Vitamin B12 trafficking and selectivity in gut bacteria

项目摘要 人类肠道微生物组由数万亿种细菌居住，这些细菌编码的基因是人类肠道微生物组的150倍以上。 人类基因组本身微生物组成的个体间差异可能是显著的， 造成这种多样性的因素还没有得到很好的理解。宏基因组学研究表明， 微生物组可能在确定个体的疾病易感性方面发挥重要作用， 对治疗的反应。缺乏对辅因子和其他因素如何影响微生物生长的了解， 组合物限制策略，以合理地改变它用于治疗目的。我们目前的理解 形成肠道微生物植物群组成的因素来自于对细菌如何产生 能量，维持氧化还原平衡，获取碳和氮。酶促反应支持 这些代谢过程通常依赖于供应不足的辅因子。维生素B12是一种 一种这种辅因子，对于许多不能生物合成它的细菌是必需的， B12独立的代谢途径，以规避其缺乏。因此，一种针对目标的方法 肠道微生物组的操纵是通过改变可用的类可丽素的水平。在这份提案中，我寻求 阐明转运系统的类咕啉选择性，以提供对肠道细菌如何 在复杂的生态系统中，它们相互竞争，争夺关键的资源。我的研究会 重点是多形拟杆菌，一种常见的肠道细菌，它缺乏发育所需的基因， 新合成维生素B12，但编码多种B12依赖性酶。5 '-脱氧腺苷钴胺素 是活性辅因子形式，被一些B12依赖性酶利用，并由BtuR合成， B。太多了。分子伴侣和催化活性未被表征，将在 目标1.它还编码三个拷贝的外膜B12转运蛋白BtuB，每个系统 显示出对类咕啉衍生物的不同偏好。该细菌还具有额外的 运输机械在E.大肠杆菌，一种模式生物，其中研究了B12在大肠杆菌中的转运， 革兰氏阴性菌一直是关注的焦点。结合生物化学和生物物理学 本文拟从B12摄取（Btu）系统的角度阐明Aim中B12转运的机制 2.目的1和2中的动力学和热力学研究将定义Btu蛋白对以下的选择性： 钴酰胺和提供深入了解蛋白质-蛋白质相互作用。结合测定的结构 在目标1和目标2中，我的研究将提供一种机制性的见解，即一种珍贵而稀有的辅因子是如何传递的 从环境中穿过两层细菌膜，以支持 常见的肠道细菌

Project Summary The human gut microbiome is inhabited by trillions of bacteria that encode over 150-fold more genes than the human genome itself. The inter-individual differences in microbial composition can be significant, and the factors contributing to this diversity are not well understood. Metagenomic studies suggest that the microbiome might play an important role in determining an individual’s predisposition to disease and responses to treatments. The paucity of understanding how cofactors and other factors influence microbial composition limit strategies to rationally alter it for therapeutic purposes. Much of our current understanding of the factors that shape the gut microbial flora composition derives form studies on how bacteria generate energy, maintain redox balance and acquire carbon and nitrogen. The enzymatic reactions that support these metabolic processes often rely on cofactors that are in short supply. Vitamin B12 is an example of one such cofactor that is essential for many bacteria that are unable to biosynthesize it and lack parallel B12-independent metabolic pathways to circumvent its absence. So, one approach to the targeted manipulation of the gut microbiome is via altering the levels of available corrinoids. In this proposal, I seek to elucidate the corrinoid selectivity of transport systems to provide needed insights into how gut bacteria compete with each other and their hosts for a critical resource in a complex ecosystem. My studies will focus on Bacteroidetes thetaiotaomicron, a common gut bacterium, which lacks the genes required for de novo synthesis of vitamin B12 but encodes multiple B12-dependent enzymes. 5’-Deoxyadenosylcobalamin is the active cofactor form that is utilized by some B12 dependent enzymes and is synthesized by BtuR in B. thetaiotaomicron. The chaperone and catalytic activities are uncharacterized and will be addressed in Aim 1. It also encodes three copies of the outer membrane B12-transporter BtuB with each system displaying a different preference for corrinoid derivatives. The bacterium also possesses additional transport machinery that is not observed in E. coli, a model organism in which studies on B12 transport in gram-negative bacteria have been focused. Using a combination of biochemical and biophysical approaches, I propose to elucidate the mechanism of B12 transport by the B12-uptake (Btu) system in Aim 2. The kinetic and thermodynamic studies in Aims 1 and 2 will define the selectivity of the Btu proteins for cobamides and provide insights into protein-protein interactions. Combined with the structures determined in Aims 1 and 2, my studies will furnish mechanistic insights into how a precious and rare cofactor is relayed from the environment across two layers of bacterial membranes to support the metabolic needs of a common gut bacterium.