Functional Analysis of a Digestive-Tract Microbiome During Dietary Changes

描述(申请人提供)：在人类中，消化道微生物区系是一个由数百个物种组成的复杂群落。这个微生物组为人类提供了一系列令人惊叹的生化过程，远远超过了动物基因组编码的那些过程。当动物成熟或改变饮食时，微生物群的组成会发生变化。这种变化如何影响微生物组的整体生理及其对宿主的贡献还知之甚少。从人类微生物组项目获得的信息已经产生了丰富的基因组信息，并提供了对可能功能的洞察，但缺乏来自动物及其本地微生物组的功能数据。微生物组的绝对复杂性阻碍了识别微生物组表达的基因--转译组学。这项应用研究了一种自然产生的简单微生物群，它位于药用水蚤的消化道中。两种细菌主导着微生物群落，其中一种是可以利用的遗传工具。在水蚤从茧中孵化出来后的头两周，以及在它们吃了一顿血后，微生物群的组成发生了变化。如果血液被部分溶解，微生物群中的一种次要成员梭状芽胞杆菌就会变得非常丰富。大规模并行测序将被用来监测随着动物成熟和改变饮食，微生物组的组成和转录组的变化。这将分别使用qPCR和qRT-PCR进行确认。这种分析使人们能够确定在特定条件下，特定的等位基因或菌株是否更丰富。利用GFP融合，表达基因的细胞的确切位置将被确定。环境参数，如电子受体，糖和短链脂肪酸将被测量。由于细菌同时存在于远洋和微集落阶段，比较生态位特异性基因表达可以通过对FACS分选细胞进行Illumina测序来确定。这项工作将使用一个简单的系统来确定适用于更复杂的人类系统的新原理，并应用随着测序吞吐量和计算能力的增加而变得更适合在高度复杂的系统中使用的技术。

DESCRIPTION (provided by applicant): In humans, the digestive-tract microbiota is a complex community composed out of hundreds of species. This microbiome provides humans with an amazing repertoire of biochemical processes that far exceed those encoded by the animal's genome. As an animal matures or switches diets the microbiome can change in composition. How this change affects the overall physiology of the microbiome and its contribution to the host is poorly understood. The information gained from the Human Microbiome Project has produced a wealth of genome information and is providing insight into possible functions, but functional data from animals with their native microbiome are lacking. The sheer complexity of the microbiome has hindered identifying the genes that the microbiome expresses, the metatranscriptome. This application investigates a naturally occurring simple microbiome that resides in the digestive tract of the medicinal leech. Two bacteria dominate the microbial community and for one genetic tools are available. The composition of the microbiome changes during the first two weeks after the leeches hatch from the cocoon and also after they consume a blood meal. If the blood was partially lysed, a Clostridium species that is minor member of the microbiome became very abundant. Massively parallel sequencing will be used to monitor changes in the composition and in the metatranscriptome of the microbiome as the animals mature and change diet. This will be confirmed using qPCR and qRT-PCR, respectively. This analysis allows one to determine if under specific conditions a particular allele or strain is more abundant. Using gfp-fusions, the exact location of the cells expressing the genes will be determined. Environmental parameter such as electron acceptors, sugars and short chained fatty acids will be measured. Because the bacteria are present both in pelagic and microcolony phases, compare niche specific gene expression can be determined by using Illumina sequencing of FACS sorted cells. This work will determine new principles using a simple system that are applicable to the more complex human system and apply technologies that will become more feasible to use in highly complex systems as the sequencing throughput and computational power increase.