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European Journal of Biochemistry Jul 2000The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced,...
Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN.
The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.
Topics: Amino Acid Sequence; Bacillaceae; Base Sequence; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Flavodoxin; Glutamic Acid; Hydrogen-Ion Concentration; Hydroquinones; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidation-Reduction; Protein Binding; Recombinant Proteins; Sequence Analysis, DNA; Temperature; Thermodynamics
PubMed: 10880967
DOI: 10.1046/j.1432-1327.2000.01490.x -
Animal Nutrition (Zhongguo Xu Mu Shou... Mar 2021This review aims to give an overview of the efficacy of yeast supplementation on growth performance, rumen pH, rumen microbiota, and their relationship to meat and milk... (Review)
Review
This review aims to give an overview of the efficacy of yeast supplementation on growth performance, rumen pH, rumen microbiota, and their relationship to meat and milk quality in ruminants. The practice of feeding high grain diets to ruminants in an effort to increase growth rate and weight gain usually results in excess deposition of saturated fatty acids in animal products and increased incidence of rumen acidosis. The supplementation of yeast at the right dose and viability level could counteract the acidotic effects of these high grain diets in the rumen and positively modify the fatty acid composition of animal products. Yeast exerts its actions by competing with lactate-producing ( and ) bacteria for available sugar and encouraging the growth of lactate-utilising bacteria (). is known to convert lactate into butyrate and propionate leading to a decrease in the accumulation of lactate thereby resulting in higher rumen pH. Interestingly, this creates a conducive environment for the proliferation of vaccenic acid-producing bacteria () and ciliate protozoa, both of which have been reported to increase the ruminal concentration of -11 and -9, -11-conjugated linoleic acid (CLA) at a pH range between 5.6 and 6.3. The addition of yeast into the diet of ruminants has also been reported to positively modify rumen biohydrogenation pathway to synthesise more of the beneficial biohydrogenation intermediates ( 11 and 9, 11). This implies that more dietary sources of linoleic acid, linolenic acid, and oleic acid along with beneficial biohydrogenation intermediates (-9, -11-CLA, and -11) would escape complete biohydrogenation in the rumen to be absorbed into milk and meat. However, further studies are required to substantiate our claim. Therefore, techniques like transcriptomics should be employed to identify the mRNA transcript expression levels of genes like stearoyl-CoA desaturase fatty acid synthase, and elongase of very long chain fatty acids 6 in the muscle. Different strains of yeast need to be tested at different doses and viability levels on the fatty acid profile of animal products as well as its vaccenic acid and rumenic acid composition.
PubMed: 33997329
DOI: 10.1016/j.aninu.2020.10.005 -
Scientific Reports Sep 2022Elevated levels of ruminal lipopolysaccharides (LPS) have been linked to ruminal acidosis; however, they result in reduced endotoxicity compared to LPS derived from...
Elevated levels of ruminal lipopolysaccharides (LPS) have been linked to ruminal acidosis; however, they result in reduced endotoxicity compared to LPS derived from species like Escherichia coli. Additionally, there is a knowledge gap on the potential effect of LPS derived from ruminal microbiome on ruminal bacteria species whose abundance is associated with ruminal acidosis. The objective of this study was to evaluate the effects of LPS-free anaerobic water (CTRL), E. coli-LPS (E. COLI), ruminal-LPS (RUM), and a 1:1 mixture of E. coli and ruminal-LPS (MIX) on the growth characteristics and fermentation end products of lactate-producing bacteria (Streptococcus bovis JB1, Selenomonas ruminantium HD4) and lactate-utilizing bacterium (Megasphaera elsdenii T81). The growth characteristics were predicted based on the logistic growth model, the ammonia concentration was determined by the phenolic acid/hypochlorite method and organic acids were analyzed with high performance liquid chromatography. Results indicate that, compared to the CTRL, the maximum specific growth rate of S. bovis JB1 decreased by approximately 19% and 23% when RUM and MIX were dosed, respectively. In addition, acetate and lactate concentrations in Se. ruminantium HD4 were reduced by approximately 30% and 18%; respectively, in response to MIX dosing. Compared to CTRL, lactate concentration from S. bovis JB1 was reduced approximately by 31% and 22% in response to RUM and MIX dosing; respectively. In summary, RUM decreased the growth and lactate production of some lactate-producing bacteria, potentially mitigating the development of subacute ruminal acidosis by restricting lactate availability to some lactate-utilizing bacteria that metabolize lactate into VFAs thus further contributing to the development of acidosis. Also, RUM did not affect Megasphaera elsdenii T81 growth.
