Biochemical and molecular analyses of plant cell wall degrading enzymes from the rumen bacterium Ruminococcus albus 8

Ruminants rely on microorganisms inhabiting the rumen to digest plant matter for subsequent fermentation to provide the host with the majority of its energy demands. Ruminococcus albus 8 is a rumen bacterium that efficiently degrades and ferments plant cell wall polysaccharides, such as cellulose and hemicelluloses. Xylan, a hemicellulosic polymer of β-1,4 linked xylose monomers may be appended with arabinose, glucuronic acid, and acetyl residues. Several classes of enzymes attack the polysaccharide to release the constitutive monosaccharides. Lichenin is a linear polymer of glucose linked together by β-1,3 and β-1,4 glycosidic bonds. Lichenin can be found in lichens and common feeds such as barley, sorghum, and wheat. A bioinformatic analysis of the genome of R. albus 8 revealed five genes encoding glycoside hydrolases predicted to hydrolyze the xylose backbone and several genes encoding putative accessory enzymes that are expected to remove side chains from xylans. The genes were cloned, heterologously expressed in E. coli, and biochemically characterized to determine how the enzymes function synergistically to release soluble sugars that can be fermented by R. albus 8. In an experiment designed to identify cellulase encoding genes in the genome of R. albus 8, four genes encoding lichenin degrading enzymes were identified. An enzyme mixture was created, and the components were shown to act synergistically to release, from the polysaccharide, cellobiose and cellotriose which are preferentially utilized by R. albus

Analysis of the ammonium assimilation pathways of the human colonic bacterium, bacteroides thetaiotaomicron

In ruminants, efficient rumen function and proper host metabolism is dependent on the nitrogen supply in the feed. Assimilated ammonium accounts for up to 70% of the microbial protein production, which satisfies up to 85% of the host protein requirements. Similar numbers for the human colon have not been determined. However, colonic bacteria are responsible for the production of ammonium, derived from host-secreted urea and endogenous and dietary proteins, that provides the preferred nitrogen source for microbial growth. Bacteroides thetaiotaomicron, a model organism for human gut Bacteroidetes, encodes genes for the capture of ammonium through the two primary pathways, the glutamate dehydrogenase (GDH) pathway and the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. To gain insight into the genomic features underlying ammonium uptake and assimilation in this bacterium, comparative transcriptomic analysis using RNA-Seq was employed on cultures growing under excess or limiting ammonium concentrations. A single genomic locus, encoding for the GS/GOGAT pathway, was identified with highly increased transcription when the organism grows under limiting ammonium concentration. The relative contribution of each gene to ammonium assimilation was assessed through construction of genomic deletion strains for each of the three GS, one GOGAT, and two GDH genes. The deletion of two genes, the NADPH-dependent glutamate dehydrogenase (gdhA) and the glutamine synthetase type 3 (glnN2) significantly impeded growth of the organisms under both nitrogen conditions. Taken together, the results demonstrate the importance of the GDH pathway for constitutive ammonium assimilation, and GlnN2 for glutamine biosynthesis. However, when the organism grows under nitrogen limitation, the GS/GOGAT pathway, including glnN1, is highly induced. To extrapolate the significance of the findings, a comparative bioinformatic analysis, using all of the available sequenced Bacteroides genomes, revealed high conservation of the critical genomic loci in gut species. Understanding of nitrogen metabolism in gut microbes is essential for a complete depiction of their ecological implications on the host´s metabolism in health and disease

Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows

Abstract Background Residual feed intake (RFI) in dairy cattle typically calculated at peak lactation is a measure of feed efficiency independent of milk production level. The objective of this study was to evaluate differences in ruminal bacteria, biopolymer hydrolyzing enzyme activities, and overall performance between the most- and the least-efficient dairy cows during the peripartal period. Twenty multiparous Holstein dairy cows with daily ad libitum access to a total mixed ration from d − 10 to d 60 relative to the calving date were used. Cows were classified into most-efficient (i.e. with low RFI, n = 10) and least-efficient (i.e. with high RFI, n = 10) based on a linear regression model involving dry matter intake (DMI), fat-corrected milk (FCM), changes in body weight (BW), and metabolic BW. Results The most-efficient cows had ~ 2.6 kg/d lower DMI at wk 4, 6, 7, and 8 compared with the least-efficient cows. In addition, the most-efficient cows had greater relative abundance of total ruminal bacterial community during the peripartal period. Compared with the least-efficient cows, the most-efficient cows had 4-fold greater relative abundance of Succinivibrio dextrinosolvens at d − 10 and d 10 around parturition and tended to have greater abundance of Fibrobacter succinogenes and Megaspheara elsdenii. In contrast, the relative abundance of Butyrivibrio proteoclasticus and Streptococcus bovis was lower and Succinimonas amylolytica and Prevotella bryantii tended to be lower in the most-efficient cows around calving. During the peripartal period, the most-efficient cows had lower enzymatic activities of cellulase, amylase, and protease compared with the least-efficient cows. Conclusions The results suggest that shifts in ruminal bacteria and digestive enzyme activities during the peripartal period could, at least in part, be part of the mechanism associated with better feed efficiency in dairy cows

Additional file 1: of Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows

Preparation of rumen contents for enzyme activities. (DOCX 16 kb