Evolving a new electron transfer pathway to nitrogenase in an alphaproteobacterium. Evolving a new electron transfer pathway to nitrogenase in an alphaproteobacterium

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin or flavodoxin to reduce dinitrogen gas (N2) to produce ammonia and hydrogen. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes many proteins that can reduce ferredoxin, the electron bifurcating FixABCX complex is the only one shown to support nitrogen fixation. To gain insight into why R. palustris is unable to use these other enzymes to reduce ferredoxin in the absence of FixABCX, we isolated a suppressor of R. palustris DfixC that allowed this strain to grow under nitrogen-fixing conditions. We found two mutations were necessary and sufficient to restore growth under nitrogen-fixing conditions in the absence of a functional FixABCX. One mutation was in the primary ferredoxin involved in nitrogen fixation, fer1, and the other mutation was in rpa0678, a homolog of NAD+-dependent ferredoxin:NADPH oxidoreductase, which carries out flavin-based electron bifurcation to generate reduced Fd. We present evidence that Rpa0678 plays a role in electron transfer to benzoyl-CoA reductase, the key enzyme involved in anaerobic aromatic compound degradation. Together these findings indicate that the electron transfer pathway for anaerobic aromatic compound degradation was re-purposed to support nitrogen fixation in the suppressor strain. Overall design: Comparative gene expression profiling analysis of RNA-seq data for R. palustris CGA753 and suppressor mutant of CGA753 with a deletion in fixC

固氮酶（Nitrogenase）是介导固氮作用的核心酶，依托铁氧还蛋白（ferredoxin）或黄素氧还蛋白（flavodoxin）传递的低电位电子，将二氮气（N₂）还原为氨与氢气。尽管光合α-变形菌沼泽红假单胞菌（Rhodopseudomonas palustris）编码多种可还原铁氧还蛋白的蛋白，但目前仅证实电子分流FixABCX复合物（electron bifurcating FixABCX complex）能够支持该菌的固氮功能。
为解析沼泽红假单胞菌在缺失FixABCX复合物时无法利用其他酶还原铁氧还蛋白的原因，本研究分离得到一株沼泽红假单胞菌DfixC的抑制突变株，该菌株可在固氮条件下正常生长。研究发现，仅需两个突变即可且必须恢复缺失功能性FixABCX复合物时的固氮生长能力：其一为参与固氮作用的主要铁氧还蛋白fer1的突变，其二为rpa0678的突变——该基因为依赖烟酰胺腺嘌呤二核苷酸（NAD+）的铁氧还蛋白:NADPH氧化还原酶（NAD+-dependent ferredoxin:NADPH oxidoreductase）的同源基因，此类酶可通过黄素介导的电子分流生成还原型铁氧还蛋白（reduced Fd）。
本研究证实，Rpa0678参与向苯甲酰辅酶A还原酶（benzoyl-CoA reductase）的电子传递，而该酶是厌氧芳香族化合物降解的关键酶。综上，本研究结果表明，在该抑制突变株中，厌氧芳香族化合物降解的电子传递通路被重新利用以支持固氮作用。
实验设计方案：对沼泽红假单胞菌CGA753及其fixC缺失的CGA753抑制突变株的RNA测序（RNA-seq）数据开展比较基因表达谱分析。