Lyophilized rumen fluid as a ruminal fermentation modifier in high grain-fed acidotic goats.

Rumen cud transfaunation re-establishes rumen micro environment and improves fermentation in recipient animals affected with digestive disorders. Preserving rumen cud or fluid will increase its availability for the treatment of rumen fermentation disorders, without having to maintain donor animals. Rumen fluid collected from healthy goats, fed standard ration having roughage 70% and concentrate 30%, was lyophilized (prefreezing -80 °C, 48 h; lyophilization -45 °C, 32 h) using 5% glycerol as cryoprotectant. The 16 S metagenome analysis of the lyophilized rumen fluid (LRF) revealed an abundance of Prevotella (33.2%). Selenomonas ruminantium (1.87%) and Megasphaera elsdenii (0.23%) were also present. Twenty-four goats having history of high grain feeding and exhibiting clinical symptoms of rumen fermentation disorders were randomly distributed into either one of the two treatment groups viz., T1 = oral administration of LRF 31 g/animal/day and T2 = oral administration of sodium bicarbonate (SB) 15 g/animal/day. Post intervention LRF and SB, improved animal body condition, feed intake, fecal consistency, elevated the ruminal pH at 48 h, reduced propionate and lactate at 48 h, reduced total volatile fatty acids (TVFA) and ammonia nitrogen at 24 h. Significant reduction in serum blood urea nitrogen (BUN) and urea levels were observed even from 24 h post intervention irrespective of the treatments. LRF significantly improved acetate and decreased propionate production compared to SB. LRF at 7.5% (v/v) can thus be used to counteract ruminal fermentation disorders in goats sequel to high grain ration.

Screening for Selenomonas noxia in a Pediatric and Adolescent Patient Population Reveals Differential Oral Prevalence across Age Groups.

Selenomonas noxia, a gram-negative anaerobe usually present in periodontitis, may be linked to overweight and obese adults. Recent advancements include a valid qPCR screening, enabling an effective prevalence study among pediatric patients aged 7 to 17 years. The aim of this study was to complete a retrospective screening of saliva samples from an existing biorepository using a validated qPCR screening protocol. The pediatric study sample (n = 87) comprised nearly equal numbers of males and females, mostly minority patients (67%), with an average age of 13.2 years. Screening for Selenomonas noxia revealed 34.4% (n = 30/87) positive samples, evenly distributed between males and females (p = 0.5478). However, an age-dependent association was observed with higher percentages of positive samples observed with higher ages (13.3% among 7 to 10 years; 34.6% among 11 to 13 years; 54.8% among 14-17 years), which was statistically significant (p = 0.0001). Although these findings revealed no noteworthy distinctions between males or females and minorities and non-minorities, the notable contrast between younger (7 to 10 years) and older (11 to 17 years) participants, possibly influenced by factors such as hormones and behavioral traits, will require further investigation of this patient population.

Anaerobic flora, Selenomonas ruminis sp. nov., and the bacteriocinogenic Ligilactobacillus salivarius strain MP3 from crossbred-lactating goats.

This study aimed to examine the distribution of anaerobic bacteria in the rumen fluid of Thai crossbred goats and to screen potential probiotic strains capable of producing antimicrobial compounds and inhibiting bacteria that cause milk fat depression. Thirty-four strains of bacteria from the rumen fluid were divided into 13 groups within 12 genera based on 16S rRNA gene sequences. The RF1-5 and RF5-12 were identified as Streptococcus luteliensis and Bacillus licheniformis, respectively, and demonstrated non-ropy exopolysaccharide. Furthermore, mPRGC5T was closely related to Selenomonas caprae JCM 33725 T (97.8% similarity) based on 16S rRNA gene sequences. It exhibited low average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values with related type strains ranging from 84.9 to 86.0%, 21.3 to 21.8%, and 73.8 to 76.1%, respectively. The genotypic and phenotypic characteristics of mPRGC5T strongly support this strain as a new species of the genus Selenomonas for which the name Selenomonas ruminis mPRGC5T was proposed. The type strain is mPRGC5T (= JCM 33724 T = KCTC 25177 T). Ligilactobacillus salivarius MP3 showed antibacterial activity against Cutibacterium acnes subsp. acnes DSM 1897 T and Kocuria rhizophila MIII. The enterolysin A cluster gene was identified in its genome. The auto-aggregation of L. salivarius MP3 was 93.6 ± 0.2%. Additionally, co-aggregation of L. salivarius MP3 with C. acnes DSM 1897 T and K. rhizophila MIII had 92.2 ± 3.4% and 87.3 ± 4.5%, respectively. The adhesion capacity of strain MP3 was 76.11 ± 2.2%. Probiogenomic analysis revealed that L. salivarius MP3 was nonhazardous to animal supplementation and included acid- and bile-tolerant ability. However, strain MP3 contained three antibiotic resistance genes. Thus, the supplementation of L. salivarius MP3 could increase the milk fat content by suppressing C. acnes DSM 1897 T with antibiotic resistance gene horizontal transfer awareness.

