Comparative Genomics of Rumen Butyrivibrio spp. Uncovers a Continuum of Polysaccharide-Degrading Capabilities.

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.IMPORTANCE Feeding a global population of 8 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Members of the genera Butyrivibrio and Pseudobutyrivibrio are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings have highlighted the immense enzymatic machinery of Butyrivibrio and Pseudobutyrivibrio species for the degradation of plant fiber, suggesting that these bacteria occupy similar niches but apply different degradation strategies in order to coexist in the competitive rumen environment.

Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet.

High-grain feeding used in the animal production is known to affect the host rumen bacterial community, but our understanding of consequent changes in goats is limited. This study was therefore aimed to evaluate bacterial population dynamics during 20 days adaptation of 4 ruminally cannulated goats to the high-grain diet (grain: hay - ratio of 40:60). The dietary transition of goats from the forage to the high-grain-diet resulted in the significant decrease of rumen fluid pH, which was however still higher than value established for acute or subacute ruminal acidosis was not diagnosed in studied animals. DGGE analysis demonstrated distinct ruminal microbial populations in hay-fed and grain-fed animals, but the substantial animal-to-animal variation were detected. Quantitative PCR showed for grain-fed animals significantly higher number of bacteria belonging to Clostridium leptum group at 10 days after the incorporation of corn into the diet and significantly lower concentration of bacteria belonging to Actinobacteria phylum at the day 20 after dietary change. Taxonomic distribution analysed by NGS at day 20 revealed the similar prevalence of the phyla Firmicutes and Bacteroidetes in all goats, significantly higher presence of the unclassified genus of groups of Bacteroidales and Ruminococcaceae in grain-fed animals and significantly higher presence the genus Prevotella and Butyrivibrio in the forage-fed animals. The three different culture-independent methods used in this study show that high proportion of concentrate in goat diet does not induce any serious disturbance of their rumen ecosystem and indicate the good adaptive response of caprine ruminal bacteria to incorporation of corn into the diet.

The fructanolytic abilities of the rumen bacterium Butyrivibrio fibrisolvens strain 3071.

AIMS: To determine if Butyrivibrio fibrisolvens strain 3071 is able to use fructose polymers for growth and to identify the enzymes involved in their digestion. METHODS AND RESULTS: Strain 3071 utilized 97, 89, 85 and 60% of sucrose, timothy grass fructan, inulin oligosaccharides and inulin, respectively, in the growth medium. A cell extract from timothy grass fructan-grown bacteria was used for identification of fructanolytic enzymes by anion exchange chromatography, gel filtration, zymography and thin-layer chromatography. The bacterium synthesizes a specific endolevanase and a nonspecific ß-fructofuranosidase. Both enzymes occurred in two forms differing in molecular weight. The ß-fructofuranosidase was not able to digest long-chain inulin or timothy grass fructan, but degraded inulin oligosaccharides and sucrose. Addition of 1,4-dithioerythritol to an enzyme solution did not affect the activity of endolevanase(s), but increased the ability of ß-fructofuranosidase to digest sucrose. The digestion of timothy grass fructan by endolevanase(s) was described by Michaelis-Menten kinetics in which Km  = 2·82 g l(-1) and Vmax  = 4·01 µmoles reducing sugar equivalents × mg(-1)  × min(-1) . CONCLUSION: Strain 3071 synthesizes enzymes enabling it to use grass fructans for growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Butyrivibrio fibrisolvens strain 3071 can be considered a member of the rumen fructanolytic guild.

Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows.

Four lactating cows fitted with ruminal cannulae and fed a grass silage-based diet were used in a 4 × 4 Latin square with 28-d periods to investigate the effects of incremental dietary fish oil (FO) supplementation (0, 75, 150, or 300 g/d) on the flow of fatty acids at the omasum and populations of rumen bacteria capable of biohydrogenation. FO decreased silage intake and ruminal volatile fatty acid concentrations and promoted an increase in molar butyrate and propionate proportions at the expense of acetate. Extensive ruminal biohydrogenation of 20:5(n-3) and 22:6(n-3) resulted in corresponding increases in numerous 20- and 22-carbon unsaturated fatty acids at the omasum. Omasal flow of several 20-, 21-, and 22-carbon all-cis (n-3) PUFA exceeded the intake from FO. Supplements of FO also induced a dose-dependent decrease in 18:0 and increased trans 18:1 and trans 18:2 flow at the omasum. Trans-11 was the major 18:1 intermediate in digesta, while FO induced quadratic increases in trans-10 18:1 flow, reaching a maximum of 300 g/d. FO had no substantial influence on omasal flow of CLA. Results suggest that one or more fatty acids in FO inhibit the reduction of trans-18:1 and trans-18:2 intermediates by ruminal microorganisms. qPCR based on 16S rRNA genes in omasal digesta indicated that key Butyrivibrio spp. declined linearly in response to FO. Dose-dependent increases in ruminal outflow of biohydrogenation intermediates containing one or more trans double bonds in response to FO has major implications for host metabolism and the nutritional quality of ruminant foods.

