To elucidate the underlying design principles of microbial community stability and health, emphasizing interactions between microorganisms for which there are established interdependencies is a challenge that requires development of cutting edge tools. Here we have developed vitamin B12 as an affinity-based probe to explore molecular interactions in a Halomonas species microbe that is opportunistic for vitamin B12, meaning it can both salvage and biosynthesize the vitamin. Vitamin B12 is an essential cofactor for several important enzymes, and also plays a key role in photosystem gene regulation. Using a chemoproteomic approach we characterized a total of 45 proteins captured by the probe. Components of all three expected B12-dependent enzymes (methionine synthase, ribonucleoside reductase, and ethanolamine ammonia-lyase) were detected. A review of the remaining proteins, suggest that B12 may act as an allosteric regulator of enzyme activity. Allosteric effects at these positions could result in redirection of metabolism between biosynthesis of heme versus B12 as well as between B12 biosynthesis versus salvage. A single DNA regulator, that we named PhrR, was also captured by B12-ABP yet lacked a recognizable B12-binding motif. Since B12 is susceptible to photolysis it can potentially control DNA binding of PhrR in the presence of light. We purified PhrR and validated that it is able to bind B12 as well as several DNA fragments that encode a palindromic binding motif that we predicted to be the target binding sites for PhrR. This motif was found at 15 sites in the genome and predicted to control 27 different genes encoding functions associated with photodamage repair, folate biosynthesis, and SAM-dependent processes. This unexpected connection between B12 and folate metabolism suggests that this vitamin may mediate a light-dependent coordination of community member metabolism.

Unanticipated interactions for vitamin B12 revealed by chemical probing

251st National Meeting (2016)

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