Biochemical, structural and dynamical characterizations of the lactate dehydrogenase from Selenomonas ruminantium provide information about an intermediate evolutionary step prior to complete allosteric regulation acquisition in the super family of lactate and malate dehydrogenases.

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant. The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.

Selenomonas caprae sp. nov., an obligately anaerobic and volatile fatty acid producing bacterium from ruminal fluid of domestic goat (Capra hircus L.).

A novel Gram-stain-negative, motile, obligately anaerobic bacterium strain mPRGC8T was isolated from the ruminal fluid of a domestic goat (Capra hircus L.) in Nakhon Pathom province, Thailand. The strain grew at 20-45 °C (optimum, 37 °C), pH 6.0-9.0 (optimum, pH 7.5) and 3 % (w/v) NaCl. It produced acetate, propionate, valerate, caproate and heptanoate from glucose. The 16S rRNA gene sequence analysis indicated that strain mPRGC8T belonged to the genus Selenomonas and was closely related to Selenomonas ruminantium subsp. ruminantium DSM 2150T (98.0 %) and Selenomonas ruminantium subsp. lactilytica JCM 6582T (97.9 %). The in silico DNA G+C content was 53.0 mol %. Strain mPRGC8T showed average nucleotide identity, digital DNA-DNA hybridization and average animo acid identity values with Selenomonas montiformis JCM 34373T, S. ruminantium subsp. lactilytica JCM 6582T and S. ruminantium subsp. ruminantium DSM 2150T ranging from 84.9 to 86.0 %, 21.3 to 21.8 % and 73.8 to 76.1 %, respectively. The predominant cellular fatty acids were C16 : 1 ω9c and C18 : 1 ω9c. Phosphatidylethanolamine, three unidentified aminophospholipids, two unidentified ninhydrin positive glycolipids, an unidentified phospholipid and an unidentified lipid were detected as polar lipids. The genomic and phenotypic characteristics of strain mPRGC8T strongly support its classification as representative of new species of the genus Selenomonas for which the name Selenomonas caprae sp. nov. is proposed. The type strain is mPRGC8T (=JCM 33725T=KCTC 25178T).

Ambient pH regulates lactate catabolism pathway of the ruminal Megasphaera elsdenii BE2-2083 and Selenomonas ruminantium HD4.

AIMS: To explore the impact of ambient pH on lactate catabolism by Megasphaera elsdenii BE2-2083 and Selenomonas ruminantium HD4 in both pure culture and in binary mixed culture. METHODS AND RESULTS: The growth rate, substrate consumption, product formation, enzymatic activity and gene expression of M. elsdenii and S. ruminantium at various pHs were examined. Furthermore, the metabolism of lactate catabolism pathways for M. elsdenii and S. ruminantium in the co-culture system was investigated by chasing the conversion of sodium L-[3-13 C]-lactate in nuclear magnetic resonance. In the pure culture systems, ambient pH had significant effects on the growth of M. elsdenii, whereas S. ruminantium was less sensitive to pH changes. In addition, lactate metabolic genes and activities of key enzymes were affected by ambient pH in M. elsdenii and S. ruminantium. In the co-culture system, low ambient pH reduced the contribution lactate catabolism by M. elsdenii. CONCLUSION: M. elsdenii BE2-2083 and S. ruminantium HD4 lactate degradation affected by ambient pH. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the regulatory mechanisms of lactate decomposing bacteria in lactate catabolism under the condition of subacute ruminal acidosis.