Pyrosequencing reveals unique microbial signatures associated with healthy and failing dental implants.

AIM: Although it is established that peri-implantitis is a bacterially induced disease, little is known about the bacterial profile of peri-implant communities in health and disease. The purpose of the present investigation was to examine the microbial signatures of the peri-implant microbiome in health and disease. MATERIALS AND METHODS: Subgingival and submucosal plaque samples were collected from forty subjects with periodontitis, peri-implantitis, periodontal and peri-implant health and analysed using 16S pyrosequencing. RESULTS: Peri-implant biofilms demonstrated significantly lower diversity than subgingival biofilms in both health and disease, however, several species, including previously unsuspected and unknown organisms, were unique to this niche. The predominant species in peri-implant communities belonged to the genera Butyrivibrio, Campylobacter, Eubacterium, Prevotella, Selenomonas, Streptococcus, Actinomyces, Leptotrichia, Propionibacterium, Peptococcus, Lactococcus and Treponema. Peri-implant disease was associated with lower levels of Prevotella and Leptotrichia and higher levels of Actinomyces, Peptococcus, Campylobacter, non-mutans Streptococcus, Butyrivibrio and Streptococcus mutans than healthy implants. These communities also demonstrated lower levels of Prevotella, non-mutans Streptococcus, Lactobacillus, Selenomonas, Leptotrichia, Actinomyces and higher levels of Peptococcus, Mycoplasma, Eubacterium, Campylobacter, Butyrivibrio, S. mutans and Treponema when compared to periodontitis-associated biofilms. CONCLUSION: The peri-implant microbiome differs significantly from the periodontal community in both health and disease. Peri-implantitis is a microbially heterogeneous infection with predominantly gram-negative species, and is less complex than periodontitis.

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen.

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation.

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

Reclassification of Clostridium proteoclasticum as Butyrivibrio proteoclasticus comb. nov., a butyrate-producing ruminal bacterium.

It is proposed that Clostridium proteoclasticum be reclassified as Butyrivibrio proteoclasticus comb. nov. on the basis of phylogenetic position, DNA G+C content and physiological traits. Phylogenetic analyses based on 16S rRNA gene sequences from an extensive range of taxa within clostridial rRNA subcluster XIVa grouped C. proteoclasticum together with isolates of the genus Butyrivibrio, though this species was genetically distinct from the extant Butyrivibrio species examined. The DNA G+C content of C. proteoclasticum was originally erroneously reported as 28 mol%. However the genome sequence of the type strain of C. proteoclasticum, strain B316(T), and HPLC analysis estimate the DNA G+C content as 40 mol%, which is within the range reported for strains of Butyrivibrio. C. proteoclasticum was distinguishable from other species of the genus Butyrivibrio as the 16S rRNA gene from strain B316(T) shared less than 97 % sequence similarity with sequences from the type strains of Butyrivibrio species. C. proteoclasticum was also able to convert linoleic acid to stearic acid, in contrast to other species of Butyrivibrio. Physiological characteristics, including carbon source utilization, volatile fatty acid production and proteinase activities, were assessed for a panel of representative strains of the genera Butyrivibrio and Pseudobutyrivibrio and C. proteoclasticum. These data, together with the phylogenetic analyses, support the reclassification of Clostridium proteoclasticum as a separate species within the genus Butyrivibrio, Butyrivibrio proteoclasticus comb. nov. (type strain B316(T)=ATCC 51982(T)=DSM 14932(T)).

Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen.

The Butyrivibrio group comprises Butyrivibrio fibrisolvens and related Gram-positive bacteria isolated mainly from the rumen of cattle and sheep. The aim of this study was to investigate phenotypic characteristics that discriminate between different phylotypes. The phylogenetic position, derived from 16S rDNA sequence data, of 45 isolates from different species and different countries was compared with their fermentation products, mechanism of butyrate formation, lipid metabolism and sensitivity to growth inhibition by linoleic acid (LA). Three clear sub-groups were evident, both phylogenetically and metabolically. Group VA1 typified most Butyrivibrio and Pseudobutyrivibrio isolates, while Groups VA2 and SA comprised Butyrivibrio hungatei and Clostridium proteoclasticum, respectively. All produced butyrate but strains of group VA1 had a butyrate kinase activity <40 U (mg protein)(-1), while strains in groups VA2 and SA all exhibited activities >600 U (mg protein)(-1). The butyrate kinase gene was present in all VA2 and SA bacteria tested but not in strains of group VA1, all of which were positive for the butyryl-CoA CoA-transferase gene. None of the bacteria tested possessed both genes. Lipase activity, measured by tributyrin hydrolysis, was high in group VA2 and SA strains and low in Group VA1 strains. Only the SA group formed stearic acid from LA. Linoleate isomerase activity, on the other hand, did not correspond with phylogenetic position. Group VA1 bacteria all grew in the presence of 200 microg LA ml(-1), while members of Groups VA2 and SA were inhibited by lower concentrations, some as low as 5 microg ml(-1). This information provides strong links between phenotypic and phylogenetic properties of this group of clostridial cluster XIVa Gram-positive bacteria.

