Zou Qian, Luo Sha, Wu Hetao, He Donglan, Li Xiaohua, Cheng Guojun
Hubei Provincial Engineering and Technology Research Center for Resources and Utilization of Microbiology, College of Life Sciences, South-Central University for Nationalities, Wuhan, China.
Front Microbiol. 2020 Mar 24;11:394. doi: 10.3389/fmicb.2020.00394. eCollection 2020.
GmcA is a FAD-containing enzyme belonging to the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. A mutation in the gene was generated by homologous recombination. The mutation in did not affect the growth of , but it displayed decreased antioxidative capacity at HO conditions higher than 5 mM. The mutant strain displayed no difference of glutathione reductase activity, but significantly lower level of the glutathione peroxidase activity than the wild type. Although the mutant was able to induce the formation of nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 30% reduction in the nitrogen fixation capacity. The observation on ultrastructure of 4-week pea nodules showed that the mutant bacteroids tended to start senescence earlier and accumulate poly-β-hydroxybutyrate (PHB) granules. In addition, the mutant was severely impaired in rhizosphere colonization. Real-time quantitative PCR showed that the gene expression was significantly up-regulated in all the detected stages of nodule development, and statistically significant decreases in the expression of the redoxin genes , and were found in mutant bacteroids. LC-MS/MS analysis quantitative proteomics techniques were employed to compare differential mutant root bacteroids in response to the wild type infection. Sixty differentially expressed proteins were identified including 33 up-regulated and 27 down-regulated proteins. By sorting the identified proteins according to metabolic function, 15 proteins were transporter protein, 12 proteins were related to stress response and virulence, and 9 proteins were related to transcription factor activity. Moreover, nine proteins related to amino acid metabolism were over-expressed.
GmcA是一种含黄素腺嘌呤二核苷酸（FAD）的酶，属于氧化还原酶的GMC（葡萄糖-甲醇-胆碱氧化酶）家族。通过同源重组产生了该基因的突变。该突变不影响其生长，但在过氧化氢（H₂O₂）浓度高于5 mM的条件下，其抗氧化能力下降。该突变菌株的谷胱甘肽还原酶活性没有差异，但其谷胱甘肽过氧化物酶活性水平显著低于野生型。尽管该突变体能够诱导根瘤形成，但其共生能力严重受损，导致根瘤形成异常表型，固氮能力降低30%。对4周龄豌豆根瘤的超微结构观察表明，突变型类菌体倾向于更早开始衰老并积累聚-β-羟基丁酸酯（PHB）颗粒。此外，该突变体在根际定殖方面严重受损。实时定量PCR表明，该基因在根瘤发育的所有检测阶段表达均显著上调，并且在突变型类菌体中发现氧化还原蛋白基因、和的表达有统计学意义的下降。采用液相色谱-串联质谱（LC-MS/MS）分析定量蛋白质组学技术比较突变型根类菌体与野生型感染后的差异。鉴定出60种差异表达蛋白，包括33种上调蛋白和27种下调蛋白。根据代谢功能对鉴定出的蛋白进行分类，15种蛋白为转运蛋白，12种蛋白与应激反应和毒力相关，9种蛋白与转录因子活性相关。此外，9种与氨基酸代谢相关的蛋白过表达。
