Multi-Omics Study of The Salivary Modulation of The Rumen Microbiome

Ruminants are able to produce large quantities of saliva which enter into the rumen. Although previous research has indicated that salivary immunoglobulins can partially modulate the rumen microbial activity, the role of the salivary components other than ions on the rumen microbial ecosystem has not been thoroughly investigated in ruminants. A total of 16 semi-continuous in vitro cultures were used to incubate rumen fluid from 4 donor goats inoculated with autoclaved saliva (AUT) as negative control, saliva from the same rumen fluid donor (OWN) as positive control, and either GOAT or SHEEP saliva as experimental interventions. Fermentation was monitored throughout the 7 days of incubation and the prokaryotic communities and metabolome were analysed at day 7 of incubation. Characterization of the salivas used prior to incubation showed a high degree of individual variability in terms of the salivary metabolites and proteins, including immunoglobulins. The prokaryotic community composition in AUT incubators was the most divergent across treatments, suggesting a modulatory effect of active salivary components, which were not affected in the other treatments (OWN, GOAT and SHEEP). The differences across treatments in microbial diversity were mostly caused by a greater abundance of Proteobacteria and Rikenellacea and lower of Prevotellaceae, a key rumen bacterium with greater abundance in GOAT and SHEEP treatments. These results suggest that specific salivary components contribute to host-associated role in selecting the rumen commensal microbiota and its activity.

PSX-B-9 Effect of trace mineral sources in the supplement for grazing cattle on ruminal bacteria diversity

The rumen soluble Cu and Zn can affect the rumen microbial populations. This study aimed to evaluate the effect of trace mineral sources (Cu and Zn) in the supplement of grazing steers on ruminal bacteria diversity. Eight rumen cannulated Nellore steers (541 kg ± 18 kg BW) were distributed in a randomized block design in individual paddocks of Urochloa brizantha cv. Marandu. Steers were supplemented during 101 days between dry to rainy season at 5 g/kg BW with commercial supplement (25% CP) containing Cu (40 mg/ kg) and Zn (148 mg/kg) in the inorganic (Control) or hydroxy (HDX; Micronutrients Inc., IN) source. Samples of ruminal content were collected before supplementation at day 97 of experimental period and the total DNA extracted by commercial kit (Quick-DNA Fecal/Soil Microbe Miniprep). The V3/V4 regions of 16SrRNA gene was sequencing using the Illumina MiSeq, using the Quantitative Insights into Microbial Ecology (QIIME v.1.9.1) to filter reads and determine Operational Taxonomic Units (OTUs). Data were compared using an unpaired Wilcoxon test in R. A total of 293 OTUs were identified at genus level. The HDX resulted in a higher ruminal abundance of Corynebacterium 1 (P = 0.01), Prevotella 1 (P = 0.01), Lachnoclostridium 10 (P = 0.02), Lachnospiraceae AC2044 group (P = 0.03), Lachnospiraceae UCG-008 (P = 0.03), Streptococcus (P = 0.02), Ruminococcaceae UCG-010 (P = 0.01), Ruminococcaceae UCG-014 (P = 0.01), Ruminococcus 1 (P = 0.04), Coprococcus 1 (P = 0.04), Mogibacterium (P = 0.02), Selenomonas 1 (P = 0.02), Anaerovibrio (P = 0.03), Methylobacterium (P = 0.02), Treponema 2 (P = 0.02), Rikenellaceae RC9 gut group (P = 0.03), Ruminococcaceae; uncultured rumen bacterium (P = 0.02) and Eubacterium hallii group (P = 0.05), and lower abundance of Fibrobacter (P = 0.04), Butyrivibrio 2 (P = 0.05), Anaerotruncus (P = 0.03), Ruminiclostridium 5 (P = 0.03), Anaerorhabdus furcosa group (P = 0.02) and Erysipelotrichaceae UCG-004 (P = 0.01). The use of HDX in the supplement for grazing cattle between dry to rainy season increase the ruminal abundance of bacteria, mainly into Firmicutes phylum with important structural and non-structural carbohydrates degradation functions.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Complete Genome Sequence of the Polysaccharide-Degrading Rumen Bacterium Pseudobutyrivibrio xylanivorans MA3014 Reveals an Incomplete Glycolytic Pathway

Bacterial species belonging to the genus Pseudobutyrivibrio are important members of the rumen microbiome contributing to the degradation of complex plant polysaccharides. Pseudobutyrivibrio xylanivorans MA3014 was selected for genome sequencing to examine its ability to breakdown and utilize plant polysaccharides. The complete genome sequence of MA3014 is 3.58 Mb, consists of three replicons (a chromosome, chromid, and plasmid), has an overall G + C content of 39.6%, and encodes 3,265 putative protein-coding genes (CDS). Comparative pan-genomic analysis of all cultivated and currently available P. xylanivorans genomes has revealed a strong correlation of orthologous genes within this rumen bacterial species. MA3014 is metabolically versatile and capable of growing on a range of simple mono- or oligosaccharides derived from complex plant polysaccharides such as pectins, mannans, starch, and hemicelluloses, with lactate, butyrate, and formate as the principal fermentation end products. The genes encoding these metabolic pathways have been identified and MA3014 is predicted to encode an extensive range of Carbohydrate-Active enZYmes with 78 glycoside hydrolases, 13 carbohydrate esterases, and 54 glycosyl transferases, suggesting an important role in solubilization of plant matter in the rumen.

