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

Abstract

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 2,000 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 (clustered regularly interspaced short palindromic repeats interference sequencing) 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 a 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 F 1 F O ATP synthase and R hodobacter n itrogen f ixation (Rnf) respiratory complex are required for the 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. 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 Zymomonas mobilis , have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is 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 of Z. mobilis to create biofuels and bioproducts.

A Targeted Genome-scale Overexpression Platform for Proteobacteria

“…We next sought to demonstrate CRISPRtOE activity in Proteobacteria with medical and industrial relevance. The Alphaproteobacterium, Zymomonas mobilis , is a promising biofuel producer and emerging model for bacteria with naturally reduced genomes 36 . To assay CRISPRtOE function in Z. mobilis , we again generated an mScarlet-I test strain and measured expression following insertion of CRISPRtOE transposons (Supplementary Fig.…”

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

“…However, for this process to be economically viable, high yields and titers are required [3][4][5]. Zymomonas mobilis, an aerotolerant anaerobic α-proteobacterium, is an attractive candidate for industrial-scale biofuel production [6] due to its high catabolic rate [7], low biomass generation, resistance against inhibitors present in lignocellulosic hydrolysates [8], and an increasing set of genetic engineering tools [9,10]. Z. mobilis utilizes the highly thermodynamically favorable Entner-Doudoroff (ED) glycolytic pathway to convert up to 95% of consumed glucose to ethanol [6,[11][12][13][14][15].…”