A Novel Endo-Hydrogenase Activity Recycles Hydrogen Produced by Nitrogen Fixation 英文参考文献.docVIP

ANovelEndo-HydrogenaseActivityRecyclesHydrogen ProducedbyNitrogenFixation GordonNg.,CurtisG.S.Tom.,AngelaS.Park,LounisZenad,RobertA.Ludwig* DepartmentofMolecular,CellularandDevelopmentalBiology,SinsheimerLaboratories,UniversityofCaliforniaSantaCruz,SantaCruz,California,UnitedStatesofAmerica Abstract Background:Nitrogen(N2)fixationalsoyieldshydrogen(H2)at1:1stoichiometricamounts.Inaerobicdiazotrophic(ableto growonN2assoleN-source)bacteria,orthodoxrespiratoryhupSL-encodedhydrogenaseactivity,associatedwiththecell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenoushydrogen. MethodsandFindings:Asshownhere,forAzorhizobiumcaulinodansdiazotrophicculturesopentotheatmosphere,exo- hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH : quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant,constructedby‘‘crossoverPCR’’,showedmarkedlydecreasedgrowthratesindiazotrophiccultures;normalgrowth was restored with added ammonium—as expected of an H2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing b-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically inducedindiazotrophicculture;aswell,hyqoperoninductionrequiredtheNIFAtranscriptionalactivator.Therefore,thehyq operonisconstituentofthenifregulon. Conclusions: Representative of aerobic N2-fixing and H2-recycling a-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H2 respiration, and Hyq endo- hydrogenaseactivityrecyclesendogenousH2,specificallythatproducedbyN2fixation.Tobene