Vitamin B12 trafficking and selectivity in gut bacteria

项目摘要 人类肠道微生物群中居住着数以万亿计的细菌，这些细菌编码的基因是 人类基因组本身。微生物组成的个体间差异可能很大，并且 造成这种多样性的因素还没有得到很好的理解。元基因组学研究表明， 微生物组可能在决定个人的疾病易感性和 对治疗的反应。缺乏对辅因子和其他因素如何影响微生物的了解 成分限制策略，以合理地改变它的治疗目的。我们目前的大部分理解 在影响肠道微生物菌群组成的因素中，有一项来自对细菌如何产生的研究 能量，保持氧化还原平衡，并获得碳和氮。支持这些反应的酶反应 这些代谢过程往往依赖于供不应求的辅因子。维生素B12就是一个例子 一种这样的辅因子，对许多细菌来说是必不可少的，这些细菌无法生物合成它，也缺乏平行的 不依赖于B12的代谢途径，以避免其缺失。因此，一种针对目标的方法 肠道微生物群的操纵是通过改变可利用的Corrinoid的水平来实现的。在这项提议中，我寻求 阐明转运系统的皮质醇选择性，以提供对肠道细菌如何 在复杂的生态系统中，相互竞争，争夺关键资源。我的学习将会 关注拟杆菌thetaiotaomicron，一种常见的肠道细菌，它缺乏De所需的基因 新合成维生素B12，但编码多种依赖B12的酶。5‘-脱氧腺苷钴胺 是一些B12依赖酶利用的活性辅因子形式，由BtuR在 B.thetaiotaomicron。伴侣和催化活性尚未确定，将在 目的1.每个系统还编码三个拷贝的外膜B12转运蛋白BtuB 对皮质激素衍生物表现出不同的偏好。细菌还拥有额外的 在大肠杆菌中没有观察到的运输机制，这是一种研究B12转运的模式生物 革兰氏阴性细菌已经成为关注的焦点。使用生化和生物物理的组合 方法，我建议阐明B12摄取(Btu)系统在AIM中转运的机制 2.AIMS 1和AIMS 2中的动力学和热力学研究将确定Btu蛋白对 并提供了对蛋白质-蛋白质相互作用的见解。结合确定的结构 在目标1和目标2中，我的研究将提供关于珍贵和稀有的辅因子如何传递的机械性见解。 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.