No abstract

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.The ruminant rumens are long believed to be the anaerobic environments efficiently degrading the plant-derived polysaccharides, which is attributed to the inhabited abundant rumen microorganisms. They implement the fibrolytic degradation by the combination of the enzymes comprising of cellulases, hemicellulases, and to a lesser extent pectinases and ligninases (12). The rumen bacteria are outnumbered of the other rumen microbes; however, only a few of cellulolytic bacteria have been isolated from rumens. Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes are considered to be the most important cellulose-degrading bacteria in the rumen (18), and they produce a set of cellulolytic enzymes, including endoglucanases, exoglucanases (generally cellobiohydrolase), and ␤-glucosidases, as well as hemicellulases. In addition, the predominant ruminal hemicellulose-digesting bacteria such as Butyrivibrio fibrisolvens and Prevotella ruminicola lack the ability to digest cellulose but degrade xylan and pectin and utilize the degraded soluble sugars as substrates (10,14). Although the robust cellulolytic species F. succinogenes degrades xylan, it cannot use the pentose product as a carbon source (24). Culture-independent approaches indicate that the three cellulolytic bacterial species represent only ϳ2% of the ruminal bacterial 16S rRNA (43). Therefore, many varieties of rumen microbes remain uncultured (2). In recent years, rumen metagenomics...

Proteiniclasticum ruminis gen. nov., sp. nov., a strictly anaerobic proteolytic bacterium isolated from yak rumen Two strictly anaerobic, proteolytic bacterial strains, designated strain D3RC-2 T and D3RC-3r, were isolated from a cellulose-degrading mixed culture enriched from yak rumen content. The strains were Gram-stain negative and non-spore-forming with cell sizes of 0.5-0.8¾0.6-2.0 mm. The temperature range for growth was 24-46 6C (optimum 38-39 6C) and the pH range was between 5.6 and 8.7 (optimum 7.0-7.3). Both strains used soya peptone, tryptone, Lphenylalanine, L-leucine, L-methionine, L-serine, L-valine, L-threonine and L-histidine as carbon and nitrogen sources, but did not use any of the saccharides tested. The major fermentation products from PY medium were acetate, propionate and iso-butyrate. The DNA G+C contents of strains D3RC-2 T and D3RC-3r were 41.0±0.1 mol% and 41.3±0.1 mol% (HPLC), respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains represented a new phyletic sublineage within the family Clostridiaceae, with ,93.8 % 16S rRNA gene sequence similarity to recognized species. On the basis of the phenotypic, genotypic and physiological evidence, strains D3RC-2 T and D3RC-3r are proposed as representing a novel species of a new genus, for which the name Proteiniclasticum ruminis gen. nov., sp. nov. is proposed. The type strain of the type species is D3RC-2 T (5AS 1.5057 T 5JCM 14817 T ).Yak (Bos grunniens) are ruminants that feed exclusively on grasses and live mainly on the Qinghai-Tibetan Plateau, China, at a height of 3000 m above sea level. Previous studies based on an uncultured approach in our laboratory have indicated that more than half of the microbes in yak rumen belong to as yet uncultured groups. The strains all showed ,90 % 16S rRNA gene sequence similarity levels with recognized species and 10.8 % of the sequences retrieved were related to known rumen fibrolytic bacteria. By using filter paper as the sole carbon source, a cellulose-degrading mixed culture enriched from yak rumen content was obtained. A few fibrolytic and nonfibrolytic bacterial strains were isolated from the mixed culture. In this paper, two proteolytic bacterial strains isolated from yak rumen are described.Strains D3RC-2 T and D3RC-3r were isolated from a fibrolytic mixed culture enriched from yak rumen. The yak rumen content was inoculated into a modified basal medium (Bryant & Burkey, 1953;Hungate, 1966) with filter paper as the sole carbon source and grown under a gas phase of N 2 /CO 2 (80 : 20). A fibrolytic mixed culture was obtained by subculturing the enrichment in the same medium. By means of serial dilution in peptone-yeast extract-glucose medium (PYG, Holdeman et al., 1977) and the Hungate roll-tube technique (Hungate, 1969), single colonies were picked and transferred to the same medium. The roll-tube procedure was repeated several times before strains D3RC-2 T and D3RC-3r were obtained. The purity of the isolates was examined by light microscopy. All i...