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Applied and Environmental Microbiology Aug 1995Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region...
A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.
Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.
Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Bacterial; Endo-1,4-beta Xylanases; Genes, Bacterial; Genetic Linkage; Molecular Sequence Data; Multigene Family; Prevotella; Rumen; Sequence Homology, Amino Acid; Xylans; Xylosidases; beta-Glucosidase
PubMed: 7487028
DOI: 10.1128/aem.61.8.2958-2964.1995 -
Journal of Dairy Science Dec 2018Cellulose acetate (CA), a derivative of cellulose in which some hydroxyl groups are substituted with acetyl groups, was evaluated as a new cellulosic feed source for...
Cellulose acetate (CA), a derivative of cellulose in which some hydroxyl groups are substituted with acetyl groups, was evaluated as a new cellulosic feed source for ruminants. In the present work, a series of in vitro studies was carried out to determine how CA supplementation affects rumen fermentation and microbiota. Batch culture studies were conducted to select the type of CA suitable for feed use and to define the optimal supplementation level. Rumen fluid from 2 Holstein cows was mixed with McDougall's buffer in test tubes into which grass hay and concentrate containing a fiber source [cellulose (control), water-soluble CA (WSCA), or insoluble CA] had been placed. Each fiber source was supplemented at 10% of total substrate. Tubes were incubated for 24 h to determine fermentation and microbial parameters. Then, the dose response of these parameters to different supplementation levels of WSCA (0, 7.5, 15, 22.5, and 30%) was tested in the same manner. We also operated a continuous culture system with WSCA supplementation and evaluated the effects on digestibility, fermentation, and microbial parameters. The supplementation level of WSCA was set at 15% of total feed. In batch culture studies, WSCA, but not insoluble CA, yielded dose-dependent increases in ruminal acetate levels. In the continuous culture system study, WSCA yielded increases in ruminal acetate levels and in the abundance of bacteria of the genus Prevotella, including Prevotella ruminicola. Dry matter digestibility and total gas production were not affected. These results suggest that WSCA supplementation at 15% of total feed yielded increased acetate levels without negatively affecting feed digestion; these effects may reflect activation of Prevotella species. As ruminal acetate is involved in milk fat synthesis, WSCA can be considered as a candidate feed additive suitable for dairy cattle.
Topics: Animal Feed; Animals; Cattle; Cellulose; Dietary Fiber; Dietary Supplements; Female; Fermentation; In Vitro Techniques; Microbiota; Prevotella; Rumen; Ruminants
PubMed: 30268629
DOI: 10.3168/jds.2018-14969 -
Applied and Environmental Microbiology May 1996In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal...
In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.
Topics: Animals; Cattle; Cellobiose; Cellulose; Dextrins; Glucosyltransferases; Prevotella
PubMed: 8633876
DOI: 10.1128/aem.62.5.1770-1773.1996 -
Frontiers in Microbiology 2021This study was conducted to examine the influences of replacing soybean meal (SBM) with fermented soybean meal (FSBM) in the diet of lactating Holstein cattle on rumen...
This study was conducted to examine the influences of replacing soybean meal (SBM) with fermented soybean meal (FSBM) in the diet of lactating Holstein cattle on rumen fermentation and ruminal bacterial microbiome. Twenty-four lactating Chinese Holstein dairy cattle were assigned to each of the two treatments in a completely randomized design: the SBM group [the basal total mixed ration (TMR) diet containing 5.77% SBM] and the FSBM group (the experimental TMR diet containing 5.55% FSBM). This trial lasted for 54 days (14 days for adjustment and 40 days for data and sample collection), and samples of rumen liquid were collected on 34 d and 54 d, respectively. The results showed that replacing SBM with FSBM significantly increased the molar percentages of propionate ( < 0.01) and valerate ( < 0.05), but reduced the total volatile fatty acid (TVFA) concentration ( < 0.05), butyrate molar proportion ( < 0.05), and the acetate to propionate ratio ( < 0.01). The copy numbers of total bacteria ( < 0.05), ( < 0.01), ( < 0.01), and spp. ( < 0.05) in the FSBM group were greater, while the density of ( < 0.05) was lower than those in the SBM treatment. Additionally, and were significantly enriched ( < 0.05) in the rumen fluid of FSBM-fed cows, despite the fact that there was no remarkable difference in the Alpha diversity indexes, structure and KEGG pathway abundances of the bacterial community across the two treatments. It could hence be concluded that the substitution of FSBM for SBM modulated rumen fermentation and rumen bacterial microbiota in lactating Holstein dairy cows. Further research is required to elucidate the relevant mechanisms of FSBM, and provide more insights into the application of FSBM in dairy cattle.
