The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Abstract

Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H 2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H 2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (⌬hupL), and we determined how this would affect the amount of H 2 produced. The ⌬hupL mutant demonstrated virtually no nitrogenase activity or H 2 production when grown under N 2 -fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O 2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N 2 -fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H 2 produced by the nitrogenase.

Distribution of Hydrogenases in Cyanobacteria: A Phylum-Wide Genomic Survey

“…In the unicellular cyanobacterium Cyanothece PCC 7822, which fixes nitrogen under aerobiosis, HupL has been shown to be essential to activity of the nitrogenase in the presence of O 2 . The authors concluded that the main function of the HupSL complex in this bacterium is the protection of the nitrogenase from O 2 (Zhang et al, 2014). The present data show that most of the strains possessing nif genes and lacking the uptake H 2 ase are unicellular [ Aphanocapsa montana BDHKU210001, Chroococcidiopsis sp.…”

[FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation

“…These organisms contain a dimeric uptake hydrogenase (HupSL) to recapture energy lost as H 2 during the reduction of N 2 [77]. Electrons from H 2 are then channeled into the quinone pool or function to reduce O 2 that deactivates the oxygen sensitive nitrogenase enzyme [78,79]. Mutation of the HupLS genes in the diazotroph Anabaena siamensis abolished H 2 uptake activity and led to an increase in light-dependent H 2 production, presumably due to the lack of recapture of H 2 produced by nitrogenase enzyme in the absence of the uptake hydrogenase [80].…”

The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5

“…This diversity of function allows hydrogenases to perform a critical role in a variety of metabolisms, including those of methanogens (Thauer et al, 2010), sulfate reducers (Baltazar et al, 2011), nitrogen fixers (Zhang et al, 2014), and photosynthesizers (Melis et al, 2004; Ghirardi et al, 2006; Tamagnini et al, 2007). …”