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.

Isolation and Characterization of the Small Subunit of the Uptake Hydrogenase from the Cyanobacterium Nostoc punctiforme

“…Most uptake hydrogenases investigated so far are rapidly inhibited by molecular oxygen (13), and a cyanobacterial uptake hydrogenase localized in the vegetative cells would undoubtedly be inactivated by photosynthetic oxygen evolution (14). Recently, it was demonstrated that the active enzyme is produced solely in heterocysts under N 2 -fixing conditions (19). The maturation system and genomic context of HupSL in N. punctiforme have been investigated extensively.…”

“…The maturation system and genomic context of HupSL in N. punctiforme have been investigated extensively. The hupSL promoter region and binding sites for the transcriptional regulator NtcA have been identified (20); the extended hyp operon region, comprising the assembly and maturation system of HupSL, has been shown to be regulated by the transcriptional regulator CalA (19); and HupW, a protease needed for the cleavage of a C-terminal peptide from the large subunit HupL, has been shown to be transcribed in N 2 -fixing cultures (20). In a related organism, Nostoc (Anabaena) sp.…”

Metalloproteins in the Biology of Heterocysts

“…Heterocysts contain two distinct O 2 -sensitive Ni–Fe hydrogenases defined by their physiological role (Table 2). One is termed uptake hydrogenase, which is encoded by the hupSL operon and catalyzes the irreversible conversion of H 2 into protons and electrons, while the second enzyme is a bidirectional hydrogenase, which catalyzes the reversible conversion of protons and electrons into H 2 and is encoded by hoxEFUYH [152,192,193,194,195,196,197].…”