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PubMed1986

31P-NMR spectra of methanogens: 2,3-cyclopyrophosphoglycerate is detectable only in methanobacteria strains.

The unique compound 2,3-cyclopyrophosphoglycerate occurs at a detectable concentration in the genera Methanobacterium and Methanobrevibacter but not in Methanococcus, Methanospirillum and Methanosarcina, as shown by a 31P-NMR survey of several different methanogens. Metabolic poisons (carbonyl cyanide m-chlorophenylhydrazone and valinomycin) do not decrease the level of the cyclic pyrophosphate in Methanobacterium thermoautotrophicum; therefore, it cannot be a phosphagen, i.e., an energy storage material. 13CO2 is rapidly incorporated into this cyclic compound which represents the major soluble carbon as well as the phosphorus component of this methanobacteria. 13C-NMR analysis demonstrates that the pKa of the 2,3-cyclopyrophosphoglycerate carboxyl group is 2.55. The unusual pseudomurein cell wall structure of methano- and methanobrevibacteria necessitates a high demand on carbohydrate metabolism. For this reason, and the fact that when its concentration is decreased no new phosphorus resonances appear in the high resolution spectra, it is suggested that 2,3-cyclopyrophosphoglycerate has a function in carbohydrate metabolism.

2,3-Diphosphoglycerate↗

PubMed2002

The effect of polymer addition on granulation in an anaerobic baffled reactor (ABR). Part II: compartmentalization of bacterial populations.

The microbial ecology of wastewater treatment plants remains one of the least understood aspects in both aerobic and anaerobic systems, despite the fact that both processes are ultimately dependent on an active biomass for operational efficiency. Ultimately, future developments in anaerobic treatment processes will require a much greater understanding of the fundamental relationships between bacterial populations within the biomass if optimum process efficiency is to be fully realised. This study assesses the influence of polymer addition on granule formation within an ABR and compares the ecology of the biomass in each compartment of two ABRs treating ice-cream wastewater. To our knowledge, this is the first reported characterisation of the microbiology of acidogenic and methanogenic bacteria in the individual compartments of an ABR. The polymer-amended reactor contained sludge that had a greater density of anaerobic bacteria and larger and denser granules than the control reactor, indicating that polymer addition possibly contributed to the retention of active biomass within the ABR. The average fraction of autofluorescent methanogens was lower, with 1.5% being in the initial compartments of the ABRs, compared to the last compartment which had 15%, showing that each compartment of an ABR had a unique microbial composition. Partial spatial separation of anaerobic bacteria appeared to have taken place with acidogenic bacteria predominating in the initial compartments and methanogenic bacteria predominating in the final compartments. Scanning electron micrographs have revealed that the dominant bacteria in the initial compartments of the ABR (Compartments 1 and 2) were those which could consume H2/CO2 and formate as substrate, i.e. Methanobrevibacter, Methanococcus, with populations shifting to acetate utilisers, i.e. Methanosaeta, Methanosarcina, in the final compartments (Compartments 3 and 4). In addition, there appeared to be a stratified structure to the bacterial genera present within the granules.

Bacteria, Anaerobic↗

PubMed2001

Steady-state and time-resolved spectroscopy of F420 extracted from methanogen cells and its utility as a marker for fecal contamination.

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) = 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material.

Animals↗

PubMed2004

Microbial monitoring by molecular tools of a two-phase anaerobic bioreactor treating fruit and vegetable wastes.

Microbial consortia in a two-phase, anaerobic bioreactor using a mixture of fruit and vegetable wastes were established. Bacterial and archaeal communities obtained by a culture-independent approach based on single strand conformation polymorphism analysis of total 16S rDNA showed the adaptation of the microflora to the process parameters. Throughout the 90 d of the study, the species composition of the bacterial community changed significantly. Bacterial 16S rDNA showed at least 7 different major species with a very prominent one corresponding to a Megasphaera elsdenii whereas bacterial 16S rDNA of a methanization bioreactor showed 10 different major species. After two weeks, Prevotella ruminicola became major and its dominance increased continuously until day 50. After an acid shock at pH 5, the 16S rDNA archaeal patterns in the acidogenic reactor showed two major prominent species corresponding to Methanosphaera stadtmanii and Methanobrevibacter wolinii, a hydrogenotrophic bacterium.

Archaea↗

PubMed2006

Salt tolerance of archaeal extremely halophilic lipid membranes.

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group.

Archaeal Proteins↗

PubMed2003

Archaeosomes as self-adjuvanting delivery systems for cancer vaccines.

