The Genetics of Aerotolerant Growth in a Naturally Reduced Genome Alphaproteobacterium

Abstract

AbstractReduced 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 ofZ. mobilisas a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterizeZ. mobilisgenes 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 betweenZ. mobilisand other Alphaproteobacteria, validatingZ. mobilisas 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 F1FOATP synthase and Rnf respiratory complex are required for anaerobic growth ofZ. 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 establishesZ. mobilisas a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informsZ. mobilisgenome manipulation for optimized production of valuable isoprenoid-based bioproducts.ImportanceThe 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,Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show thatZ. mobilisis a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use ofZ. mobilisto create biofuels and bioproducts.