Molecular Analysis of Corrinoid Specificity

项目摘要 驻留在人体肠道中的微生物（肠道微生物群）的组成对 对人类健康的影响。尽管最近在理解肠道微生物群在健康中的作用方面取得了进展， 仍然需要操纵肠道微生物群以治疗和预防疾病的有效机制。一 操纵肠道群落的潜在靶点是辅助因子（维生素B12和相关的 含钴的改性四吡咯），这是肠道中大多数细菌所需要的，尽管它们 只由一小部分人口生产。存在多种结构形式的类咕啉， 功能相当，但对细菌基因组序列和适应性之间的联系知之甚少 用于特定的类可丽素。这项提案的目标是确定基因组之间的关系， 肠道微生物对类可啉的偏好。这一目标将通过检查细菌 corrinoid偏好在分子，有机体，和社区水平。a同源物的类柯啉特异性 一种依赖于类可丽素的模型酶将通过生物化学、遗传学和生物信息学的结合来剖析。 接近。与此同时，核糖开关RNA的类corrinoid特异性，存在于大多数基因组中， 将通过使用基于荧光的体外结合测定和体内结合测定来检查人类微生物组。 报告基因测定。这些数据将用于预测类可啉特异性的序列特征，并测试这些特征。 通过靶向诱变进行预测。此外，将在培养的人肠道中检查类可丽素偏好。 通过使用高通量机器人平台测量在需要类可丽素的条件下的生长来检测细菌。 这项工作将提高解释基因组序列信息的能力，并提供详细的 对大多数人类肠道微生物至关重要的代谢过程的表征。结果将促进 开发新的策略来预防和治疗与肠道微生物群相关的疾病。

Project Summary The composition of microorganisms that reside in the human intestine (gut microbiota) has a significant impact on human health. Despite recent advances in understanding the role of the gut microbiota in health, there remains a need for effective mechanisms of manipulating the gut microbiota to treat and prevent disease. One potential target for manipulation of the gut community is the corrinoid family of cofactors (vitamin B12 and related cobalt-containing modified tetrapyrroles), which are required by the majority of bacteria in the gut, though they are produced only by a fraction of the population. A variety of structural forms of corrinoids exist that are not functionally equivalent, yet little is known about the link between bacterial genome sequences and adaptations for the use of particular corrinoids. The goal of this proposal is to determine the relationship between genome sequence and corrinoid preferences in gut microbes. This goal will be accomplished by examining bacterial corrinoid preferences at the molecular, organismal, and community levels. Corrinoid specificity of homologs of a model corrinoid-dependent enzyme will be dissected by a combination of biochemical, genetic, and bioinformatic approaches. In parallel, corrinoid specificity of riboswitch RNAs, which are present in the majority of genomes in the human microbiome, will be examined by using an in vitro fluorescence-based binding assay and in vivo reporter assays. These data will be used to predict sequence signatures of corrinoid specificity and test these predictions by targeted mutagenesis. Additionally, corrinoid preferences will be examined in cultured human gut bacteria by measuring growth under corrinoid-requiring conditions using a high-throughput robotics platform. This work will lead to an improved ability to interpret genome sequence information and provide a detailed characterization of metabolic processes critical to the majority of human gut microbes. The results will promote the development of novel strategies to prevent and treat diseases associated with the gut microbiota.