Archaeal ether glycerolipid vesicles (archaeosomes) efficiently deliver exogenous antigen for induction of humoral and cell-mediated immunity. Because induction of CD8 cytotoxic T cells is critical for protective vaccination against tumors, we compared the ability of various archaeosome lipid compositions to evoke a strong CD8 CTL response to entrapped antigen. Subcutaneous immunization of mice with ovalbumin (OVA) entrapped in all archaeosome lipid compositions evoked a primary (day 10) splenic CTL response indicating processing for MHC class I presentation. Interestingly, several polar lipid compositions from halophilic archaea were very potent to adjuvant this early CTL response. Despite this, the lytic units reduced substantially by weeks 6-7. More importantly, at >50 weeks, only Methanobrevibacter smithii and Thermoplasma acidophilum both rich in bipolar membrane-spanning caldarchaeols, demonstrated recall memory CTLs. Immunization of mice with OVA entrapped in M. smithii, Halobacterium salinarum or T. acidophilum vesicles provided prophylactic protection against challenge with OVA-expressing solid tumors at 6 weeks. Even a dose of 3 microg OVA in archaeosomes significantly delayed tumor growth. Tumor protection was also noted in a therapeutic design wherein OVA-archaeosomes were injected concurrent with the tumor challenge. Interestingly, antigen-free T. acidophilum but not antigen-free H. salinarum archaeosomes provided innate therapeutic protection. Vaccination with a CTL peptide epitope from the melanoma differentiation antigen, tyrosinase-related protein 2, in archaeosomes induced a protective CD8 response against B16OVA metastasis, indicating potential for targeting self, tumor antigens. Thus, lipid structural properties of archaea may differentially modulate primary, long-term and/or innate immunity, impacting adjuvant choice for vaccine design.

Animals↗

PubMed2002

Safety of archaeosome adjuvants evaluated in a mouse model.

Archaeosomes, liposomes prepared from the polar ether lipids extracted from Archaea, demonstrate great potential as immunomodulating carriers of soluble antigens, promoting humoral and cell mediated immunity in the vaccinated host. The safety of unilamellar archaeosomes prepared from the total polar lipids (TPL) of Halobacterium salinarum, Methanobrevibacter smithii or Thermoplasma acidophilum was evaluated in female BALB/c mice using ovalbumin (OVA) as the model antigen. Groups of 6-8 mice were injected (0.1 mL final volume) subcutaneously at 0 and 21 days, with phosphate buffered saline (PBS), 11 microg OVA in PBS, 1.25 mg of antigen-free archaeosomes in PBS (ca 70 mg/kg body wt), or PBS containing 11-20 microg OVA encapsulated in 1.25mg archaeosomes. Animals were monitored daily for injection site reactions, body weight,temperature and clinical signs of adverse reactions. Sera were collected on days 1, 2, 22, and 39 for analyses of creatine phosphokinase. Mice were sacrificed on 39 d, sera were collected for biochemical analyses, and major organs (liver, spleen, kidneys, heart, lungs) were weighed and examined macroscopically. There were no indications of adverse reactions or toxicity associated with any of the archaeosome adjuvants. None of the antigen-free archaeosomes elicited significant anti lipid antibodies when subcutaneously injected (1 mg each at 0, 1, 2, and 4 weeks) in mice, although anti H. salinarum lipid antibodies were detected. These antilipid antibodies cross-reacted with the TPL of T. acidophilum archaeosomes but not with the TPL of M. smithii archaeosomes nor with lipids of ester liposomes made from L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dimyristoylphosphatidylglycerol (DMPG), and cholesterol (CHOL). In vitro hemolysis assay on mouse erythrocytes indicated no lysis with M. smithii or T. acidophilum archaeosomes at up to 2.5 mg/mL concentration. At this concentration, H. salinarum archaeosomes and DMPC/DMPG/CHOL ester liposomes caused about 2% and 4% hemolysis, respectively. Based on this mouse model evaluation, archaeosomes are well-tolerated and appear relatively safe for potential vaccine applications.

Adjuvants, Immunologic↗

PubMed2026

Contribution of a combined approach using refined enterotyping and non-negative matrix factorization (NMF) to the characterization of the gut microbiota in Tunisia, North Africa.

