Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen
Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen
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DOI：
10.1128/aem.02606-09
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发表时间：
2010-03-01
影响因子：
4.4
通讯作者：
Kobayashi, Y.
中科院分区：
文献类型：
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作者：
Koike, S.;Handa, Y.;Kobayashi, Y.
为了评估未培养菌群对纤维降解的贡献，我们试图检索瘤胃中未培养菌群的生态和功能信息。在之前报道的未培养细菌中，属于低GC革兰氏阳性细菌组的纤维相关组U2和U3是目标。设计针对16S rRNA基因的聚合酶链式反应(PCR)引物和荧光原位杂交(FISH)探针，用于监测靶标的分布。U2组瘤胃内种群数高达1.87%，而U3组仅为0.03%。瘤胃内附着于植物纤维上的U2细胞有较强的荧光信号。这些发现表明了U2组在瘤胃中的生态学意义。我们成功地用瘤胃培养的稻草作为接种物，然后在适当的含有革兰氏阴性菌抑制剂的培养基中培养U2组。因此，我们成功地分离到两株菌株，命名为B76和R-25，属于U2群。两株都是革兰氏阳性短杆菌或球菌，大小为0.5~0.8微米。菌株B76具有木聚糖酶和α-L-阿拉伯呋喃糖苷酶活性。其中，菌株B76的木聚糖酶活性高于纤维二糖上生长的木聚糖酶降解丁状弧菌H17c。菌株R-25的α-L-阿拉伯糖苷酶活性高于菌株B76。这些结果表明，菌株B76和R-25有助于瘤胃内半纤维素的降解。
To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 mu m in size. Strain B76 possessed xylanase and alpha-L-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an alpha-L-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.