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Frontiers in Microbiology 2018Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics...
Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that was identified as a predominant in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 10 CFU/ml) containing the by oral gavage once a day during the period of 6-20 days of age prior to early weaning. Our data demonstrated that oral administration of significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by administration. Furthermore, our data revealed that oral administration of significantly increased the relative abundances of health-promoting microbes (including , LA39, , and ) and decreased the relative abundances of opportunistic pathogens (including and ). Functional alteration of the intestinal bacterial community by administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that facilitates intestinal epithelial barrier function maintenance in early-weaned piglets and may be a promising antibiotic alternative used for intestinal epithelial barrier function damage prevention in mammals.
PubMed: 29867808
DOI: 10.3389/fmicb.2018.00897 -
Communications Biology Jun 2020Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and...
Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling Hg, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.
Topics: Bacterial Proteins; Corrinoids; Desulfovibrio desulfuricans; Mercury; Metagenome; Methylation; Models, Molecular; Multiprotein Complexes; Phylogeny; Protein Conformation; Protein Domains; Spectrophotometry, Ultraviolet
PubMed: 32561885
DOI: 10.1038/s42003-020-1047-5 -
Environmental Science & Technology Sep 2017Natural dissolved organic matter (DOM) affects mercury (Hg) redox reactions and anaerobic microbial methylation in the environment. Several studies have shown that DOM...
Natural dissolved organic matter (DOM) affects mercury (Hg) redox reactions and anaerobic microbial methylation in the environment. Several studies have shown that DOM can enhance Hg methylation, especially under sulfidic conditions, whereas others show that DOM inhibits Hg methylation due to strong Hg-DOM complexation. In this study, we investigated and compared the effects of DOM on Hg methylation by an iron-reducing bacterium Geobacter sulfurreducens PCA and a sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 under nonsulfidic conditions. The methylation experiment was performed with washed cells either in the absence or presence of DOM or glutathione, both of which form strong complexes with Hg via thiol-functional groups. DOM was found to greatly inhibit Hg methylation by G. Sulfurreducens PCA but enhance Hg methylation by D. desulfuricans ND132 cells with increasing DOM concentration. These strain-dependent opposing effects of DOM were also observed with glutathione, suggesting that thiols in DOM likely played an essential role in affecting microbial Hg uptake and methylation. Additionally, DOM and glutathione greatly decreased Hg sorption by G. sulfurreducens PCA but showed little effect on D. desulfuricans ND132 cells, demonstrating that ND132 has a higher affinity to sorb or take up Hg than the PCA strain. These observations indicate that DOM effects on Hg methylation are bacterial strain specific, depend on the DOM:Hg ratio or site-specific conditions, and may thus offer new insights into the role of DOM in methylmercury production in the environment.
Topics: Desulfovibrio desulfuricans; Geobacter; Mercury; Methylation; Methylmercury Compounds
PubMed: 28806071
DOI: 10.1021/acs.est.7b02518 -
The FEBS Journal Dec 2021Hybrid cluster protein (HCP) is a unique Fe-S-O-type metallocluster-containing enzyme present in many anaerobic organisms and is categorized into three distinct classes...
Hybrid cluster protein (HCP) is a unique Fe-S-O-type metallocluster-containing enzyme present in many anaerobic organisms and is categorized into three distinct classes (I, II, and III). The class II HCP uniquely utilizes hybrid cluster protein reductase (HCR), unlike the other classes of HCPs. To gain structural insights into the electron transfer system between the class II HCP and HCR, we elucidated the X-ray crystal structure of Escherichia coli HCP (Ec HCP), representing the first report of a class II HCP structure. Surprisingly, Ec HCP was found to harbor a [4Fe-4S] cluster rather than a [2Fe-2S] cluster at the N-terminal Cys-rich region, similar to class I HCPs. It was also found that the Cys-rich motif forms a unique protrusion and that the surrounding charge distributions on the surface of class II Ec HCP are distinct from those of class I HCPs. The functional significance of the Cys-rich region was investigated using an Ec HCP variant (chimeric HCP) containing a class I HCP Cys-rich motif from Desulfovibrio desulfuricans. The biochemical analyses showed that the chimeric HCP lacks the hybrid cluster and the electron-accepting function from HCR despite the formation of the chimeric HCP-HCR complex. Furthermore, HCP-HCR molecular docking analysis suggested that the protrusion area serves as an HCR-binding region. Therefore, the protrusion of the unique Cys-rich motif and the surrounding area of class II HCP are likely important for maturation of Ec HCP and orienting HCR onto the surface of HCP to facilitate electron transfer in the HCP-HCR complex.