This pilot study aimed to assess the enhanced capabilities of a combined approach using refined enterotyping and non-negative matrix factorization (NMF) for identifying specific microbiota patterns in healthy adults in Tunisia. Shotgun metagenomic sequencing was performed on 21 stool samples. Taxonomic classification was carried out using Kraken2, followed by Bracken analysis. Enterotype (ET) assignment was performed using a publicly available, reference-based classification tool involving Fuzzy-k-means (FKM) clustering. Next, NMF was applied to identify 'enterosignatures' (ESs). The FKM approach revealed a co-dominance of Prevotella-ET (P-ET, 57%) and Firmicutes-ET (F-ET, 38%) with 41% of P-ET samples exhibiting a significant deviation from the reference enterotype center. These latter had a lower proportion of Prevotella-ES and a higher proportion of Bacteroides/Phocaeicola-, Firmicutes- and/or Bifidobacterium-enriched ESs. The F-ET samples were differentially enriched by Blautia (P = 0.007) and Vescimonas (P = 0.007). NMF revealed within this group, a candidate Firmicutes-associated ES driven by Blautia and encompassing Vescimonas, Akkermansia, and Methanobrevibacter. These findings demonstrate the combined power of refined enterotyping and NMF in characterizing gut microbiota, providing a key methodology for future large-scale research. However, our relatively small sample size limits statistical power and biological interpretation, making this study exploratory in nature. Candidate ES requires validation in larger independent datasets.

Humans↗

PubMed1988

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids.

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.

Archaea↗

PubMed2005

The first true obligately syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum.

A Gram-positive, spore-forming, syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov. strain HH(T), was isolated as a co-culture with Methanospirillum hungatei JF-1(T) from anaerobic, freeze-dried granular sludge obtained from an upflow anaerobic sludge bed reactor treating sugar beet wastewater. The bacterium converted propionate to acetate in co-culture with Methanospirillum hungatei JF-1(T) or Methanobacterium formicicum MF(NT), but not in co-culture with Methanobrevibacter arboriphilus AZ. The organism could not be cultured axenically with any of the substrates tested and therefore can be considered as a (the first) true anaerobic syntrophic bacterium. The bacterium contained two distinct 16S rRNA gene sequences, with 96.8% sequence similarity, which were both expressed during syntrophic growth on propionate as revealed by fluorescent in situ hybridization. The most closely related organisms are Cryptanaerobacter phenolicus LR7.2(T), a bacterium that transforms phenol into benzoate, and Pelotomaculum thermopropionicum SI(T), a thermophilic, syntrophic propionate-oxidizing bacterium. Other related species belong to the Gram-positive, sulfate-reducing genus Desulfotomaculum. The type strain of Pelotomaculum schinkii is strain HH(T) (=ATCC BAA-615(T)=DSM 15200(T)).

Anaerobiosis↗

PubMed2007

Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections.

Phylogenetic analysis of bacterial and archaeal 16S rRNA was used to examine polymicrobial communities within infected root canals of 20 symptomatic and 14 asymptomatic patients. Nucleotide sequences from approximately 750 clones amplified from each patient group with universal bacterial primers were matched to the Ribosomal Database Project II database. Phylotypes from 37 genera representing Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were identified. Results were compared to those obtained with species-specific primers designed to detect Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Peptostreptococcus micros, Enterococcus sp., Streptococcus sp., Fusobacterium nucleatum, Tannerella forsythensis and Treponema denticola. Since members of the domain Archaea have been implicated in the severity of periodontal disease, and a recent report confirms that archaea are present in endodontic infections, 16S archaeal primers were also used to detect which patients carried these prokaryotes, to determine if their presence correlated with severity of the clinical symptoms. A Methanobrevibacter oralis-like species was detected in one asymptomatic and one symptomatic patient. DNA from root canals of these two patients was further analysed using species-specific primers to determine bacterial cohabitants. Trep. denticola was detected in the asymptomatic but not the symptomatic patient. Conversely, Porph. endodontalis was found in the symptomatic but not the asymptomatic patient. All other species except enterococci were detected with the species-specific primers in both patients. These results confirm the presence of archaea in root canals and provide additional insights into the polymicrobial communities in endodontic infections associated with clinical symptoms.

Archaea↗

PubMed1989

Enhanced resistance of anaerobic rumen fungi to the ionophores monensin and lasalocid in the presence of methanogenic bacteria.

The presence of Methanobrevibacter smithii altered the susceptibility of the anaerobic fungi Neocallimastix frontalis and Piromonas communis to the carboxylic ionophores monensin and lasalocid. The ionophores depressed growth (measured by chitin accretion), the uptake of glucose and the production of H2, formate and acetate by the fungi growing axenically in semi-solid medium. In the presence of M. smithii, the sensitivity of the fungi to monensin and lasalocid was decreased. For example, the uptake of glucose by N. frontalis strain RE1 in the culture was reduced to 50% of the control value by monensin at 0.5 microgram/ml. In the presence of M. smithii strain PS, approximately three times as much monensin was needed to bring about the same effect. In similar tests, the sensitivity of strain RE1 to lasalocid was decreased about nine-fold in the presence of M. smithii. The effect was not observed if the methanogens were killed by autoclaving before inoculation. It is suggested that the enhanced resistance to ionophores in the presence of M. smithii is a consequence of changes in the energy metabolism of the fungi growing in co-culture.

