Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions

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 B7 have 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.微生物基因组的快速组装完成，正给功能基因的发掘带来难解的困境。亟需开发新方法，以缩小微生物基因组功能注释与生物信息学预测功能的实验验证之间的差距。其中尤为关键的是转运机制：这类机制负责将B族维生素、代谢物等营养物质跨细胞膜转运，且从环境微生物群落成员到致病菌的各类微生物生存均依赖此类机制。目前亟需开发可精准为大量未通过实验鉴定、或仅为预测的膜内嵌转运蛋白赋予功能与特异性的方法，同时解析胞内酶-辅因子结合关联，以此大幅深化我们对微生物生物化学与生理学、微生物互作以及微生物对环境扰动的响应的理解。我们基于B1、B2、B7族维生素开发的化学探针，通过对丝状无氧化能光合异养菌（filamentous anoxygenic photoheterotroph）橙色绿弯菌（Chloroflexus aurantiacus）J-10-fl进行活细胞标记，成功鉴定出B族维生素转运蛋白与胞内酶-辅因子结合关系，从而从实验层面解决了前述需求；该菌株已知同时具备B族维生素生物合成与环境补救摄取两种机制。本研究开发的探针可通过鉴定微生物生存必需的B族维生素转运过程及辅因子依赖型互作关系，为直接将细胞活动与蛋白质功能关联至生态系统或宿主动态提供了独特契机。