Characterization and transcription of the genes involved in butyrate production in Butyrivibrio fibrisolvens type I and II strains.

The genes in Butyrivibrio fibrisolvens that encode the enzymes involved in butyrate production were sequenced. In a type I strain (ATCC 19171(T)), the genes coding for the enzymes that catalyze the conversion from acetyl-CoA to butyryl-CoA, thl (thiolase), crt (crotonase), hbd (beta-hydroxybutyryl-CoA dehydrogenase), bcd (butyryl-CoA dehydrogenase), etfB (electron transfer flavoprotein [ETF]-beta), and etfA (ETF-alpha), were found to be clustered and arranged in this order. A type II strain (ATCC 51255) had the same clustered genes with the same arrangement, except that crt was not present in the clustered genes. The deduced amino acid sequences of these enzymes did not greatly differ between the two strains, and even between B. fibrisolvens and clostridia. Amino acid identity appeared to be higher within the same type than between types I and II. The clustered genes were shown to be cotranscribed, and constitutively transcribed without being affected significantly by culture conditions.

A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid.

A new strain of Butyrivibrio fibrisolvens (TH1) that has high potential to produce conjugated linoleic acid (CLA) was isolated. Strain TH1 had higher LA isomerase (LA-I) activity, and was much more tolerant to linoleic acid (LA) than other strains examined. However, high CLA reductase (CLA-R) activity resulted in the temporary accumulation of CLA and subsequent conversion to trans-vaccenic acid (t-VA). When LA was added to growing TH1 cultures in a solution with dimethylsulfoxide (LA/DMSO), CLA produced was greater than when LA was added in a mixture with bovine serum albumin (BSA). The number of viable cells decreased upon addition of LA/DMSO, but then increased as the CLA decreased upon its conversion to t-VA. This result suggests that B. fibrisolvens can resume growing by the removal of CLA from the cells. Most CLA was released from B. fibrisolvens cells by gentle washing with BSA, suggesting that CLA bound to the cells might be removed in the rumen and large intestine. Thus, CLA production by B. fibrisolvens in the digestive tract could be increased by a reduction in CLA-R activity without accompanying an overall decrease in the cell number of B. fibrisolvens. Fatty acids (FAs) with 18 carbon backbone inducted LA-I activity, whereas unsaturated FAs induced CLA-R activity, suggesting that FAs stimulate the synthesis of LA-I and CLA-R. Providing a diet with a low ratio of unsaturated to saturated FAs may favor CLA production.

The use of PCR for the identification and characterisation of bacteriocin genes from bacterial strains isolated from rumen or caecal contents of cattle and sheep.

PCR primers were designed to amplify the gene that encodes bovicin 255 from Streptococcus gallolyticus LRC0255 and the bacteriocin genes from Butyrivibrio fibrisolvens strains AR10 and OR79A (bviD and bvi79A) in order to screen for their incidence in rumen and caecal B. fibrisolvens and Streptococcus bovis-like isolates from New Zealand and North American ruminants. None of the B. fibrisolvens-like strains (n=34) isolated from New Zealand or North America had the genes encoding for butyrivibriocins AR10 (bviD) or OR79 (bvi79A). However, seven S. bovis isolates from New Zealand ruminants and three from North American animals had the bovicin 255 gene. Sequence comparison of cloned bovicin 255 PCR products indicated a 92.9-95.7% similarity to that of the corresponding bovicin 255 gene sequence of S. gallolyticus. Four of the New Zealand bovicin 255 positive S. bovis isolates were from the caecal contents of the same sheep and had identical PFGE profiles. Two other S. bovis isolates sharing the same PFGE profile were isolated from a separate animal from the same flock. PFGE analysis of the North American strains indicated that all three were closely related as two of three had identical PFGE profiles with the remaining isolate differing only by a single band position. The 16S rRNA gene sequences of the 10 isolates were at least 99.8% identical to S. bovis. All 10 S. bovis isolates having the gene for bovicin 255 produced bacteriocin activity that inhibited the growth of Peptostreptococcus anaerobius D1 in a deferred antagonism plating (DAP) assay. Certain S. bovis isolates obtained from ruminants have bacteriocin activity associated with a distinct bovicin 255 gene sequence but it appears that bacteriocin production by the rumen anaerobe B. fibrisolvens may be uncommon in strains isolated from cattle and sheep in New Zealand.