Genome analysis of bacteria inhabiting the mucosal surface of intestine

肠道粘膜表面细菌的基因组分析

基本信息

批准号：
18310132
负责人：
HAYASHI Tetsuya
金额：
$ 11.19万
依托单位：
University of Miyazaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18310132/
关键词：
Genetics Gut microbiota Genome Metagenome Unculturable bacteria 難培養性

项目摘要

The final goal of our research is to clarify the compositional and genomic features of gut microbiota and their positional differences. However, it is impossible to know the positional differences by metagenomic analysis of fecal samples. We need to analyze the samples that are obtained directly from intestine, but such sampling in human is accompanied by many technical and ethical problems. Therefore, the mouse intestine was used as a model system in our study. In the present research project, we focused on the microbiota residing on the mucosal surface, which are obviously playing most important roles in term of host-resident microbe interaction, particularly on genome sequence determination of segmented filamentous bacterium (SFB). SFB is an unculturable bacterium which appears on the mucosal surface as a dominant bacterial species during the period of weaning.We first isolated about 5,000 SFB cells from the intestine of weanling mice by a micromanipulator, amplified their genomic DNA by rolling circle amplification, and tried to determine the sequence by the random shotgun sequencing method. However, we found that plasmid DNA was preferentially amplified, and thus, it is very hard to determine the whole genome sequence by this strategy. Therefore, we cultivated SFB cells in gnotobiotic mice, and prepare their DNA for genome sequencing. We then constructed a random shotgun library of SFB, and generated about 50,000 shotgun reads. Contamination of DNA derived from other bacterial species severely disturbed our analysis, but by PCR-based gap closing, we have obtained 50 super-contigs. For the final finishing process, we have re-isolated SFB cells and prepared a BAC library. Currently, we are determining the end-sequences of 1,200 BAC clones for the final finishing process. The approach we employed in this study is a new strategy for the genome analysis of unculturable bacteria in gut microbiota.

我们研究的最终目标是阐明肠道微生物群的组成和基因组特征及其位置差异。然而，通过粪便样本的宏基因组分析不可能知道位置差异。我们需要分析直接从肠道获得的样本，但这种人体采样伴随着许多技术和伦理问题。因此，我们的研究中使用小鼠肠道作为模型系统。在本研究项目中，我们重点关注粘膜表面的微生物群，它们在宿主与微生物相互作用方面显然发挥着最重要的作用，特别是在分段丝状细菌（SFB）的基因组序列测定方面。 SFB是一种不可培养的细菌，在断奶期间作为优势菌种出现在粘膜表面。我们首先通过显微操作器从断奶小鼠的肠道中分离出约5000个SFB细胞，通过滚环扩增法扩增其基因组DNA，并尝试通过随机鸟枪测序法确定序列。然而，我们发现质粒DNA被优先扩增，因此，通过这种策略很难确定整个基因组序列。因此，我们在限生小鼠中培养了 SFB 细胞，并准备其 DNA 进行基因组测序。然后，我们构建了 SFB 的随机霰弹枪库，并生成了大约 50,000 个霰弹枪读数。来自其他细菌物种的 DNA 污染严重干扰了我们的分析，但通过基于 PCR 的间隙闭合，我们获得了 50 个超级重叠群。对于最终的整理过程，我们重新分离了SFB细胞并准备了BAC文库。目前，我们正在确定 1,200 个 BAC 克隆的末端序列，以用于最终的整理过程。我们在本研究中采用的方法是对肠道微生物群中不可培养细菌进行基因组分析的新策略。