Challenges to explore uncultivable but functional bacteria in the rumen

本研究旨在通过分子生物学技术探索瘤胃细菌的生态和功能。通过对纤维伴生菌(紧密附着在稻草茎上的细菌)的16SrDNA序列比较分析，得到以下结果：1.迄今发现未鉴定的瘤胃细菌，主要分为3个类群(U1~U3)。U1属于PFB门，由与拟杆菌密切相关的细菌组成，而U2与木纤维消化细菌艾氏梭状芽孢杆菌关系密切。U3的半纤维瘤胃细菌与瘤胃真细菌的亲缘关系最近。2.针对U2和U3设计了针对U2和U3的特异的聚合酶链式反应(PCR)引物，建立了实时荧光定量分析方法。该方法灵敏度高，重现性好，可快速监测每组细菌。在紧密附着的细菌中，琥珀酸纤维杆菌是一种有效的纤维分解细菌，其生物量最大，其次是U2。此外，U2与产琥珀酸假单胞菌一样，在茎上表现出时间进程。这些结果有力地表明了U2对瘤胃纤维消化的重要贡献。4.在扫描电子显微镜下观察到茎上有许多具有突起结构的类梭状芽孢杆菌，这些细菌可能是U2细菌。因此，U2可以在秸秆茎上浓缩，并在培养前通过热处理接种来分离。

The present study is to explore ecology and function of uncultivated rumen bacteria through molecular biological techniques. By comparative 16SrDNA sequencing analysis of fiber-associated bacteria (bacteria tightly adherent to rice straw stems), the following results were obtained.1.So far uncharacterized rumen bacteria were found and mainly grouped into 3 clusters (U1 through U3). U1 was in PFB phylum comprising bacteria closely related to Bacteroides, while U2 was the group close to the wood fiber digesting bacterium Clostridium aldrichii. U3 had the hemicellulolytic rumen bacterium Eubacterium ruminantium as a closest relative.2.PCR primers specific to U2 and U3 were designed for developing real-time PCR assays to quantitate these groups. The developed assays were highly sensitive, reliable in reproducibility, and allowed rapid monitoring of each bacterial group. Such reliable assays were established for total 14 bacterial species or groups that were associated with ruminally incubated straw stems.3.Of tightly adherent bacteria, Fibrobacter succinogenes, a potent fibrolytic species, was major in its biomass followed by U2. Also, U2 showed time-course on stems just as F.succinogenes. These results strongly suggest significant contribution of U2 to rumen fiber digestion.4.Since many Clostridium-like bacteria with protuberance structure were observed on stems under scanning electronic microscopy, these might be U2 bacteria. Therefore, U2 could be enriched on straw sterns and isolated by using heat treatment to inoculum before cultivation.