The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f-1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.

gThe bovine rumen represents a highly specialized bioreactor where plant cell wall polysaccharides (PCWPs) are efficiently deconstructed via numerous enzymes produced by resident microorganisms. Although a large number of fibrolytic genes from rumen microorganisms have been identified, it remains unclear how they are expressed in a coordinated manner to efficiently degrade PCWPs. In this study, we performed a metatranscriptomic analysis of the rumen microbiomes of adult Holstein cows fed a fiber diet and obtained a total of 1,107,083 high-quality non-rRNA reads with an average length of 483 nucleotides. Transcripts encoding glycoside hydrolases (GHs) and carbohydrate binding modules (CBMs) accounted for ϳ1% and ϳ0.1% of the total non-rRNAs, respectively. The majority (ϳ98%) of the putative cellulases belonged to four GH families (i.e., GH5, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus and Fibrobacter. Notably, transcripts for GH48 cellobiohydrolases were relatively abundant compared to the abundance of transcripts for other cellulases. Two-thirds of the putative hemicellulases were of the GH10, GH11, and GH26 types and were produced by members of the genera Ruminococcus, Prevotella, and Fibrobacter. Most (ϳ82%) predicted oligosaccharide-degrading enzymes were GH1, GH2, GH3, and GH43 proteins and were from a diverse group of microorganisms. Transcripts for CBM10 and dockerin, key components of the cellulosome, were also relatively abundant. Our results provide metatranscriptomic evidence in support of the notion that members of the genera Ruminococcus, Fibrobacter, and Prevotella are predominant PCWP degraders and point to the significant contribution of GH48 cellobiohydrolases and cellulosome-like structures to efficient PCWP degradation in the cow rumen. In nature, the cow rumen represents a highly specialized bioreactor wherein plant cell wall polysaccharides (PCWPs) are efficiently deconstructed. The extraordinary efficiency results from the concerted action of various enzymes produced by rumen-resident bacteria, archaea, fungi, and protozoa. Three rumen bacteria, i.e., Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes, which can be isolated and cultivated in the laboratory, have been thought to serve a predominant role in the degradation of cellulosic PCWPs in this niche (1, 2). However, metagenomic quantitations based on 16S rRNA gene analysis indicate that these three species of bacteria account for only less than 5% of the total rumen microorganisms (3). In addition, Koike et al. estimated that ϳ77% of the rumen microorganisms attached to solid fibers are uncultured, as their 16S rRNA gene sequences share less than 97% similarity with those of known isolates (4).To bypass the cultivation step, metagenomic approaches involving the direct analysis of total DNA sequences have been extensively used to investigate the PCWP-degrading gastrointestinal microbes in a variety of herbivores, such as termite hindguts (5); cow (6-8), yak (9), and Svalbard reindeer (10) ...

The rumen hosts one of the most efficient microbial systems for degrading plant cell walls, yet the predominant cellulolytic proteins and fibrolytic mechanism(s) remain elusive. Here we investigated the cellulolytic microbiome of the yak rumen by using a combination of metagenome-based and bacterial artificial chromosome (BAC)-based functional screening approaches. Totally 223 fibrolytic BAC clones were pyrosequenced and 10,070 ORFs were identified. Among them 150 were annotated as the glycoside hydrolase (GH) genes for fibrolytic proteins, and the majority (69%) of them were clustered or linked with genes encoding related functions. Among the 35 fibrolytic contigs of >10 Kb in length, 25 were derived from Bacteroidetes and four from Firmicutes. Coverage analysis indicated that the fibrolytic genes on most Bacteroidetes-contigs were abundantly represented in the metagenomic sequences, and they were frequently linked with genes encoding SusC/SusD-type outer-membrane proteins. GH5, GH9, and GH10 cellulase/hemicellulase genes were predominant, but no GH48 exocellulase gene was found. Most (85%) of the cellulase and hemicellulase proteins possessed a signal peptide; only a few carried carbohydrate-binding modules, and no cellulosomal domains were detected. These findings suggest that the SucC/SucD-involving mechanism, instead of one based on cellulosomes or the free-enzyme system, serves a major role in lignocellulose degradation in yak rumen. Genes encoding an endoglucanase of a novel GH5 subfamily occurred frequently in the metagenome, and the recombinant proteins encoded by the genes displayed moderate Avicelase in addition to endoglucanase activities, suggesting their important contribution to lignocellulose degradation in the exocellulase-scarce rumen.