Topics: Binding Sites; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; Electron Transport; Escherichia coli; Escherichia coli Proteins; Iron-Sulfur Proteins; Molecular Docking Simulation; Mutation; Oxidoreductases; Protein Binding; Protein Conformation; Recombinant Fusion Proteins; Spectrophotometry
PubMed: 34101368
DOI: 10.1111/febs.16062 -
Biomedicines Nov 2022Chronic diarrhea is associated with enteric dysbiosis and provokes the overuse of antibiotics. Fecal microbiota transplantation (FMT) is a promising therapy, but it...
Chronic diarrhea is associated with enteric dysbiosis and provokes the overuse of antibiotics. Fecal microbiota transplantation (FMT) is a promising therapy, but it shows discrepant clinical efficacy. Bacterial colonization in recipients has been studied, although little is known about the role of gut fungi and Archaea after FMT. In this study, we evaluated the efficacy of human-derived FMT on spontaneous chronic diarrhea cynomolgus monkeys and revealed the effector mechanisms. We demonstrated that FMT can mitigate the appearance of diarrheal symptoms and inhibit the increase in interleukin-6, interleukin-8, interleukin-1β, and interferon-γ and the decrease in interleukin-10 in serum. We confirmed that FMT restored the disturbance of gut bacteria by reducing the relative abundances of potential pathogens, including 5_2_54FAA, and 21_3, and increasing the levels of and CAG_367 in diarrheal monkeys. The metabolic pathways of healthy and FMT monkeys' gut bacteria were enriched in amino acid metabolism, carbohydrate metabolism, and lipid metabolism, while the metabolic pathways of pre-FMT monkeys' gut bacteria were enriched in antibiotic production. Moreover, a higher Ascomycota/Basidiomycota ratio, higher levels, and lower abundance were present in intestinal fungi after FMT. Although the abundance of the Archaea did not change significantly, it was inversely correlated with the anti-inflammatory factor IL-4 after FMT. These results support the further development and application of FMT for chronic diarrhea.
PubMed: 36551772
DOI: 10.3390/biomedicines10123016 -
Journal of Animal Science and... 2019Several studies have evaluated the effects of live yeast supplementation on rumen microbial population; however, its effect on differential microbial genes and their...
Several studies have evaluated the effects of live yeast supplementation on rumen microbial population; however, its effect on differential microbial genes and their functional potential has not been described. Thus, this study applied shotgun metagenomic sequencing to evaluate the effects of live yeast supplementation on genetic and functional potential of the rumen microbiota in beef cattle. Eight rumen-cannulated Holstein steers were randomly assigned to two treatments in a cross-over design with two 25-day experimental periods and a 10-day wash-out between the two periods. The steers were housed in individual pens and fed 50% concentrate-mix and 50% red clover/orchard hay . Treatments were (1) control (CON; basal diet without additive) and (2) yeast (YEA; basal diet plus 15 g/d of live yeast product). Rumen fluid samples were collected at 3, 6, and 9 h after feeding on the last d of each period. Sequencing was done on an Illumina HiSeq 2500 platform. Dietary yeast supplementation increased the relative abundance of carbohydrate-fermenting bacteria (such as , . , , and ) and lactate-utilizing bacteria (such as , , and ). A total of 154 differentially abundant genes (DEGs) were obtained (false discovery rate < 0.01). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis of the DEGs revealed that 10 pathways, including amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, lipopolysaccharide biosynthesis, pantothenate and coenzyme A biosynthesis, glutathione metabolism, beta-alanine metabolism, polyketide sugar unit biosynthesis, protein export, ribosome, and bacterial secretory system, were enriched in steers fed YEA. Annotation analysis of the DEGs in the carbohydrate-active enzymes (CAZy) database revealed that the abundance of genes coding for enzymes belonging to glycoside hydrolases, glycosyltransferases, and carbohydrate binding modules were enriched in steers fed YEA. These results confirm the effectiveness of a live product for improving rumen function in beef steers by increasing the abundance of cellulolytic bacteria, lactic acid-utilizing bacteria, and carbohydrate-active enzymes in the rumen.
