authors.initials: JD
authors.lastname: Lipscomb
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Randall T
authors.initials: RT
authors.lastname: Irvin
authors.affiliation: Department of Microbiology, University of Colorado, Aurora, Colorado, United States of America.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Schurr
authors.affiliation: Departments of Anesthesiology, Cell, Developmental and Integrative Biology, and Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Lancaster
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Rhett A
authors.initials: RA
authors.lastname: Kovall
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, United States of America.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Hassett
mesh_terms: D000693:Anaerobiosis / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002374:Catalase / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007501:Iron / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009589:Nitrogen Oxides / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D009573:Nitrites; D009589:Nitrogen Oxides; D009569:Nitric Oxide; C073661:dinitrosyl iron complex; D007501:Iron; D002374:Catalase