The phylum Actinobacteria has been reported to be common or even abundant in deep marine sediments, however, knowledge about the diversity, distribution, and function of actinobacteria is limited. In this study, actinobacterial diversity in the deep sea along the Southwest Indian Ridge (SWIR) was investigated using both 16S rRNA gene pyrosequencing and culture-based methods. The samples were collected at depths of 1662–4000 m below water surface. Actinobacterial sequences represented 1.2–9.1% of all microbial 16S rRNA gene amplicon sequences in each sample. A total of 5 actinobacterial classes, 17 orders, 28 families, and 52 genera were detected by pyrosequencing, dominated by the classes Acidimicrobiia and Actinobacteria. Differences in actinobacterial community compositions were found among the samples. The community structure showed significant correlations to geochemical factors, notably pH, calcium, total organic carbon, total phosphorus, and total nitrogen, rather than to spatial distance at the scale of the investigation. In addition, 176 strains of the Actinobacteria class, belonging to 9 known orders, 18 families, and 29 genera, were isolated. Among these cultivated taxa, 8 orders, 13 families, and 15 genera were also recovered by pyrosequencing. At a 97% 16S rRNA gene sequence similarity, the pyrosequencing data encompassed 77.3% of the isolates but the isolates represented only 10.3% of the actinobacterial reads. Phylogenetic analysis of all the representative actinobacterial sequences and isolates indicated that at least four new orders within the phylum Actinobacteria were detected by pyrosequencing. More than half of the isolates spanning 23 genera and all samples demonstrated activity in the degradation of refractory organics, including polycyclic aromatic hydrocarbons and polysaccharides, suggesting their potential ecological functions and biotechnological applications for carbon recycling.

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The objective of this study was to determine whether feeding tannin-containing hays to heifers and mature beef cows influences enteric methane (CH4) emissions and nitrogen (N) excretion relative to feeding traditional legume and grass hays. Fifteen mature beef cows (Exp. 1) and 9 yearling heifers (Exp. 2) were each randomly assigned to treatment groups in an incomplete bock design with 2 periods and 6 types of hays with 3 hays fed each period (n = 5 cows and 3 heifers per treatment). Groups were fed tannin-containing [birdsfoot trefoil (BFT), sainfoin (SAN), small burnet (SML)] or non-tannin-containing [alfalfa (ALF), cicer milkvetch (CMV), meadow bromegrass (MB)] hays. Each period consisted of 14 d of adjustment followed by 5 d of sample collection. Nine cows and 9 heifers were selected for the measurement of enteric CH4 emissions (sulfur hexafluoride tracer gas technique), and excretion of feces and urine, while dry matter intake (DMI) was measured for all animals. The concentration of condensed tannins in SAN and BFT was 2.5 ± 0.50% and 0.6 ± 0.09% of dry matter (DM), respectively, while SML contained hydrolyzable tannins (4.5 ± 0.55% of DM). Cows and heifers fed tannin-containing hays excreted less urinary urea N (g/d; P < 0.001) and showed lower concentrations of blood urea N (mg/dL; P < 0.001) than animals fed ALF or CMV, indicating that tannins led to a shift in route of N excretion from urine to feces. Additionally, cows fed either BFT or CMV showed the greatest percentage of retained N (P < 0.001). Enteric CH4 yield (g/kg of DMI) from heifers (P = 0.089) was greatest for MB, while daily CH4 production (g/d) from heifers (P = 0.054) was least for SML. However, digestibility of crude protein was reduced for cows (P < 0.001) and heifers (P < 0.001) consuming SML. The results suggest that tannin-containing hays have the potential to reduce urinary urea N excretion, increase N retention, and reduce enteric CH4 emissions from beef cattle. The non-bloating tannin-free legume CMV may also reduce environmental impacts relative to ALF and MB hays by reducing N excretion in urine and increasing N retention.

An aerobic, yellow-pigmented, Gram-negative bacterium, designated strain S13 T , was isolated from freshwater sediment of Taihu Lake in central China. The taxonomy of strain S13 T was studied by using phenotypic and phylogenetic methods. Cells of strain S13 T were rod-shaped, non-motile and with a size range of 0?35-0?5561?5-2?5 mm. The nearly complete 16S rRNA gene of strain S13 T was amplified and sequenced. A BLAST search and phylogenetic analysis based on 16S rRNA gene sequence similarity showed that strain S13 T was related to members of the genus Flavobacterium, with the highest sequence similarity of 93?8 % to Flavobacterium columnare (ATCC 23463 T ). Cells contained menaquinone-6 (MK-6) as the major respiratory quinone and the genomic DNA G+C content was 41 mol%. The major fatty acids were iso-C 15 : 0 (28?2 %) and iso-C 17 : 1 v9c (19?0 %). It is proposed that S13 T (=CGMCC 1.3801 T =JCM 13331 T ) represents the type strain of a novel species, Flavobacterium saliperosum sp. nov.

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