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

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 (Delta hupL), and we determined how this would affect the amount of H-2 produced. The Delta 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.