Laboratory evolution of E. coli with a natural vitamin B12 analog reveals roles for cobamide uptake and adenosylation in methionine synthase-dependent growth.

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Bacteria encounter chemically similar nutrients in their environment, which impact their growth in distinct ways. Among such nutrients are cobamides, the structurally diverse family of cofactors related to vitamin B12 (cobalamin), which function as cofactors for diverse metabolic processes. Given that different environments contain varying abundances of different cobamides, bacteria are likely to encounter cobamides that enable them to grow robustly and also those that do not function efficiently for their metabolism. To gain insights into how bacteria might respond under the latter conditions, we performed a laboratory evolution of a cobamide-dependent strain of Escherichia coli with pseudocobalamin (pCbl), a cobamide that E. coli uses less effectively than cobalamin for MetH-dependent methionine synthesis. Propagation and sequencing of nine independent lines identified potential genetic adaptations in cobamide-related genes that improved growth with less-preferred cobamides. We constructed targeted mutants to validate these findings and found that increasing the expression of the outer membrane cobamide transporter BtuB is beneficial during growth under cobamide-limiting conditions. Unexpectedly, we also found that overexpression of the cobamide adenosyltransferase BtuR confers a specific growth advantage with pCbl. Characterization of the latter phenotype revealed that BtuR and adenosylated cobamides contribute to optimal MetH-dependent growth. Together, these findings improve our understanding of how bacteria expand their cobamide-dependent metabolic potential.IMPORTANCEIn nature, bacteria commonly experience fluctuations in the availability of required nutrients. Thus, their environment often contains nutrients that are insufficient in quantity or that function poorly in their metabolism. Cobamides, the vitamin B12 family of cofactors, are ideal for investigating the influence of nutrient quality on bacterial growth. We performed a laboratory evolution experiment in E. coli with a less-preferred cobamide to examine whether and how bacteria can improve their growth with less ideal nutrients. We found that overexpression of genes for cobamide uptake and modification are genetic adaptations that improve growth under these conditions. Given that cobamides are key shared metabolites in microbial communities, our results reveal insights into bacterial interactions and competition for nutrients.