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Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen

机译:绵羊瘤胃中以前未经培养的细菌菌株的分子监测和分离

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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 μm in size. Strain B76 possessed xylanase and α-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 α-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.
机译:为了评估未培养细菌群体对纤维降解的贡献,我们尝试检索瘤胃中未培养细菌群体的生态和功能信息。在先前报道的未培养细菌中,与低GC革兰氏阳性细菌组相关的与纤维相关的组U2和U3被靶向。设计了针对16S rRNA基因或rRNA的PCR引物和荧光原位杂交(FISH)探针,并用于监测靶标的分布。瘤胃中U2组的人口规模高达1.87%,而U3组的人口规模仅为0.03%。从瘤胃中附着于植物纤维的U2组细胞观察到强荧光信号。这些发现表明瘤胃中U2族的生态意义。我们成功地使用瘤胃培养的稻草作为接种物富集了U2组,然后在合适的培养基中与抑制革兰氏阴性细菌的药剂一起进行了培养。因此,我们成功分离出两个属于U2组的菌株,分别命名为B76和R-25。两种菌株均为革兰氏阳性短棒或球菌,大小为0.5至0.8μm。菌株B76具有木聚糖酶和α-1-阿拉伯呋喃糖苷酶活性。特别地,菌株B76的木聚糖酶活性高于在纤维二糖上生长的木聚糖水解纤维状丁酸梭菌H17c的木聚糖酶活性。菌株R-25显示出比菌株B76更高的α-1-阿拉伯呋喃糖苷酶活性。这些结果表明,菌株B76和R-25促成瘤胃中半纤维素的降解。

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