Abstract 1963 Bacterial Organelles: Encapsulation of Anaerobic Metabolic Pathways within Bacterial Microcompartments in the Human Microbiome

Despite living in an oxygen-filled world, anaerobic bacteria are abundant; there are many oxygen-scarce sites in the human microbiome, most notably the human gut. Under low-oxygen conditions, anaerobic bacteria rely on oxygen-sensitive enzymes to carry out metabolic processes critical for survival. Our lab explores molecular tactics that human gut anaerobes rely on to protect such pathways vulnerable to oxidative damage and thrive amidst oxidative stress, potentially providing a competitive advantage for these microbes. One potential strategy is physically encapsulating oxygen-sensitive enzymes within bacterial microcompartments (BMCs) to shield them from molecular oxygen and reactive oxidants. BMCs are highly ordered, self-assembling, protein-based organelles made to optimize metabolic pathways. They form a semipermeable protein shell that encapsulate associated catabolic enzymes and their intermediates to make an ideal catalytic environment. Because the associated enzymes are often oxygen-sensitive enzymes containing glycyl radical, Fe-S cluster, or vitamin B12 cofactors, it has been proposed that BMCs might shield these reactions from oxidative damage in anaerobic bacteria, and also contain toxic intermediates. The human gut is a highly anaerobic environment enriched in ethanolamine, a nutrient and substrate of ethanolamine ammonia lyase (EAL), a B12-dependent enzyme. EAL and other ethanolamine utilization (EUT) enzymes are encapsulated within BMCs in many gut anaerobes, and these BMCs are linked to pathogenesis in certain bacteria. Here we aim to structurally and biochemically characterize EUT BMCs, under aerobic, anaerobic, and micro-anaerobic conditions. We have optimized a protocol to express and purify EUT BMCs in E. coli strains and are working towards elucidating the macromolecular architecture of these organelles via electron microscopy approaches. We are investigating how BMCs differ under oxidative conditions, and we are exploring whether the BMC shell provides a competitive advantage upon oxygen exposure via genetic approaches. Furthermore, we are working towards solving the structures of BMC-associated EALs, including structurally elusive auto-encapsulation domains. Altogether these experiments will add to our basic biological understanding of these abundant bacterial organelles and will provide insight into whether BMCs provide a competitive advantage to microbes that utilize them, potentially leading to new therapeutic targets. This research was funded by the Whitehead Institute for Biomedical Research, the ValhallaFoundation, and a gift from Jim and Cathy Stone.