Topics: Acetates; Acidosis; Ammonia; Animals; Bacteria; Escherichia coli; Fermentation; Hydrogen-Ion Concentration; Hypochlorous Acid; Lactic Acid; Lipopolysaccharides; Rumen; Water
PubMed: 36151241
DOI: 10.1038/s41598-022-20073-2 -
Journal of Animal Science and... Jan 2022Methane production and fatty acids (FA) biohydrogenation in the rumen are two main constraints in ruminant production causing environmental burden and reducing food...
Dietary Coleus amboinicus Lour. decreases ruminal methanogenesis and biohydrogenation, and improves meat quality and fatty acid composition in longissimus thoracis muscle of lambs.
BACKGROUND
Methane production and fatty acids (FA) biohydrogenation in the rumen are two main constraints in ruminant production causing environmental burden and reducing food product quality. Rumen functions can be modulated by the biologically active compounds (BACs) of plant origins as shown in several studies e.g. reduction in methane emission, modulation of FA composition with positive impact on the ruminant products. Coleus amboinicus Lour. (CAL) contains high concentration of polyphenols that may potentially reduce methane production and modulate ruminal biohydrogenation of unsaturated FA. This study aimed to investigate the effect of BAC of Coleus amboinicus Lour. (CAL) fed to growing lambs on ruminal methane production, biohydrogenation of unsaturated FA and meat characteristics. In this study, the in vitro experiment aiming at determining the most effective CAL dose for in vivo experiments was followed by two in vivo experiments in rumen-cannulated rams and growing lambs. Experiment 1 (RUSITEC) comprised of control and three experimental diets differing in CAL content (10%, 15%, and 20% of the total diet). The two in vivo experiments were conducted on six growing, rumen-cannulated lambs (Exp. 2) and 16 growing lambs (Exp. 3). Animals were assigned into the control (CON) and experimental (20% of CAL) groups. Several parameters were examined in vitro (pH, ammonia and VFA concentrations, protozoa, methanogens and select bacteria populations) and in vivo (methane production, digestibility, ruminal microorganism populations, meat quality, fatty acids profiles in rumen fluid and meat, transcript expression of 5 genes in meat).
RESULTS
CAL lowered in vitro methane production by 51%. In the in vivo Exp. 3, CAL decreased methane production by 20% compared with the CON group, which corresponded to reduction of total methanogen counts by up to 28% in all experiments, notably Methanobacteriales. In Exp. 3, CAL increased or tended to increase populations of some rumen bacteria (Ruminococcus albus, Megasphaera elsdenii, Butyrivibrio proteoclasticus, and Butyrivibrio fibrisolvens). Dietary CAL suppressed the Holotricha population, but increased or tended to increase Entodiniomorpha population in vivo. An increase in the polyunsaturated fatty acid (PUFA) proportion in the rumen of lambs was noted in response to the CAL diet, which was mainly attributable to the increase in C18:3 cis-9 cis-12 cis-15 (LNA) proportion. CAL reduced the mRNA expression of four out of five genes investigated in meat (fatty acid synthase, stearoyl-CoA desaturase, lipoprotein lipase, and fatty acid desaturase 1).
CONCLUSIONS
Summarizing, polyphenols of CAL origin (20% in diet) mitigated ruminal methane production by inhibiting the methanogen communities. CAL supplementation also improved ruminal environment by modulating ruminal bacteria involved in fermentation and biohydrogenation of FA. Besides, CAL elevated the LNA concentration, which improved meat quality through increased deposition of n-3 PUFA.
PubMed: 35027089
DOI: 10.1186/s40104-021-00654-3 -
Biomedicines Nov 2022The role of gut microbes has been suggested in type 2 diabetes (T2DM) risk. However, their results remain controversial. We hypothesized that Asians with T2DM had...