PubMed: 33584627
DOI: 10.3389/fmicb.2021.625857 -
Frontiers in Microbiology 2022Mastitis, mainly caused by bacterial intramammary infections, is the main problem in the breeding of dairy animals. The inflammations of the mammary gland is separated...
Mastitis, mainly caused by bacterial intramammary infections, is the main problem in the breeding of dairy animals. The inflammations of the mammary gland is separated by types of mastitis, being subclinical, clinical, and the most severe, gangrenous mastitis. Here, we used 16S rRNA amplicon sequencing to characterize the bacterial microbiota of goat milk in the different types of goat mastitis caused by bacteria. We used 72 goat milk samples from a region of the state of Minas Gerais in Brazil, of which 12 were from clinically healthy animals, 42 from animals diagnosed with subclinical mastitis, 16 from animals with clinical mastitis, and 2 from animals with gangrenous mastitis. The group related to gangrenous mastitis was the most divergent in terms of alpha and beta diversity. The most abundant genus among samples of the groups was spp., and we found a high abundance of sp. in the milk of animals diagnosed with clinical mastitis. The most statistically relevant microorganisms among the groups were sp., , sp., and sp. We highlight a new association of bacterial agents in gangrenous mastitis among sp./ sp. and sp. and provide the second report of the genus sp., in milk samples. Only the taxa sp., sp., , and sp., were present in all groups. The superpathway of L-tryptophan biosynthesis metabolites and the sucrose degradation III (sucrose invertase) pathway were the most prominent ones among the groups. In this study, we demonstrate how a rich microbiota of goat milk from healthy animals can be altered during the aggravation of different types of mastitis, in addition to demonstrating new bacterial genera in milk not previously detected in other studies as well as new associations between agents.
PubMed: 36090116
DOI: 10.3389/fmicb.2022.918706 -
Animals : An Open Access Journal From... Jul 2021We evaluated whether olive leaves (OLs) are effective as feed additives and supplements for ruminants and the potential methane reduction effects during in vitro...
We evaluated whether olive leaves (OLs) are effective as feed additives and supplements for ruminants and the potential methane reduction effects during in vitro fermentation. Two Hanwoo cows (460 ± 20 kg) equipped with cannula were fed Timothy hay and corn-based feed 3% of the body weight at a ratio of 6:4 (8:30 a.m. and 5:00 p.m.). Ruminal fluid from the cows was collected and mixed before morning feeding. In vitro batch fermentation was monitored after 12 and 24 h of incubation at 39 °C, and OLs were used as supplements to achieve the concentration of 5% in the basal diet. At 12 h of fermentation, methane production decreased in the 5% OLs group compared to that in the control group, but not at 24 h. The proportion of cellulose-degrading bacteria, , and , tended to increase in the 5% OLs group at 12 h. The amount of ammonia produced was the same as the polymerase chain reaction result for . At 12 h, the proportion of was significantly higher in the 5% OLs group. OLs may be used incorporated with protein byproducts or other methane-reducing agents in animal feed.
PubMed: 34359136
DOI: 10.3390/ani11072008 -
Applied and Environmental Microbiology Jun 2001A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter...
A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.