Isolation and description of Selenomonas timonae sp. nov., a novel Selenomonas species detected in a gingivitis patient.

A Gram-stain-negative bacterium, designated strain Marseille-Q3039T, was isolated from subgingival dental plaque of a woman with gingivitis in Marseille, France. Strain Marseille-Q3039T was found to be an anaerobic, motile and spore-forming crescent-shaped bacterium that grew at 25-41.5 °C (optimum, 37 °C), pH 5.5-8.5 (optimum, pH 7.5) and salinity of 5.0 g l-1 NaCl. The results of 16S rRNA gene sequence analysis revealed that strain Marseille-Q3039T was closely related to Selenomonas infelix ATCC 43532T (98.42 % similarity), Selenomonas dianae ATCC 43527T (97.25 %) and Centipedia periodontii DSM 2778T (97.19 %). The orthologous average nucleotide identity and digital DNA-DNA hybridization relatedness between strain Q3039T and its closest phylogenetic neighbours were respectively 84.57 and 28.2 % for S. infelix ATCC 43532T and 83.93 and 27.2 % for C. periodontii DSM 2778T. The major fatty acids were identified as C13 : 0 (27.7 %), C15 : 0 (24.4 %) and specific C13 : 0 3-OH (12.3 %). Genome sequencing revealed a genome size of 2 351 779 bp and a G+C content of 57.2 mol%. On the basis of the results from phenotypic, chemotaxonomic, genomic and phylogenetic analyses and data, we concluded that strain Marseille-Q3039T represents a novel species of the genus Selenomonas, for which the name Selenomonas timonae sp. nov. is proposed (=CSUR Q3039=CECT 30128).

Microbial Screening Reveals Oral Site-Specific Locations of the Periodontal Pathogen Selenomonas noxia.

INTRODUCTION: Selenomonas noxia (SN) is an important periodontal pathogen, associated with gingivitis and periodontitis. Many studies have found associations between SN and indicators of poor health outcomes, such as smoking, low socioeconomic status and obesity. However, less is known about the prevalence of this organism and more specifically about other oral site-specific locations that may harbor this organism. METHODS: Using an existing patient repository (n = 47) of DNA isolated from saliva and other oral sites (n = 235), including the dorsum of the tongue, lower lingual incisor, upper buccal molar and gingival crevicular fluid (GCF), molecular screening for SN was performed. Screening results were analyzed for associations between demographic variables (age, sex, race/ethnicity) and clinical information (body mass index or BMI, presence of orthodontic brackets, primary/mixed/permanent dentition). RESULTS: qPCR screening revealed a total of n = 62/235 sites or 26.3% harboring SN with saliva and GCF (either alone or in combination with one or more sites) most often observed (Saliva, n = 23/27 or 85.18%, GCF, n = 14/27 or 51%). Analysis of site-specific data revealed most positive results were found among saliva and GCF alone or in combination, with fewer positive results observed among the tongue (33.3%), lower lingual incisor (29.6%), and upper buccal molar (25.9%). No significant associations were found between demographic or clinical variables and presence of SN at any site. CONCLUSIONS: These results may be among the first to describe site-specific locations of S. noxia among various additional oral biofilm sites. These data may represent a significant advancement in our understanding of the sites and locations that harbor this organism, which may be important for our understanding of the prevalence and distribution of these organisms among patients of different ages undergoing different types of oral treatments, such as orthodontic treatment or therapy.

The impact of caries status on supragingival plaque and salivary microbiome in children with mixed dentition: a cross-sectional survey.