The role of gut microbes has been suggested in type 2 diabetes (T2DM) risk. However, their results remain controversial. We hypothesized that Asians with T2DM had different fecal bacterial compositions, co-abundance networks, and metagenome functions compared to healthy individuals, according to enterotypes. This hypothesis was examined using the combined gut microbiota data from human fecal samples from previous studies. The human fecal bacterial FASTA/Q files from 36 different T2DM studies in Asians were combined (healthy, n = 3378; T2DM, n = 551), and operational taxonomic units (OTUs) and their counts were obtained using qiime2 tools. In the machine learning approaches, fecal bacteria rich in T2DM were found. They were separated into two enterotypes, Lachnospiraceae (ET-L) and Prevotellaceae (ET-P). The Shannon and Chao1 indices, representing α-diversity, were significantly lower in the T2DM group compared to the healthy group in ET-L (p < 0.05) but not in ET-P. In the Shapley additive explanations analysis of ET-L, Escherichia fergusonii, Collinsella aerofaciens, Streptococcus vestibularis, and Bifidobacterium longum were higher (p < 0.001), while Phocaeicola vulgatus, Bacteroides uniformis, and Faecalibacterium prausnitzii were lower in the T2DM group than in the healthy group (p < 0.00005). In ET-P, Escherichia fergusonii, Megasphaera elsdenii, and Oscillibacter valericigenes were higher, and Bacteroides koreensis and Faecalibacterium prausnitzii were lower in the T2DM group than in the healthy group. In ET-L and ET-P, bacteria in the healthy and T2DM groups positively interacted with each other within each group (p < 0.0001) but negatively interacted between the T2DM and healthy groups in the network analysis (p < 0.0001). In the metagenome functions of the fecal bacteria, the gluconeogenesis, glycolysis, and amino acid metabolism pathways were higher, whereas insulin signaling and adenosine 5′ monophosphate-activated protein kinase (AMPK) signaling pathways were lower in the T2DM group than in the healthy group for both enterotypes (p < 0.00005). In conclusion, Asians with T2DM exhibited gut dysbiosis, potentially linked to intestinal permeability and the enteric vagus nervous system.
PubMed: 36428566
DOI: 10.3390/biomedicines10112998 -
Veterinary Sciences Dec 2022Diarrhea is one of the most commonly reported diseases in young farm animals. () F17 is one of the major pathogenic bacteria responsible for diarrhea. However, the...
Diarrhea is one of the most commonly reported diseases in young farm animals. () F17 is one of the major pathogenic bacteria responsible for diarrhea. However, the pathogenicity of diarrhea in lambs involving F17 strains and how F17 infection modifies lambs' intestinal microbiota are largely unknown. To evaluate diarrhea in newborn lambs with an infection of F17, 50 lambs were selected for challenge experiments and divided into four groups, namely, a high-dose challenge group, low-dose challenge group, positive control group, and negative control group. The F17 challenge experiments caused diarrhea and increased mortality in the experimental lamb population, with a higher prevalence (90%), mortality (35%), and rapid onset time (4-12 h) being observed in the high-dose challenge group than the results observed in the low-dose challenge group (75%, 10%, 6-24 h, respectively). After the challenge experiment, healthy lambs in the high-dose challenge group and severely diarrheic lamb in the low-dose challenge group were identified as lambs sensitive/resistant to F17 ( F17 -resistant/-sensitive candidate, AN/SE) according to the histopathological detection. Results of intestinal contents bacteria plate counting revealed that the number of bacteria in the intestinal contents of SE lambs was 10-fold greater than that of the AN lambs, especially in the jejunum. Then, 16S rRNA sequencing was conducted to profile the intestinal microbiota using the jejunal contents, and the results showed that SE lambs had higher and a lower ratio and intestinal microbiota diversity in the jejunum than AN lambs. Notably, high abundance of was revealed in AN lambs, which indicated that may serve as a potential probiotic for F17 infection. Our study provides an alternative challenge model for the identification of F17-sensitive/-resistant lambs and contributes to the basic understandings of intestinal microbiota in lambs with different susceptibilities to F17.
PubMed: 36548832
DOI: 10.3390/vetsci9120670 -
Applied and Environmental Microbiology Jul 2003Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline....
Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.
