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

Abstract

Bacteria encounter chemically similar nutrients in their environment that 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 as well as those that do not function efficiently for their metabolism. Here, 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, to identify genetic adaptations that lead to improved growth with less-preferred cobamides. After propagating and sequencing nine independent lines and validating the results by constructing targeted mutations, we found that mutations that increase expression of the outer membrane cobamide transporter BtuB are beneficial during growth under cobamide-limiting conditions. Unexpectedly, we also found that overexpression of the cobamide adenosyltransferase BtuR confers a specific growth advantage in 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.