Complete Genome Sequence of the Polysaccharide-Degrading Rumen Bacterium Pseudobutyrivibrio xylanivorans MA3014

Ruminants are essential for maintaining the global population and managing greenhouse gas emissions. In the rumen, bacterial species belonging to the genera rumen Butyrivibrio and Pseudobutyrivibrio constitute the core bacterial rumen microbiome and are important degraders of plant-derived complex polysaccharides. Pseudobutyrivibrio xylanivorans MA3014 was selected for genome sequencing in order to examine its ability to breakdown and utilize plant polysaccharides. The complete genome sequence of MA3014 is 3.58 Mb, consists of three replicons (a chromosome, chromid and plasmid), has an overall G+C content of 39.6% and encodes 3,265 putative protein-coding genes (PCGs). Comparative pan-genomics of all cultivated and currently available P. xylanivorans genomes has revealed highly open genomes and a strong correlation of orthologous genes within this species of rumen bacteria. MA3014 is metabolically versatile and capable of utilizing a range of simple mono-or oligosaccharides to complex plant polysaccharides such as pectins, mannans, starch and hemicelluloses for growth, with lactate, butyrate and formate as the principal fermentation end-products. The genes encoding these metabolic pathways have been identified and MA3014 is predicted to encode an extensive repertoire of Carbohydrate-Active enZYmes (CAZymes) with 80 Glycoside Hydrolases (GHs), 28 Carbohydrate Esterases (CEs) and 51 Glycosyl Transferases (GTs), that suggest its role as an initiator of primary solubilization of plant matter in the rumen.

A Genomic Perspective on the Potential of Wild-Type Rumen Bacterium Enterobacter sp. LU1 as an Industrial Platform for Bio-Based Succinate Production

Enterobacter sp. LU1, a wild-type bacterium originating from goat rumen, proved to be a potential succinic acid producer in previous studies. Here, the first complete genome of this strain was obtained and analyzed from a biotechnological perspective. A hybrid sequencing approach combining short (Illumina MiSeq) and long (ONT MinION) reads allowed us to obtain a single continuous chromosome 4,636,526 bp in size, with an average 55.6% GC content that lacked plasmids. A total of 4425 genes, including 4283 protein-coding genes, 25 ribosomal RNA (rRNA)-, 84 transfer RNA (tRNA)-, and 5 non-coding RNA (ncRNA)-encoding genes and 49 pseudogenes, were predicted. It has been shown that genes involved in transport and metabolism of carbohydrates and amino acids and the transcription process constitute the major group of genes, according to the Clusters of Orthologous Groups of proteins (COGs) database. The genetic ability of the LU1 strain to metabolize a wide range of industrially relevant carbon sources has been confirmed. The genome exploration indicated that Enterobacter sp. LU1 possesses all genes that encode the enzymes involved in the glycerol metabolism pathway. It has also been shown that succinate can be produced as an end product of fermentation via the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. The transport system involved in succinate excretion into the growth medium and the genes involved in the response to osmotic and oxidative stress have also been recognized. Furthermore, three intact prophage regions ~70.3 kb, ~20.9 kb, and ~49.8 kb in length, 45 genomic islands (GIs), and two clustered regularly interspaced short palindromic repeats (CRISPR) were recognized in the genome. Sequencing and genome analysis of Enterobacter sp. LU1 confirms many earlier results based on physiological experiments and provides insight into their genetic background. All of these findings illustrate that the LU1 strain has great potential to be an efficient platform for bio-based succinate production.

Characterization and survey in cattle of a rumen Pyrimadobacter sp. which degrades the plant toxin fluoroacetate

ABSTRACT Among the natural halogenic compounds, the plant toxin fluoroacetate (FA) causes livestock fatalities in southern hemisphere countries. Here, we report on the isolation of a rumen bacterium, strain C12–8 that degrades FA under anaerobic conditions. 16S rRNA gene sequence analysis showed this bacterium belonged to the Pyramidobacter genus within the Synergistetes phylum and was 98% similar to Pyramidobacter piscolens W5455 isolated from the human oral cavity. Transmission electron microscopy showed the cell envelope to be unusual, with only one membrane and no obvious external wall. Growth and FA degradation were enhanced by peptide-rich protein hydrolysates but not carbohydrates. End products of metabolism were mainly acetate, propionate/isovalerate and isobutyrate. Strain C12-8 preferentially used peptide-bound amino acids rather than free amino acids. Glycine, serine, threonine, leucine, histidine and isoleucine were utilized as free and peptide-bound amino acids, but there was minimal utilization of alanine, proline, methionine, aspartic acid, lysine and arginine in either form. A survey of several cattle properties in northern Australia showed that strain C12-8 and other FA degrading bacteria affiliated with Cloacibacillus porcorum strain MFA1 were endemic to cattle in the northern beef herd and may help to reduce toxicity.

Energy conservation involving 2 respiratory circuits

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of H+, but it can also be composed of Na+. Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:NAD+ oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is H+-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis. Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden–Meyerhof–Parnas or the pentose–phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for H+ bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.