Topics: Animals; Anti-Bacterial Agents; Bacteria, Anaerobic; Bacterial Proteins; Carrier Proteins; Cecum; Chlortetracycline; Culture Media; DNA, Ribosomal; Evolution, Molecular; Gram-Positive Cocci; Lactates; Microbial Sensitivity Tests; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Recombination, Genetic; Sequence Analysis, DNA; Swine; Tetracycline Resistance
PubMed: 12839756
DOI: 10.1128/AEM.69.7.3874-3882.2003 -
MSystems Dec 2021Piglets are often weaned between 19 and 22 days of age in North America, although in some swine operations this may occur at 14 days or less. Piglets are abruptly...
Piglets are often weaned between 19 and 22 days of age in North America, although in some swine operations this may occur at 14 days or less. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. The effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Here, pigs were weaned at either 14, 21, or 28 days of age, and fecal samples were collected 20 times from day 4 (neonatal) through marketing at day 140. The fecal microbiome was characterized using 16S rRNA gene and shotgun metagenomic sequencing. The fecal microbiome of all piglets shifted significantly 3 to 7 days postweaning, with an increase in microbial diversity. Several spp. increased in relative abundance immediately after weaning, as did butyrate-producing species such as Butyricicoccus porcorum, Faecalibacterium prausnitzii, and Megasphaera elsdenii. Within 7 days of weaning, the gut microbiome of pigs weaned at 21 and 28 days of age resembled that of pigs weaned at 14 days. Resistance genes to most antimicrobial classes decreased in relative abundance postweaning, with the exception of those conferring resistance to tetracyclines and macrolides-lincosamides-streptogramin B. The relative abundance of microbial carbohydrate-active enzymes (CAZymes) changed significantly in the postweaning period, with an enrichment of CAZymes involved in degradation of plant-derived polysaccharides. These results demonstrate that the pig gut microbiome tends change in a predictable manner postweaning and that weaning age has only a temporary effect on this microbiome. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. This is the most important period in commercial swine production, yet the effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Metagenomic sequencing allows for a higher-resolution assessment of the pig gut microbiome and enables characterization of the resistome. Here, we used metagenomic sequencing to identify bacterial species that were enriched postweaning and therefore may provide targets for future manipulation studies. In addition, functional profiling of the microbiome indicated that many carbohydrate and metabolic enzymes decrease in relative abundance after weaning. This study also highlights the challenges faced in reducing antimicrobial resistance in pigs, as genes conferring tetracycline and macrolide resistance remained relatively stable from 7 days of age through to market weight at 140 days despite no exposure to antimicrobials.
PubMed: 34812652
DOI: 10.1128/mSystems.00682-21 -
Applied and Environmental Microbiology Jan 1993Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from... (Comparative Study)
Comparative Study
Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.
Topics: Acetates; Acrylates; Animals; Chromatography, DEAE-Cellulose; Culture Media; Enzyme Activation; Fatty Acids, Volatile; Glucose; L-Lactate Dehydrogenase; Lactates; Racemases and Epimerases; Structure-Activity Relationship; Veillonellaceae
PubMed: 8439152
DOI: 10.1128/aem.59.1.255-259.1993 -
Veterinary Sciences Nov 2022The gut microbiome exerts important functions on host health maintenance, whereas excessive antibiotic use may cause gut flora dysfunction resulting in serious disease...
The gut microbiome exerts important functions on host health maintenance, whereas excessive antibiotic use may cause gut flora dysfunction resulting in serious disease and dysbiosis. Colistin is a broad-spectrum antibiotic with serious resistance phenomena. However, it is unclear whether colistin alters the gastrointestinal tract microbiome in piglets. In this study, 16s rDNA-based metagenome analyses were used to assess the effects of colistin on the modification of the piglet microbiome in the stomach, duodenum, jejunum, cecum, and feces. Both α- and β-diversity indices showed that colistin modified microbiome composition in these gastrointestinal areas. In addition, colistin influenced microbiome composition at the phylum and genus levels. At the species level, colistin upregulated , , , , and abundance, but downregulated , , , , , , and abundance in stomachs. Colistin-fed piglets showed an increased abundance of , , , and , but , , , , and abundance was decreased in the cecum. In feces, colistin promoted , , , , and abundance when compared with controls. Taken together, colistin modified the microbiome composition of gastrointestinal areas in piglets. This study provides new clinical rationalization strategies for colistin on the maintenance of animal gut balance and human public health.
PubMed: 36548827
DOI: 10.3390/vetsci9120666