BACKGROUND: Supragingival plaque and saliva are commonly used for microbiome analysis. Many epidemiological studies have identified deciduous teeth caries as a risk factor for caries development in first permanent molar (FPM); nevertheless, to the best of our knowledge, there are no reports on the effects of deciduous teeth caries on the microbiome of healthy FPM. Additionally, it remains unclear whether saliva can be used instead of supragingival plaque for caries microbial studies. Therefore, we aimed to elucidate this issue, and to characterize and compare the oral microbiome of healthy FPMs in children with different caries statuses and that from children with and without caries in a similar microhabitat, by PacBio sequencing. Currently, few studies have investigated the oral microbiome of children using this technique. METHODS: Thirty children (aged 7-9 years) with mixed dentition were enrolled; 15 had dental caries, and 15 did not. Supragingival plaques of deciduous molars and maxillary FPMs, and non-stimulating saliva samples were collected. DNA was extracted and the v1-v9 regions of 16S rRNA were amplified. Subsequently, PacBio sequencing and bioinformatic analyses were performed for microbiome identification. RESULTS: The microbial alpha diversity of the saliva samples was lower than that of the supragingival plaque (p < 0.05); however, no differences were detected between deciduous teeth and FPMs (p > 0.05). In addition, the alpha and beta diversity of children with and without caries was also similar (p > 0.05). Nonmetric multidimensional scaling and Adonis analyses indicated that the microbial structure of salivary and supragingival plaque samples differ (p < 0.05). Further analysis of deciduous teeth plaque showed that Streptococcus mutans, Propionibacterium acidifaciens, and Veillonella dispar were more abundant in children with caries than in those without (p < 0.05); while in FPMs plaque, Selenomonas noxia was more abundant in healthy children (p < 0.05). No differences in microorganisms abundance were found in the saliva subgroups (p > 0.05). CONCLUSION: We have determined that supragingival plaque was the best candidate for studying carious microbiome. Furthermore, S. mutans, V. dispar, and P. acidifaciens were highly associated with deciduous teeth caries. S. noxia may be associated with the abiding health of FPM; however, this requires additional studies.

Cecal microbiota contribute to the development of woody breast myopathy.

The objective of this study was to characterize the bacterial diversity of cecal microbiota in broilers related to breast phenotype, diet, and genetic strain. Broilers from 2 genetic strains (120 birds/strain) were fed a control diet (15 birds/pen) and an amino acid reduced diet (15 birds/pen, digestible lysine, total sulfur amino acids, and threonine reduced by 20% compared to the control diet). At 8 wk of age, 4 male broilers with normal breast (NB, 1 chick per pen) and 4 male broilers with woody breast (WB, 1 chick per pen) were selected for each treatment (strain × diet). The DNA of cecal samples was extracted and the 16S rRNA genes were sequenced and analyzed. There were no differences (P > 0.05) in the alpha diversity of gut microbiota between 2 phenotypes (NB vs. WB), 2 strains, or 2 diets (control vs. reduced). However, principal coordinate analysis plots (beta diversity) revealed that there were composition differences in samples between the 2 phenotypes (P = 0.001) and the 2 diets (P = 0.024). The most abundant phyla in all samples were Firmicutes, followed by Bacteroidetes and Proteobacteria. There were differences (false discovery rate, FDR < 0.05) in bacterial relative abundance between phenotypes and between diet treatments, but not (FDR > 0.05) between the 2 genetic strains. Selenomonas bovis (12.6%) and Bacteroides plebeius (12.3%) were the top 2 predominant bacteria in the ceca of WB birds; however, the relative abundances of these 2 bacteria were only 5.1% and 1.2% in NB birds, respectively. Function analysis predicted that the metabolic activities differed (q < 0.05) only between phenotypes. The microbiota of WB birds was characterized as reduced glycolysis and urea cycle but increased tricarboxylic acid (TCA) cycles, sugar degradation, and purine and pyrimidine nucleotides biosynthesis. Further studies are needed to investigate if WB incidence could be reduced by regulating gut microbiota and the potential mechanism that leads to decreased WB incidence.

The relationship between oral-origin bacteria in the fecal microbiome and albumin-bilirubin grade in patients with hepatitis C.

