Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
Appl Environ Microbiol. 2018 Aug 31;84(18). doi: 10.1128/AEM.00955-18. Print 2018 Sep 15.
Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B). Our knowledge of the roles of vitamin B is limited, because technologies that enable characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B supports the growth of B-auxotrophic bacteria and archaea. We demonstrate that a B activity-based probe (B-ABP) is actively transported into cells and converted to adenosyl-B-ABP akin to native B Identification of the proteins that bind the B-ABP in , a sp. and , demonstrate the specificity for known and novel B protein targets. The B-ABP also regulates the B dependent RNA riboswitch and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B interactions in living cells and can be used to discover the role of B in diverse microbial systems. We demonstrate that a cobalamin chemical probe can be used to investigate roles of vitamin B in microbial growth and regulation by supporting the growth of B auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B-protein interactions. The B-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B The application of the B-ABP at biologically relevant concentrations facilitates a unique way to measure B microbial dynamics and identify new B protein targets in bacteria and archaea. We demonstrate that the B-ABP can be used to identify protein interactions across diverse microbes, from to microbes isolated from naturally occurring phototrophic biofilms to the salt-tolerant archaea .
理解调节微生物功能和微生物群落组装、功能和适应性的因素是一个巨大的挑战。钴胺素（维生素 B）是一个关键因素，也是重要的酶辅因子和基因表达调节剂。我们对维生素 B 作用的了解是有限的，因为需要能够在最小影响细胞生理学的情况下对微生物代谢和基因调控进行特征描述的技术。为了满足这一需求，我们展示了一种合成的 B 探针类似物可以支持 B 营养缺陷型细菌和古菌的生长。我们证明了一种 B 活性探针（B-ABP）可以被主动转运到细胞中，并转化为类似于天然 B 的腺苷酰基-B-ABP。在 Sp. 和 中，鉴定与 B-ABP 结合的蛋白质，证明了对已知和新型 B 蛋白靶标的特异性。B-ABP 还调节 B 依赖性 RNA 核糖开关和转录因子 EutR。我们的结果展示了一种新的方法来了解 B 在微生物功能中的作用。我们的方法提供了一种强大的非破坏性工具，可用于分析活细胞中 B 的相互作用，并可用于发现 B 在各种微生物系统中的作用。我们证明，一种钴胺素化学探针可以通过支持 B 营养缺陷型细菌和古菌的生长来用于研究维生素 B 在微生物生长和调控中的作用，使三种不同的细胞大分子（RNA、DNA 和蛋白质）具有生物活性，并促进功能蛋白质组学以表征 B-蛋白相互作用。B-ABP 能够转录和翻译调控基因表达，类似于天然维生素 B。B-ABP 在生物相关浓度下的应用为测量 B 微生物动力学和在细菌和古菌中识别新的 B 蛋白靶标提供了一种独特的方法。我们证明，B-ABP 可用于识别来自不同微生物的蛋白质相互作用，从到从自然发生的光养生物膜中分离出来的微生物到耐盐古菌。
