Advances in high throughput, next generation sequencing technologies have allowed an in-depth examination of biological environments and phenomena, and are particularly useful for culture-independent microbial community studies. Recently the use of RNA for metatranscriptomic studies has been used to elucidate the role of active microbes in the environment. Extraction of RNA of appropriate quality is critical in these experiments and TRIzol reagent is often used for maintaining stability of RNA molecules during extraction. However, for studies using rumen content there is no consensus on (1) the amount of rumen digesta to use or (2) the amount of TRIzol reagent to be used in RNA extraction procedures. This study evaluated the effect of using various quantities of ground rumen digesta and of TRIzol reagent on the yield and quality of extracted RNA. It also investigated the possibility of using lower masses of solid-phase rumen digesta and lower amounts of TRIzol reagent than is used currently, for extraction of RNA for metatranscriptomic studies. We found that high quality RNA could be isolated from 2 g of ground rumen digesta sample, whilst using 0.6 g of ground matter for RNA extraction and using 3 mL (a 5:1 TRIzol : extraction mass ratio) of TRIzol reagent. This represents a significant savings in the cost of RNA isolation. These lower masses and volumes were then applied in the RNA-Seq analysis of solid-phase rumen samples obtained from 6 Angus X Hereford beef heifers which had been fed a high forage diet (comprised of barley straw in a forage-to-concentrate ratio of 70:30) for 102 days. A bioinformatics analysis pipeline was developed in-house that generated relative abundance values of archaea, protozoa, fungi and bacteria in the rumen and also allowed the extraction of individual rRNA variable regions that could be analyzed in downstream molecular ecology programs. The average relative abundances of rRNA transcripts of archaea, bacteria, protozoa and fungi in our samples were 1.4 ± 0.06, 44.16 ± 1.55, 35.38 ± 1.64, and 16.37 ± 0.65% respectively. This represents the first study to define the relative active contributions of these populations to the rumen ecosystem and is especially important in defining the role of the anaerobic fungi and protozoa.

We isolated a novel gram-positive bacterium, Brevibacillus texasporus, that produces an antibiotic, BT. BT is a group of related peptides that are produced by B. texasporus cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and D-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me 2 -4-methyl-4-In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.Novel antibiotics are needed to combat infections caused by bacteria that are resistant to conventional antibiotics. It is well known that microbes produce a huge variety of antibiotics to wage chemical warfare against competing microbes. We screened soil microorganisms for strains that produce novel antibiotics. Bacillus sp. strain E58 (ϭ ATCC PTA-5854) was isolated for its ability to produce the antibiotic BT against Staphylococcus aureus strains that cause life-threatening infections (Jiang and Munoz-Romero, unpublished results). This strain was named Brevibacillus texasporus based on its relatedness to Brevibacillus laterosporus.Many peptide antibiotics of microbial origin (mostly from actinomycetes, bacilli, and fungi) are synthesized by nonribosomal peptide synthetases (NRPS), and they contain unusual amino acids. NRPS usually have a colinear modular architecture (15). The N-terminal to C-terminal order and the specificities of the individual modules correspond to the sequential order and identities of the amino acid residues in the peptide product. Each NRPS module recognizes a specific amino acid and catalyzes stepwise condensation to form a growing peptide chain. The identity of the amino acid recognized by a particular module can be predicted by comparisons to other modules having known specificities (3). Such strict correlation made it possible to identify genes encoding the NRPS enzymes for a number of microbial nonribosomal peptides with known structures, as demonstrated by identification of the mycobactin biosynthesis operon in the genome of Mycobacterium tuberculosis (18). Conversely, an NRPS operon may be a source of information that allows researchers to determine certain structural details of the peptide product. ...

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Muskox (Ovibos moschatus), as the biggest herbivore in the High Arctic, has been enduring the austere arctic nutritional conditions and has evolved to ingest and digest scarce and high lignified forages to support the growth and reproduce, implying probably harbor a distinct microbial reservoir for the deconstruction of plant biomass. Therefore, metagenomics approach was applied to characterize the rumen microbial community and understand the alteration in rumen microbiome of muskoxen fed either triticale straw or brome hay. The difference in the structure of microbial communities including bacteria, archaea, fungi, and protozoa between the two forages was observed at the taxonomic level of genus. Further, although the highly abundant phylotypes in muskoxen rumen fed either triticale straw or brome hay were almost the same, the selective enrichment different phylotypes for fiber degrading, soluble substrates fermenting, electron and hydrogen scavenging through methanogenesis, acetogenesis, propionogenesis, and sulfur-reducing was also noticed. Specifically, triticale straw with higher content of fiber, cellulose selectively enriched more lignocellulolytic taxa and electron transferring taxa, while brome hay with higher nitrogen content selectively enriched more families and genera for degradable substrates-digesting. Intriguingly, the carbohydrate-active enzyme profile suggested an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on triticale straw. The majority of the cellulases belonged to fiver GH families (i.e., GH5, GH6, GH9, GH45, and GH48) and were primarily synthesized by Ruminococcus, Piromyces, Neocallimastix, and Fibrobacter. Abundance of major genes coding for hemicellulose digestion was higher than cellulose mainly including GH8, GH10, GH16, GH26, and GH30, and these enzymes were produced by members of the genera Fibrobacter, Ruminococcus, and Clostridium. Oligosaccharides were mainly of the GH1, GH2, GH3, and GH31 types and were associated with the genera Prevotella and Piromyces. Our results strengthen metatranscriptomic evidence in support of the understanding of the microbial community and plant polysaccharide response to changes in the feed type and host animal. The study also establishes these specific microbial consortia procured from triticale straw group can be used further for efficient plant biomass hydrolysis.