BACKGROUND: Bacteria of oral origin (BO) in the gut are associated with prognosis in patients with cirrhosis. The Greengenes database (gg_13_8) is widely used in microbiome analysis, but the expanded Human Oral Microbiome Database (eHOMD), a specialized database for BO, can add more detailed information. We used each database to evaluate the relationship between the albumin-bilirubin grade (ALBI) and the microbiome in patients with hepatitis C. METHODS: Eighty patients were classified into the low ALBI group (LA; n = 34) or high ALBI group (HA; n = 46). Isolated DNA from stool was amplified to target the V3-4 regions of 16S rRNA. The microbiomes of the two groups were compared using gg_13_8 or eHOMD. We evaluated the associations between microbiomes and prognoses using Cox proportional hazards models. RESULTS: At the genus level, the two groups differed significantly regarding 6 (gg_13_8) and 7 (eHOMD) types of bacteria. All types except Akkermansia are classified as BO. Both databases showed an increase in Streptococcus and Veillonella. eHOMD showed a decrease in Fusobacterium and an increase in Fretibacterium; both produce various types of short-chain fatty acids. At the species level, the two groups demonstrated significant differences in 2 (gg_13_8) and 6 (eHOMD) bacterial types. Selenomonas noxia and Streptococcus salivarius were related to poor prognosis in univariate analysis. CONCLUSION: The HA group demonstrated increased BO, most of which produce lactic acid or acetic acid. The correlation between the microbiome and metabolism might be related to prognosis. eHOMD was a useful database for analyzing BO.

Calcium salts of long-chain fatty acids from linseed oil decrease methane production by altering the rumen microbiome in vitro.

Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH4) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH4 production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH4 production, and rumen microbiome in vitro. We compared five treatments: three CSFA concentrations-0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis-15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P < 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P < 0.05), DM digestibility-adjusted CH4 production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P < 0.05). The genera Ruminobacter, Succinivibrio, Succiniclasticum, Streptococcus, Selenomonas.1, and Megasphaera, which are related to propionate production, were increased (P < 0.05), while Methanobrevibacter and protozoa counts, which are associated with CH4 production, were decreased in FAH, compared with CON (P < 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH4 production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH4 production, CSFA may be a promising candidate for reduction of CH4 emission from ruminants.

Effects of dietary grape pomace on the intestinal microbiota and growth performance of weaned piglets.

Grape pomace (GP) is an abundant by-product from wine production and is rich in phenolic compounds, unsaturated fatty acids, dietary fibre and beneficial bacteria. In this study, weaned piglets were fed a basic diet supplemented with 5% GP for 4 weeks. Compared with those in the control (CON) group, it was found that the proportion of Lactobacillus delbrueckii, Olsenella umbonata and Selenomonas bovis in the caecum and the villus height and villus height/crypt depth ratio (VCR) of the jejunum were both significantly increased in the GP group (p < 0.05). Meanwhile, at the mRNA expression level, several proinflammatory cytokines (IL-1ß, IL-8, IL-6 and TNF-α) were significantly downregulated (p < 0.05) in piglet caecal tissue, and the short-chain fatty acid receptors (GPR41 and GPR43) were not significantly upregulated. In contrast, the levels of IgG was significantly increased (p < 0.05) in the sera of weaned piglets in the GP group. However, no difference in growth performance between the two groups of piglets was detected. These results show that GP had no adverse effects on the growth performance of piglets, but GP can promote the content of some beneficial bacteria in the caecum; this effect is conducive to improving the disease resistance potential of piglets.

Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

Improvement of Selenomonas ruminantium ß-xylosidase thermal stability by replacing buried free cysteines via site directed mutagenesis.

ß-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium ß-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results.

Flagellin Glycoproteomics of the Periodontitis Associated Pathogen Selenomonas sputigena Reveals Previously Not Described O-glycans and Rhamnose Fragment Rearrangement Occurring on the Glycopeptides.

Flagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859.

Partial characterization of phylogeny, ecology and function of the fibrolytic bacterium Ruminococcus flavefaciens OS14, newly isolated from the rumen of swamp buffalo.

