title: A HupS-GFP fusion protein demonstrates a heterocyst-specific localization of the uptake hydrogenase in Nostoc punctiforme.
abstract: All diazotrophic filamentous cyanobacteria contain an uptake hydrogenase that is involved in the reoxidation of H(2) produced during N(2) -fixation. In Nostoc punctiforme ATCC 29133, N(2) -fixation takes place in the microaerobic heterocysts, catalysed by a nitrogenase. Although the function of the uptake hydrogenase may be closely connected to that of nitrogenase, the localization in cyanobacteria has been under debate. Moreover, the subcellular localization is not understood. To investigate the cellular and subcellular localization of the uptake hydrogenase in N. punctiforme, a reporter construct consisting of the green fluorescent protein (GFP) translationally fused to HupS, within the complete hupSL operon, was constructed and transferred into N. punctiforme on a self-replicative vector by electroporation. Expression of the complete HupS-GFP fusion protein was confirmed by Western blotting using GFP antibodies. The N. punctiforme culture expressing HupS-GFP was examined using laser scanning confocal microscopy, and fluorescence was exclusively detected in the heterocysts. Furthermore, the fluorescence in mature heterocysts was localized to several small or fewer large clusters, which indicates a specificity of the subcellular localization of the uptake hydrogenase.
authors.affiliation: Ångström Laboratories, Department of Photochemistry and Molecular Science, Uppsala University, Uppsala, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Camsund
authors.forename: Ellenor
authors.initials: E
authors.lastname: Devine
authors.forename: Marie
authors.initials: M
authors.lastname: Holmqvist
authors.forename: Peter
authors.initials: P
authors.lastname: Yohanoun
authors.forename: Peter
authors.initials: P
authors.lastname: Lindblad
authors.forename: Karin
authors.initials: K
authors.lastname: Stensjö
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D046937:Nostoc / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C029746:uptake hydrogenase