The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, 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 as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize 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 and other Alphaproteobacteria, validating as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, , was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F F ATP synthase and itrogen ixation (Rnf) respiratory complex are required for the anaerobic growth of . 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 as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium , have fewer genes but remain environmentally robust. In this study, we show that is a simplified genetic model for Alphaproteobacteria, a class with impor