Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

Genes from Bacillus subtilis predicted to encode a phosphate-specific transport (Pst) system were shown by mutation to affect high-affinity P i uptake but not arsenate resistance or phosphate (Pho) regulation. The transcription start of the promoter upstream of the pstS gene was defined by primer extension. The promoter contains structural features analogous to the Escherichia coli pst promoter but not sequence similarity. Expression from this promoter was induced >5,000-fold upon phosphate starvation and regulated by the PhoP-PhoR two-component regulatory system. These data indicate that the pst operon is involved in phosphate transport and is a member of the Pho regulon but is not involved in P i regulation.Escherichia coli has two major P i transport systems (24,29,30,35,44). The Pst (phosphate-specific transport) system is a high-affinity, low-velocity, free-P i transport system which is structurally similar to ABC transporters (3). The Pit (phosphate inorganic transport) system is a divalent metal transporter for which P i (40) or arsenate (48) can serve as the anion (39). Additionally, several organophosphate transport systems result in P i uptake in E. coli (42).Recently, the genes encoding proteins homologous to those of the E. coli pst gene products were cloned and sequenced in Bacillus subtilis (36). Five cistrons with homology to genes in the E. coli pst operon were named pstS, pstC, pstA, pstB1, and pstB2. A putative promoter sequence was located 5Ј of the pstS gene, and a potential rho-independent transcription terminator (⌬G ϭ Ϫ18.4 kcal/mol) was observed 3Ј of pstB2.The pst operon of E. coli and the proposed pst operon of B. subtilis encode proteins similar to ABC transporters (3, 45), which are composed of a binding protein (similar to PstS), two integral inner membrane proteins (similar to PstC and PstA), and an ATP binding protein (similar to PstB for E. coli or PstB1 and PstB2 for B. subtilis). The pstB2 gene is at the 3Ј end of the operon in the position of phoU in E. coli. PhoU, which has no similarity to ABC proteins, is not in the pst operon of B. subtilis.The first four genes in the pst operon of E. coli, i.e., pstS, pstC, pstA, and pstB, are required for phosphate transport; mutations in any of these genes abolish P i uptake (6,33,46) when the concentration of P i is low. The pst operon also has a role in the regulation of Pho regulon genes (1,43,46) in that phoU is required for the repression of the Pho regulon but is apparently not required for phosphate transport through the Pst system (25,34,50).E. coli genes induced by phosphate starvation and controlled by phoB-phoR constitute the Pho regulon (21,22,37,38,41). During P i limitation, PhoR (histidine kinase) turns on the Pho regulon genes by phosphorylating PhoB, a response regulator. The activated phoB in turn activates transcription by binding specifically to a DNA sequence of 18 nucleotides (the Pho box) (20), which is part of the promoter of each Pho regulon gene, including the pst operon.In B. subtilis, Pho regulon genes are exp...

The fiber-associated rumen bacterial community was phylogenetically examined by analysis of 16S rRNA gene (16S rDNA) sequences. Hay stems of orchardgrass and alfalfa were incubated for 6 and 20 h, respectively in the rumen of two different sheep, and total DNA was extracted from the incubated stems to clone bacterial 16S rDNAs using polymerase chain reaction (PCR). Of 91 such clones, 21 showed more than 97% sequence similarity with known isolates, 32 clones had 90^97% similarity with known sequences, and for the remaining 38 clones, the similarity was less than 90%. The majority of clones fell into the Cytophaga^Flavobacter^Bacteroides and low G+C Grampositive bacterial phyla (43 and 44%, respectively). Prevotella-related and Butyrivibrio fibrisolvens-related sequences formed large clusters in the phylogenetic tree. Unknown sequences were found to form three unique clusters, one of which was suggested by semi-quantitative PCR to be more prevalent in the rumen receiving a high alfalfa diet.

Cashew nut shell liquid (CNSL) containing antibacterial phenolic compounds was evaluated for its potency as a feed additive for ruminants. In experiment 1, ruminal responses to CNSL supplementation were assessed using a batch culture system. Rumen fluid from cattle was diluted with artificial saliva and incubated for 18h in a batch culture with a mixed diet containing a 30:70 hay:concentrate diet to which raw or heated CNSL was added at a final concentration of 500 μg/mL. In experiment 2, a Rusitec, using rumen fluid from the same cattle, was operated over a period of 7 d during which only raw CNSL was tested at concentrations of 0, 50, 100, or 200 μg/mL, and variations in fermentation and bacterial population were assessed. In experiment 3, a pure culture study was conducted using selected bacteria to determine their susceptibility to CNSL. In experiment 1, methane production was inhibited by raw CNSL (56.9% inhibition) but not by heated CNSL. Total volatile fatty acid concentration was not affected, whereas increased concentrations of propionate and decreased concentrations of acetate and butyrate were observed using either raw or heated CNSL. These changes were more obvious when raw CNSL was tested. In experiment 2, raw CNSL inhibited methanogenesis and increased propionate production in a dose-dependent manner, showing maximum methane inhibition (70.1%) and propionate enhancement (44.4%) at 200 μg/mL supplementation. Raw CNSL increased total volatile fatty acid concentration and dry matter digestibility. Raw CNSL also appeared to induce a dramatic shift in the population of rumen microbiota, based on decreased protozoa numbers and changes in quantitative PCR assay values for representative bacterial species. In experiment 3, using pure cultures, raw CNSL prevented the growth of hydrogen-, formate-, and butyrate-producing rumen bacteria, but not the growth of bacteria involved in propionate production. Based on these data, raw CNSL, rich in the antibacterial phenolic compound anacardic acid, is a potential candidate feed additive with selective activity against rumen microbes, leading to fermentation that results in decreased methane and enhanced propionate production.

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.

16S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was extracted from the rumen digesta of three sheep fed two diets with different hay-to-concentrate ratios (10 : 1 and 1 : 2). Real-time PCR quantification of Prevotella revealed that the relative abundance of this genus in the total rumen bacteria was up to 19.7%, while the representative species Prevotella bryantii and Prevotella ruminicola accounted for only 0.6% and 3.8%, respectively. Denaturing gradient gel electrophoresis analysis for Prevotella revealed shifts in the community composition with the diet. Analysis of 16S rRNA gene clone libraries showed significant differences (P=0.001) between clones detected from the sheep on the diets with different hay-to-concentrate ratios. The majority (87.8%) of Prevotella clones had <97% sequence similarity with known rumen Prevotella. These data suggest that uncultured Prevotella is more abundant than known Prevotella and that members of this genus appear to have specific metabolic niches.

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