The fibrolytic rumen bacterium Ruminococcus flavefaciensOS14 was isolated from swamp buffalo and its phylogenetic, ecological and digestive properties were partially characterized. Isolates from rumen contents of four swamp buffalo were screened for fibrolytic bacteria; one of the 40 isolates showed a distinctive feature of solubilizing cellulose powder in liquid culture and was identified as R. flavefaciens based on its 16S ribosomal DNA sequence. This isolate, OS14, was employed for detection and digestion studies, for which a quantitative PCR assay was developed and defined cultures were tested with representative forages in Thailand. OS14 was phylogenetically distant from other isolated and uncultured R. flavefaciens and showed limited distribution among Thai ruminants but was absent in Japanese cattle. OS14 digested rice straw and other tropical forage to a greater extent than the type strain C94 of R. flavefaciens. OS14 produced more lactate than C94, and digested para grass to produce propionate more extensively in co-culture with lactate-utilizing Selenomonas ruminantium S137 than a co-culture of C94 with S137. These results indicate that phylogenetically distinct OS14 could digest Thai local forage more efficiently than the type strain, possibly forming a symbiotic cross-feeding relationship with lactate-utilizing bacteria. This strain might be useful for future animal and other industrial applications.

Differences Regarding the Molecular Features and Gut Microbiota Between Right and Left Colon Cancer

For many years, developmental and physiological differences have been known to exist between anatomic segments of the colorectum. Because of different outcomes, prognoses, and clinical responses to chemotherapy, the distinction between right colon cancer (RCC) and left colon cancer (LCC) has gained attention. Furthermore, variations in the molecular features and gut microbiota between right and LCCs have recently been a hot research topic. CpG island methylator phenotype-high, microsatellite instability-high colorectal cancers are more likely to occur on the right side whereas tumors with chromosomal instability have been detected in approximately 75% of LCC patients and 30% of RCC patients. The mutation rates of oncogenes and tumor suppressor genes also differ between RCC and LCC patients. Biofilm is more abundant in RCC patients than LLC patients, as are Prevotella, Selenomonas, and Peptostreptococcus. Conversely, Fusobacterium, Escherichia/Shigella, and Leptotrichia are more abundant in LCC patients compared to RCC patients. Distinctive characteristics are apparent in terms of molecular features and gut microbiota between right and LCC. However, how or to what extent these differences influence diverging oncologic outcomes remains unclear. Further clinical and translational studies are needed to elucidate the causative relationship between primary tumor location and prognosis.

[High-level expression and characterization of Selenomonas ruminantium ß-xylosidase in Pichia pastoris].

ß-xylosidase (EC 3.2.1.37) is an important part of the xylanolytic enzymes system. In the present research, ß-xylosidase gene Sxa derived from Selenomonas ruminantium was expressed in Pichia pastoris GS115. According to the codon bias and rare codons of P. pastoris, mRNA secondary structure and GC content, Sxa gene was optimized. The optimized full-length gene mSxa was obtained by gene synthesis technique and the recombinant yeast expression vector pPIC9K-mSxa was constructed. After being digested by restriction enzyme BglⅡ, the mSxa gene was transformed into P. pastoris GS115. Then, phenotype and geneticin G418 resistance screening, and PCR were adopted to identify the positive transformants. Finally, the recombinant P. pastoris GS115-pPIC9K-mSxa was obtained. Based on enzymatic activity assay, a high-level expression clone was picked up and then the enzymatic characteristics of the recombinant ß-xylosidase were studied. The results showed that the molecular weight of the mSxa expressed in P. pastoris G115 was about 66 kDa. The maximum activity was achieved 287.61 IU/mL at fermenter level. Enzymatic characterization showed the ß-xylosidase was stable between 40 ℃ and 60 ℃, and pH between 5.0 and 7.0. The optimal reaction temperature and pH were 55 ℃ and 6.0, and preferentially degrading the ß-xylose glycosidic bond. The enzymatic activity was activated by Mn²âº and Ca²âº, and inhibited by Fe³âº, Cu²âº, Co²âº, Mg²âº, EDTA and SDS. The study indicates that the modified ß-xylosidase gene mSxa from Selenomonas ruminantium can express successfully with high activity in P. pastoris. The study lays a foundation for further industrial application of the ß-xylosidase.