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.

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.

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.

Cloning and high-level expression of ß-xylosidase from Selenomonas ruminantium in Pichia pastoris by optimizing of pH, methanol concentration and temperature conditions.

ß-xylosidase and several other glycoside hydrolase family members, including xylanase, cooperate together to degrade hemicelluloses, a commonly found xylan polymer of plant-cell wall. ß-d-xylosidase/α-l-arabinofuranosidase from the ruminal anaerobic bacterium Selenomonas ruminantium (SXA) has potential utility in industrial processes such as production of fuel ethanol and other bioproducts. The optimized synthetic SXA gene was overexpressed in methylotrophic Pichia pastoris under the control of alcohol oxidase I (AOX1) promoter and secreted into the medium. Recombinant protein showed an optimum pH 4.8 and optimum temperature 50 °C. Furthermore, optimization of growth and induction conditions in shake flask was carried out. Using the optimum expression condition (pH 6, temperature 20 °C and 1% methanol induction), protein production was increased by about three times in comparison to the control. The recombinant SXA we have expressed here showed higher turnover frequency using ρ-nitrophenyl ß-xylopyranoside (PNPX) substrate, in contrast to most xylosidase experiments reported previously. This is the first report on the cloning and expression of a ß-xylosidase gene from glycoside hydrolase (GH) family 43 in Pichia pastoris. Our results confirm that P. pastoris is an appropriate host for high level expression and production of SXA for industrial applications.

Development of a polymerase chain reaction assay for the rapid detection of the oral pathogenic bacterium, Selenomonas noxia.

BACKGROUND: In recent studies, periodontal health has been linked to being overweight and/or obese. Among common oral bacteria, Selenomonas noxia has been implicated in converting periodontal health to disease, and Selenomonas species have also been found in gastric ulcers. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of S. noxia. METHODS: Two oligonucleotide primer pairs and one probe were designed and tested to determine optimal amplification signal with three strains of S. noxia. The PCR assay was tested against fourteen non-target organisms, including closely related oral Selenomonads, one phylogenetically closely related bacterium, and two commonly isolated oral bacteria. RESULTS: One of the primer sets was more sensitive at detecting the target organism and was selected for optimization and validation experiments. The designed primers and probe amplified the target organism with 100% specificity. PCR inhibition was observed with an internal positive control, and inhibition was resolved by diluting the DNA extract. CONCLUSIONS: The qPCR assay designed in this study can be used to specifically detect S. noxia in the clinical setting and in future research involving the enhanced detection of S. noxia. The assay can also be used in epidemiological studies for understanding the role of S. noxia in disease processes including, but not limited to, oral health and obesity of infectious origin.

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.

A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia.

The class Clostridia in the phylum Firmicutes (formerly low-G+C Gram-positive bacteria) includes diverse bacteria of medical, environmental and biotechnological importance. The Selenomonas-Megasphaera-Sporomusa branch, which unifies members of the Firmicutes with Gram-negative-type cell envelopes, was recently moved from Clostridia to a separate class Negativicutes. However, draft genome sequences of the spore-forming members of the Negativicutes revealed typically clostridial sets of sporulation genes. To address this and other questions in clostridial phylogeny, we have compared a phylogenetic tree for a concatenated set of 50 widespread ribosomal proteins with the trees for beta subunits of the RNA polymerase (RpoB) and DNA gyrase (GyrB) and with the 16S rRNA-based phylogeny. The results obtained by these methods showed remarkable consistency, suggesting that they reflect the true evolutionary history of these bacteria. These data put the Selenomonas-Megasphaera-Sporomusa group back within the Clostridia. They also support placement of Clostridium difficile and its close relatives within the family Peptostreptococcaceae; we suggest resolving the long-standing naming conundrum by renaming it Peptoclostridium difficile. These data also indicate the existence of a group of cellulolytic clostridia that belong to the family Ruminococcaceae. As a tentative solution to resolve the current taxonomical problems, we propose assigning 78 validly described Clostridium species that clearly fall outside the family Clostridiaceae to six new genera: Peptoclostridium, Lachnoclostridium, Ruminiclostridium, Erysipelatoclostridium, Gottschalkia and Tyzzerella. This work reaffirms that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits, even such key ones as the structure of the cell envelope and Gram-staining pattern.

Coverage theories for metagenomic DNA sequencing based on a generalization of Stevens' theorem.

Metagenomic project design has relied variously upon speculation, semi-empirical and ad hoc heuristic models, and elementary extensions of single-sample Lander-Waterman expectation theory, all of which are demonstrably inadequate. Here, we propose an approach based upon a generalization of Stevens' Theorem for randomly covering a domain. We extend this result to account for the presence of multiple species, from which are derived useful probabilities for fully recovering a particular target microbe of interest and for average contig length. These show improved specificities compared to older measures and recommend deeper data generation than the levels chosen by some early studies, supporting the view that poor assemblies were due at least somewhat to insufficient data. We assess predictions empirically by generating roughly 4.5 Gb of sequence from a twelve member bacterial community, comparing coverage for two particular members, Selenomonas artemidis and Enterococcus faecium, which are the least ([Formula: see text]3 %) and most ([Formula: see text]12 %) abundant species, respectively. Agreement is reasonable, with differences likely attributable to coverage biases. We show that, in some cases, bias is simple in the sense that a small reduction in read length to simulate less efficient covering brings data and theory into essentially complete accord. Finally, we describe two applications of the theory. One plots coverage probability over the relevant parameter space, constructing essentially a "metagenomic design map" to enable straightforward analysis and design of future projects. The other gives an overview of the data requirements for various types of sequencing milestones, including a desired number of contact reads and contig length, for detection of a rare viral species.

