Analysis of <i>Escherichia coli</i> Mutants with a Linear Respiratory Chain

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.大肠杆菌（E. coli）的呼吸链呈分支构型，赋予细胞灵活适应环境条件动态变化的能力。该呼吸链包含NADH泛醌氧化还原酶（NADH:ubiquinone oxidoreductases）家族的NADH脱氢酶I（NADH dehydrogenase I）与II（NADH dehydrogenase II），以及三类末端氧化酶。不同组分的能量效率（质子转运效率）、对各类醌/氢醌物质及氧气的亲和力均存在显著差异。为探究分支型电子传递链相较于线性链的生物学优势，并明确不同末端氧化酶的使用如何调控不同氧浓度下的菌株生长行为，研究人员构建了一组同基因诱变菌株：通过敲除NADH脱氢酶I与两类末端氧化酶，最终获得仅携带线性呼吸链的工程菌株。采用葡萄糖限制恒化培养实验对上述菌株开展分析，实验通过精准控制供氧条件，覆盖了有氧、无氧及多种微有氧培养环境。与野生型菌株MG1655不同，所有诱变菌株即使在有氧条件下也会分泌乙酸。其中菌株TBE032缺失NADH脱氢酶I，且仅以细胞色素bd-II（cytochrome bd-II）作为唯一末端氧化酶，在有氧培养条件下展现出最高的乙酸生成速率。该结果佐证了如下假说：在较高氧浓度环境中，细胞色素bd-II末端氧化酶无法高效催化氢醌池（quinol pool）的氧化反应，其主要功能仅在限氧条件下得以发挥。研究同时分析了双组分系统ArcBA（two-component system ArcBA）的调控因子ArcA的磷酸化状态——除转录因子Fnr外，ArcA是响应氧浓度变化的核心转录因子。结果显示，诱变菌株中ArcA的磷酸化模式发生显著改变：其去磷酸化（进而导致蛋白失活）过程起始的有氧水平显著低于野生型菌株。值得关注的是，此次基因诱变不仅影响了微有氧与有氧代谢途径，还波及了本应不依赖呼吸链的无氧代谢过程。