Genome analysis of bacteria inhabiting the mucosal surface of intestine

我们研究的最终目的是澄清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是不可能知道位置差异的。我们需要对直接从肠道中获取的样本进行分析，但这种对人体的采样伴随着许多技术和伦理问题。因此，本研究以小鼠肠道为模型系统。在本研究项目中，我们重点研究了粘膜表面的微生物群，这些微生物群在宿主-驻留微生物相互作用方面显然起着最重要的作用，特别是在片段丝状细菌（SFB）的基因组序列测定中。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。我们首先用微操作器从断奶小鼠肠道中分离出约5000个SFB细胞，用滚动圈扩增法扩增其基因组DNA，并尝试用随机霰弹枪测序法确定序列。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，我们在非生物小鼠中培养SFB细胞，并制备其DNA进行基因组测序。然后，我们构建了一个随机的SFB霰弹枪库，并生成了大约50,000个霰弹枪读取。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的间隙关闭，我们获得了50个超contigs。在最后的整理过程中，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1200个BAC克隆的末端序列，以进行最后的精加工。我们在这项研究中采用的方法是肠道微生物群中不可培养细菌基因组分析的新策略。

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.