Effects of extracts of Humulus lupulus (hops) and Yucca schidigera applied alone or in combination with monensin on rumen fermentation and microbial populations in vitro.

BACKGROUND: ß-Acids in hops (Humulus lupulus) and saponins in yucca (Yucca schidigera) have been found to possess antimicrobial properties similar to that of monensin and could be an alternative to in-feed antibiotics. The effects of monensin (MON) and ethanol extracts of hops (HE) and Y. schidigera (YE) alone and in combination with MON were assessed on ruminal microbial composition and fermentation in vitro of a barley-based diet. RESULTS: All treatments decreased (P < 0.05) CH4 production (per unit of dry matter), microbial protein (mg), and NH3 -N accumulation. All treatments reduced (P < 0.01) the acetate:propionate (A:P) ratio and molar proportions of butyrate, but increased (P < 0.01) those of propionate, whereas those of acetate decreased (P < 0.001) with addition of MON (10 µg mL(-1)) and combined with HE or YE. Methane produced per unit of true digested dry matter decreased (P < 0.001) with all treatments except YE. Monensin reduced (P < 0.001) proportions of 16S rRNA copies of Ruminococcus flavefaciens, but increased (P < 0.01) those of Selenomonas ruminantium. Hops extract alone or combined with MON reduced (P < 0.01) proportions of R. flavefaciens but combined with MON tended (P < 0.1) to increase those of S. ruminantium. Yucca extract combined with MON increased (P < 0.01) the proportions of R. flavefaciens and S. ruminantium. All treatments except MON (2.5 µg mL(-1)) reduced (P < 0.01) the relative abundance of methanogens. CONCLUSION: Hops extract and YE altered rumen microbes and fermentation in a manner similar to MON with many responses being additive when applied in combination.

An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand.

Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100 % similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02 g/l) was obtained when the cells were grown on minimal salt medium containing 15 g/l molasses and 1 g/l commercial monosodium glutamate supplemented with 1 g/l NaCl, 0.1 g/l leucine, 5 % (v/v) inoculum size at 30 °C and 150 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small CMC value (8 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional ß-D-xylosidase/α-L-arabinofuranosidase.

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-ß-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional ß-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.

Correlation between composition of the bacterial community and concentration of volatile fatty acids in the rumen during the transition period and ketosis in dairy cows.

The transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity of Veillonellaceae organisms was reduced and that of Streptococcaceae organisms was increased in rumen samples from the transition period and ketosis groups, with the number of Lactobacillaceae organisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes, Megasphaera elsdenii and Selenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium, Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number of Lactobacillus sp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers of Selenomonas ruminantium and Megasphaera elsdenii organisms in the rumen.

Levels of Selenomonas species in generalized aggressive periodontitis.

BACKGROUND AND OBJECTIVE: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. MATERIAL AND METHODS: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the Mann-Whitney U-test. RESULTS: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects (p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P. gingivalis and S. sputigena were higher in deep sites (probing depth ≥ 5 mm) than in shallow sites (probing depth ≤ 3 mm) (p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of ≤ 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P. gingivalis (r = 0.77; p < 0.01), S. sputigena (r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) (r = 0.42, p < 0.05). CONCLUSION: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further.

Cadaverine covalently linked to the peptidoglycan serves as the correct constituent for the anchoring mechanism between the outer membrane and peptidoglycan in Selenomonas ruminantium.

In Selenomonas ruminantium, a strictly anaerobic and gram-negative bacterium, cadaverine covalently linked to the peptidoglycan is required for the interaction between the peptidoglycan and the S-layer homologous (SLH) domain of the major outer membrane protein Mep45. Here, using a series of diamines with a general structure of NH(3)(+)(CH(2))(n)NH(3)(+) (n = 3 to 6), we found that cadaverine (n = 5) specifically serves as the most efficient constituent of the peptidoglycan in acquiring the high resistance of the cell to external damage agents and is required for effective interaction between the SLH domain of Mep45 and the peptidoglycan, facilitating the correct anchoring of the outer membrane to the peptidoglycan.

Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal ß-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal ß-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded ß-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

Structural instability of small rolling circle replication plasmids from Selenomonas ruminantium.

The complete nucleotide sequence of pSRD192 plasmid from Selenomonas ruminantium 19D has been obtained and analyzed. The plasmid, 2334bp in length, was shown to replicate by rolling circle replication mechanism. By PCR method variability of pSRD192-like plasmids was investigated and another variant of pSRD192-like plasmid; the pSRM22 plasmid 2338bp in length; was detected and characterized. Both pSRD192 and pSRM22 plasmids share an identical rep gene and origins of replication to that of another S. ruminantium pONE429 plasmid. Other than that there are additional regions of sequence similarity between the three plasmids, interspersed with divergent regions. The sequence comparisons suggest structural instability of pSRD192-like rolling circle replication plasmids.