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The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.