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首页> 美国卫生研究院文献>Journal of Bacteriology >The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity
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The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

机译：单细胞重氮蓝藻蓝藻sp。中的吸收氢酶。 PCC 7822菌株可保护氮酶免受氧气中毒

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Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-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 O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-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 H2 produced by the nitrogenase.
机译：蓝藻PCC 7822菌株是单细胞的重氮蓝藻细菌，当重氮生长时可以产生大量H2。该菌株也能够进行遗传操作，并且可以代表一个改进的模型，用于改善蓝藻产生的H2。为此，在hupL基因（ΔhupL）中产生了敲除突变，我们确定了这将如何影响所产生的H2量。在N2固定条件下生长时，ΔhupL突变体几乎没有显示出固氮酶活性或H2产生。为确保该突变仅影响hupL基因，可轻易构建具有野生型特性的互补菌株。这表明原始插入仅在hupL中。该突变体没有摄取氢化酶活性，但是具有增加的双向氢化酶（Hox）活性。电子显微镜下的蛋白质印迹和免疫细胞化学分析表明，尽管该nif基因被转录，但该突变体既没有HupL也没有NifHDK。有趣的是，生化分析表明HupL和NifH都可能与膜相关。结果表明，nif基因被转录，但NifHDK要么不翻译，要么被翻译，但迅速降解。我们假设Nif蛋白已制成，但异常易受O2破坏。因此，我们使突变细胞在厌氧条件下生长，发现它们在固氮条件下生长良好。我们得出的结论是，在单细胞重氮菌中，如Cyanothece sp。 HupLS复合物是PCC 7822的变种，它有助于从固氮酶中除去氧气，这是比仅氧化固氮酶产生的H2更重要的功能。

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