The Genetics of Aerotolerant Growth in Zymomonas mobilis

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living Alphaproteobacterium, Zymomonas mobilis, has a naturally reduced genome containing fewer than 2000 protein coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth, or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rnf respiratory complex are required for anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts.

基因组简化细菌（reduced genome bacteria）是经遗传精简的系统，可助力生物学研究与工业应用。自由生活的α变形菌门（Alphaproteobacteria）菌株运动发酵单胞菌（Zymomonas mobilis，以下简称Z. mobilis）拥有天然简化的基因组，其蛋白编码基因不足2000个。尽管基因组规模小巧，运动发酵单胞菌可在多种环境条件下存活，包括有氧与无氧环境。然而，由于对其必需基因与条件必需基因的功能表征不足，运动发酵单胞菌作为α变形菌门模式菌株的推广应用受到了限制。本研究采用全基因组范围的CRISPR干扰测序（CRISPRi-seq）技术，系统鉴定并表征了运动发酵单胞菌中对耐氧生长或厌氧生长具有条件必需性，或是在两种条件下均为普遍必需的基因。比较基因组学分析显示，运动发酵单胞菌与其他α变形菌门物种的多数普遍必需基因具有共有的必需性特征，证实了运动发酵单胞菌可作为基因组简化型模式菌株。在条件必需基因中，本研究发现DNA修复基因recJ仅对有氧生长至关重要，却可在两种培养条件下降低突变率。此外，本研究证实，编码F1FO ATP合酶（F1FO ATP synthase）与Rnf呼吸复合物（Rnf respiratory complex）的基因是运动发酵单胞菌厌氧生长所必需的。本研究将CRISPRi部分敲低实验与代谢组学、膜电位检测技术相结合，探明了F1FO ATP合酶可产生膜电位，而该膜电位可被Rnf复合物消耗以驱动下游生理过程。Rnf复合物敲低菌株会积累异戊二烯类生物合成中间体，这表明Rnf复合物在驱动关键生物合成反应中发挥核心作用。本研究确立了运动发酵单胞菌作为α变形菌门遗传学研究的精简模式系统，其研究成果对细菌能量耦合机制研究具有广泛参考价值，同时可为通过基因组编辑优化生产高价值异戊二烯类生物制品提供理论指导。