Acetates↗

PubMed2004

Phylogenetic analysis of methyl coenzyme-M reductase detected from the bovine rumen.

AIMS: The object of the present study is isolation of methyl coenzyme-M reductase (MCR) genes (mcrA) from the bovine rumen fluid and determination of phylogenetical placements of the genes to investigate mechanisms of methanogenesis in the rumen from a point of view of mcrA genes. METHODS: Genes for methanogen-specific MCR were isolated from the bovine rumen by PCR amplification. The deduced amino acid sequences were fitted to the alignments of mcrA gene products from the referred sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the deduced amino acid sequences of mcrA genes, isolated from the bovine rumen in the present study, were close to that of Methanobrevibacter ruminantium, these amino acid sequences did not fall into known clusters of MCR. The findings suggest that methanogenesis in the rumen would be partially carried out by unknown methanogens.

Animals↗

PubMed2000

Molecular phylogeny of methanogens associated with flagellated protists in the gut and with the gut epithelium of termites.

The molecular phylogeny of methanogenic archaea associated with the flagellated protist species Dinenympha and Microjoenia in the gut of termites, Reticulitermes speratus and Hodotermopsis sjoestedti, and those attached to the gut epithelium was examined based on PCR-amplified small-subunit ribosomal RNA genes. The sequences identified were classified into six groups within the genus Methanobrevibacter, including groups of yet uncharacterized novel species. Closely related methanogens were shared between Microjoenia and some Dinenympha cells in each termite. The methanogens harbored by the flagellates were phylogenetically different from the methanogens associated with the gut epithelium, suggesting that distinct methanogen species showed distinct spatial distributions in the termite gut.

Journal Article↗

PubMed1995

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2.

The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H2 and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.

Acetates↗

PubMed1997

Archaeosomes as novel antigen delivery systems.

The humoral immune response mounted in BALB/c mice against bovine serum albumin or cholera toxin B subunit was compared when the antigens were associated with liposomes composed of either archaeal ether lipids or conventional lipids. Antibody titres in sera from mice immunised intraperitoneally were elevated to an extent comparable to those achieved with Freund's adjuvant by encapsulating bovine serum albumin in archaeal lipid vesicles (archaeosomes) of about 200 nm diameter. Comparison among six archaeosome and three conventional liposome compositions established that archaeosomes were generally much superior in potentiating an immune response. Further, only two immunisations, at the most, were needed to achieve close to the maximum antibody titre, as shown with archaeosomes composed of the polar lipids from Methanobrevibacter smithii, an inhabitant of the human colon. A similar positive response to presenting the more immunogenic cholera B subunit protein to the immune system of mice was shown for M. smithii archaeosomes. Encapsulation of the antigen in the archaeosome was necessary to achieve the full humoral response. This represents the first study to our knowledge where archaeosomes have been evaluated in vivo as possible antigen carriers.

Animals↗

PubMed1999

Phylogenetic diversity of methanogenic archaea in swine waste storage pits.

Total DNA was isolated from swine feces and a swine waste storage pit and used as templates for PCR amplification of archaeal 16 rDNA using specific primers. Only the sample from the center of the waste pit produced a PCR product. DNA sequence analyses of random clones demonstrated a variety of methanogenic archaea. Six groups of sequences were identified, including those similar to Methanobrevibacter sp., Methanocorpusculum sp., and Methanoculleus sp. Three groups of sequences represented unidentified organisms. These data suggest that swine waste storage pits may represent an untapped source of novel methanogenic archaea.

Animals↗

PubMed1999

Phylogenetic relationships of symbiotic methanogens in diverse termites.

Termites harbor symbiotic microorganisms in their gut which emit methane. The phylogeny of the termite methanogens was inferred without cultivation based on nucleotide sequences of PCR-amplified 16S ribosomal RNA genes. Seven methanogen sequences from four termite species were newly isolated, and together with those previously published, these sequences were phylogenetically compared. The termite methanogen sequences were divided into three clusters. Two clusters of sequences, derived from the gut DNA of so-called higher termites, were related to methanogens in the orders Methanosarcinales or Methanomicrobiales. All of the sequences in the case of lower termites were closely related to the genus Methanobrevibacter. However, most of the termite symbionts were found to be distinct from known methanogens. They are not dispersed among diverse methanogen species, but rather formed unique lineages in the phylogenetic trees.

Animals↗