Topics: Animal Feed; Animals; Bacteria; Cattle; DNA Primers; DNA, Ribosomal; Diet; Ecosystem; Female; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Rhinitis, Allergic, Seasonal; Rumen; Sequence Analysis, DNA
PubMed: 11375193
DOI: 10.1128/AEM.67.6.2766-2774.2001 -
Applied and Environmental Microbiology Feb 1997A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity.... (Comparative Study)
Comparative Study
A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.
Topics: 2-Naphthylamine; Adhesins, Bacterial; Ammonia; Animals; Cysteine Endopeptidases; Dipeptides; Gingipain Cysteine Endopeptidases; Hemagglutinins; Mutagenesis; Prevotella; Rumen; Subcellular Fractions
PubMed: 9023945
DOI: 10.1128/aem.63.2.670-675.1997 -
Frontiers in Microbiology 2019The objective of this study was to examine the association between bacterial community structure and the utilization efficiency of nitrogen (UEN) phenotypes by...
The objective of this study was to examine the association between bacterial community structure and the utilization efficiency of nitrogen (UEN) phenotypes by determining the bacterial community in the gastrointestinal tract (GIT) of goats that differ in UEN using high-throughput 16S rRNA gene sequencing. Thirty Nubian goats were selected as experimental animals, and their UEN was determined in a metabolic experiment. Subsequently, eight individuals were grouped into the high nitrogen utilization (HNU) phenotype, and seven were grouped into the low nitrogen utilization (LNU) phenotype. The bacterial 16S rRNA gene amplicons from the rumen, abomasum, jejunum, cecum and colon contents of these animals were sequenced using next-generation high-throughput sequencing technology. Two hundred thirty-nine genera belonging to 23 phyla in the rumen, 319 genera belonging to 30 phyla in the abomasum, 248 genera belonging to 36 phyla in the jejunum, 248 genera belonging to 25 phyla in the colon and 246 genera belonging to 23 phyla in the cecum were detected, with Bacteroidetes and Firmicutes predominating. In addition, a significant correlation was observed between the UEN and the genera and in the rumen; and in the abomasum; and in the jejunum; and in the colon; and in the cecum. Furthermore, the real-time PCR results showed that the ruminal copies of , , _sp._HUN007, and in the HNU animals were significantly higher than those in the LNU animals. This study suggests an association of GIT microbial communities as a factor that influences UEN in goats.
PubMed: 30873128
DOI: 10.3389/fmicb.2019.00239 -
Animals : An Open Access Journal From... Aug 2020The yak rumen microflora has more efficient fiber-degrading and energy-harvesting abilities than that of low-altitude cattle; however, few studies have investigated the...
The yak rumen microflora has more efficient fiber-degrading and energy-harvesting abilities than that of low-altitude cattle; however, few studies have investigated the effects of dietary energy levels on the rumen bacterial populations and the relationship between rumen bacteria and the intramuscular fatty acid profile of fattening yaks. In this study, thirty yaks were randomly assigned to three groups. Each group received one of the three isonitrogenous diets with low (3.72 MJ/kg), medium (4.52 MJ/kg), and high (5.32 MJ/kg) levels of net energy for maintenance and fattening. After 120 days of feeding, results showed that increasing dietary energy significantly increased ruminal propionate fermentation and reduced ammonia concentration. The 16S rDNA sequencing results showed that increasing dietary energy significantly increased the ratio of to and stimulated the relative abundance of , , , and populations. The quantitative real-time PCR analysis showed that high dietary energy increased the abundances of , , and at the species level. Association analysis showed that ruminal acetate was positively related to some intramuscular saturated fatty acid (SFA) contents, and was significantly positively related to intramuscular total polyunsaturated fatty acid content and negatively related to intramuscular total SFA content. This study showed that high dietary energy mainly increased ruminal amylolytic and propionate-producing bacteria populations, which gave insights into how the effects of dietary energy on rumen bacteria are related to intramuscular fat fatty acids of fattening yaks.
PubMed: 32842565
DOI: 10.3390/ani10091474