PubMed: 31508230
DOI: 10.1186/s40104-019-0378-x -
Proceedings of the National Academy of... Feb 2019Hydrogen sulfide (HS) production in the intestinal microbiota has many contributions to human health and disease. An important source of HS in the human gut is anaerobic...
Hydrogen sulfide (HS) production in the intestinal microbiota has many contributions to human health and disease. An important source of HS in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to HS by dissimilatory sulfite reductase. This unique GRE is also found in DSM642 and G20, which use isethionate but not taurine; corresponding knockout mutants of G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal HS production.
Topics: Alcohol Oxidoreductases; Anaerobiosis; Bilophila; Gastrointestinal Microbiome; Humans; Hydrogen Sulfide; Oxidation-Reduction; Proteomics; Taurine
PubMed: 30718429
DOI: 10.1073/pnas.1815661116 -
Biochimica Et Biophysica Acta Sep 2016A linear cluster formulated as [S2MoS2CuS2MoS2](3-), a unique heterometallic cluster found in biological systems, was identified in a small monomeric protein (named as...
A linear cluster formulated as [S2MoS2CuS2MoS2](3-), a unique heterometallic cluster found in biological systems, was identified in a small monomeric protein (named as Orange Protein). The gene coding for this protein is part of an operon mainly present in strict anaerobic bacteria, which is composed (in its core) by genes coding for the Orange Protein and two ATPase proposed to contain Fe-S clusters. In Desulfovibrio desulfuricans G20, there is an ORF, Dde_3197 that encodes a small protein containing several cysteine residues in its primary sequence. The heterologously produced Dde_3197 aggregates mostly in inclusion bodies and was isolated by unfolding with a chaotropic agent and refolding by dialysis. The refolded protein contained sub-stoichiometric amounts of iron atoms/protein (0.5±0.2), but after reconstitution with iron and sulfide, high iron load contents were detected (1.8±0.1 or 3.4±0.2) using 2- and 4-fold iron excess. The visible absorption spectral features of the iron-sulfur clusters in refolded and reconstituted Dde_3197 are similar and resemble the ones of [2Fe-2S] cluster containing proteins. The refolded and reconstituted [2Fe-2S] Dde_3197 are EPR silent, but after reduction with dithionite, a rhombic signal is observed with gmax=2.00, gmed=1.95 and gmin=1.92, consistent with a one-electron reduction of a [2Fe-2S](2+) cluster into a [2Fe-2S](1+) state, with an electron spin of S=½. The data suggests that Dde_3197 can harbor one or two [2Fe-2S] clusters, one being stable and the other labile, with quite identical spectroscopic properties, but stable to oxygen.
Topics: Amino Acid Sequence; Bacterial Proteins; Desulfovibrio gigas; Electron Spin Resonance Spectroscopy; Iron-Sulfur Proteins; Operon; Protein Folding
PubMed: 27240719
DOI: 10.1016/j.bbabio.2016.05.006 -
Journal of the American Chemical Society Nov 2021[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H). Their active site H-cluster is formed of a canonical [4Fe-4S]...
[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]) covalently attached to a unique [2Fe] subcluster ([2Fe]), where both sites are redox active. Heterolytic splitting and formation of H takes place at [2Fe], while [4Fe-4S] stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state H, named HH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HH was previously suggested to have a protonated [4Fe-4S]. Here, we show that HH forms by simple addition of sodium sulfite (NaSO, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO) is the species involved. Spectroscopy supports binding at or near [4Fe-4S], causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HH and its one-electron reduced counterpart H'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states H-DT and H-DT. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.
Topics: Algal Proteins; Bacterial Proteins; Biocatalysis; Chlamydomonas reinhardtii; Clostridium; Desulfovibrio desulfuricans; Dithionite; Hydrogen; Hydrogenase; Iron-Sulfur Proteins; Oxidation-Reduction; Reducing Agents; Sulfites; Sulfur Dioxide
PubMed: 34668697
DOI: 10.1021/jacs.1c07322 -
Scientific Reports Oct 2015The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans...
The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.
Topics: Amino Acid Motifs; Bacterial Proteins; Computational Biology; Data Mining; Genome, Bacterial; Genomics; Glycoproteins; Glycosylation; Hexosyltransferases; Humans; Immunoglobulin Fc Fragments; Membrane Proteins; Models, Molecular; Molecular Conformation; Phylogeny; Polysaccharides; Polysaccharides, Bacterial; Substrate Specificity; Sweetening Agents
PubMed: 26482295
DOI: 10.1038/srep15237