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

The majority of bacteria use cobamides as cofactors for methionine synthesis or other diverse metabolic processes. Cobamides are a structurally diverse family of cofactors related to vitamin B12 (cobalamin), and most bacteria studied to date grow most robustly with particular cobamides. Because different environments contain varying abundances of distinct cobamides, bacteria are likely to encounter cobamides 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 increasing 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 in pCbl. Characterization of this 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.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

IMPORTANCE: In addition to proteins, microbes can use structured RNAs such as riboswitches for the important task of regulating gene expression. Riboswitches control gene expression by changing their structure in response to binding a small molecule and are widespread among bacteria. Here we determine the mechanism of regulation in a riboswitch that responds to corrinoids-a family of coenzymes related to vitamin B12. We report the alternative RNA secondary structures that couple corrinoid sensing with response in a repressing and novel activating corrinoid riboswitch. We then applied this knowledge to flipping the regulatory sign by constructing synthetic riboswitches that activate expression to a higher level than the natural one. In the process, we observed patterns in which sequence, in addition to structure, impacts function in paired RNA regions. The synthetic riboswitches we describe here have potential applications as biosensors.

Genetic dissection of regulation by a repressing and novel activating corrinoid riboswitch enables engineering of synthetic riboswitches.

The ability to sense and respond to intracellular metabolite levels enables cells to adapt to environmental conditions. Many prokaryotes use riboswitches - structured RNA elements usually located in the 5' untranslated region of mRNAs - to sense intracellular metabolites and respond by modulating gene expression. The corrinoid riboswitch class, which responds to adenosylcobalamin (coenzyme B12) and related metabolites, is among the most widespread in bacteria. The structural elements for corrinoid binding and the requirement for a kissing loop interaction between the aptamer and expression platform domains have been established for several corrinoid riboswitches. However, the conformational changes in the expression platform that modulate gene expression in response to corrinoid binding remain unknown. Here, we employ an in vivo GFP reporter system in Bacillus subtilis to define alternative secondary structures in the expression platform of a corrinoid riboswitch from Priestia megaterium by disrupting and restoring base-pairing interactions. Moreover, we report the discovery and characterization of the first riboswitch known to activate gene expression in response to corrinoids. In both cases, mutually exclusive RNA secondary structures are responsible for promoting or preventing the formation of an intrinsic transcription terminator in response to the corrinoid binding state of the aptamer domain. Knowledge of these regulatory mechanisms allowed us to develop synthetic corrinoid riboswitches that convert repressing riboswitches to riboswitches that robustly induce gene expression in response to corrinoids. Due to their high expression levels, low background, and over 100-fold level of induction, these synthetic riboswitches have potential use as biosensors or genetic tools.

Cobalamin Riboswitches Are Broadly Sensitive to Corrinoid Cofactors to Enable an Efficient Gene Regulatory Strategy.

In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found, among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semiselective. Analyses of chimeric riboswitches and structural models indicate, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites. IMPORTANCE Some bacterial mRNAs contain a region called a riboswitch which controls gene expression by binding to a metabolite in the cell. Typically, riboswitches sense and respond to a limited range of cellular metabolites, often just one type. In this work, we found the cobalamin (vitamin B12) riboswitch class is an exception, capable of sensing and responding to multiple variants of B12-collectively called corrinoids. We found cobalamin riboswitches vary in corrinoid specificity with some riboswitches responding to each of the corrinoids we tested, while others responding only to a subset of corrinoids. Our results suggest the latter class of riboswitches sense intrinsic conformational differences among corrinoids in order to support the corrinoid-specific needs of the cell. These findings provide insight into how bacteria sense and respond to an exceptionally diverse, often essential set of enzyme cofactors.