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

Abstract

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 m in size. Strain B76 possessed xylanase and ␣-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 ␣-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.

Bacterial community in the rumen of Tibetan sheep and Gansu alpine fine-wool sheep grazing on the Qinghai-Tibetan Plateau, China

“…The roles of uncultured bacteria in plant fiber digestion have yet to be determined, but recent advances in molecular techniques have improved our understanding of this topic. Research has shown that approximately 77% of fiber-associated community members were uncultured bacteria; two previously uncultured strains were isolated from the sheep rumens and confirmed to degrade hemicellulose in the rumen (Koike et al, 2004(Koike et al, , 2010. The high proportion of uncultured bacteria in TS and GS indicated that these bacteria may be involved in ruminal fiber degradation; however, further research is necessary to confirm this.…”

Genome sequencing of rumen bacteria and archaea and its application to methane mitigation strategies

“…The development of cultivation-independent techniques led to a decline in isolation and cultivation studies, and results from 16S ribosomal RNA gene sequencing and metagenomic studies consistently emphasize that the majority of sequences are derived from organisms that are phylogenetically distinct from currently cultivated species Fouts et al, 2012). In recent years, there have been few attempts to systematically bring additional organisms into cultivation; however, recent studies in Japan (Koike et al, 2010;Nyonyo et al, 2013) and New Zealand (Kenters et al, 2011; S.J. Noel et al, unpublished) have successfully increased the number of taxa of rumen bacteria that have cultured representatives.…”

Poster presentations (Monday)

“…While in recent years there have been periodic reports of novel bacteria isolated from the rumen of domesticated and wild ruminants, there have been few attempts to systematically bring additional organisms into cultivation. Recent studies in Japan (Koike et al, 2010, Nyonyo et al, 2013 and NZ (Kenters et al, 2011, Noel et al, unpublished) have successfully increased the number of taxa of rumen bacteria that have cultured representatives. The rumen methanogenic archaea are poorly represented in culture collections, and work is in progress to isolate and genome sequence additional rumen methanogen cultures.…”