RESUMEN
The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.
Asunto(s)
Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Methanobacteriaceae/genética , Adhesión Bacteriana/efectos de los fármacos , Proteínas Bacterianas/química , Cationes Bivalentes/farmacología , Endopeptidasa K/farmacología , Genoma Bacteriano/genética , Calor , Metano/metabolismo , Methanobacteriaceae/clasificación , Methanobacteriaceae/ultraestructura , Mutación , Filogenia , Dominios Proteicos , Análisis de Secuencia de ADNRESUMEN
Several thermophilic hydrogenotrophic methanogens naturally aggregate in their habitats in association with hydrogen-producing bacteria for efficient transfer of the methane fermentation intermediates to produce methane. However, physiology of aggregation and the identity of aggregation-specific genes remain to be elucidated. Here, we isolated and characterized a hydrogen and formate-utilizing Methanothermobacter sp. CaT2 that is capable of self-aggregation and utilizing formate. CaT2 produced methane from propionate oxidation in association with a syntrophic propionate-oxidizing bacterium faster than other methanogens, including Methanothermobacter thermautotrophicus ΔH and Methanothermobacter thermautotrophicusâ Z-245. CaT2 also aggregated throughout the culture period and was coated with polysaccharides, which was not found on the ΔH and Z-245 cells. Sugar content (particularly of rhamnose and mannose) was also higher in the CaT2 cells than the ΔH and Z-245 cells. Comparative genomic analysis of CaT2 indicated that four candidate genes, all of which encode glycosyltransferase, were involved in aggregation of CaT2. Transcriptional analysis showed that one glycosyltransferase gene was expressed at relatively high levels under normal growth conditions. The polysaccharide layer on the CaT2 cell surface, which is probably assembled by these glycosyltransferases, may be involved in cell aggregation.
Asunto(s)
Methanobacteriaceae/fisiología , Propionatos/metabolismo , Adhesión Bacteriana/genética , Perfilación de la Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Hidrógeno/metabolismo , Metano/metabolismo , Methanobacteriaceae/aislamiento & purificación , Methanobacteriaceae/ultraestructura , Monosacáridos/metabolismo , Oxidación-Reducción , Transcripción GenéticaRESUMEN
BACKGROUND: Several methanogenic archaea have been detected in the human intestinal microbiota. These intestinal archaea may contaminate medical devices such as colonoscopes. However, no biocide activity has been reported among these human-associated archaea. METHODOLOGY: The minimal archaeacidal concentration (MAC) of peracetic acid, chlorhexidine, squalamine and twelve parent synthetic derivatives reported in this study was determined against five human-associated methanogenic archaea including Methanobrevibacter smithii, Methanobrevibacter oralis, Methanobrevibacter arboriphilicus, Methanosphaera stadtmanae, Methanomassiliicoccus luminyensis and two environmental methanogens Methanobacterium beijingense and Methanosaeta concilii by using a serial dilution technique in Hungates tubes. PRINCIPAL FINDINGS: MAC of squalamine derivative S1 was 0.05 mg/L against M. smithii strains, M. oralis, M. arboriphilicus, M. concilii and M. beijingense whereas MAC of squalamine and derivatives S2-S12 varied from 0.5 to 5 mg/L. For M. stadtmanae and M. luminyensis, MAC of derivative S1 was 0.1 mg/L and varied from 1 to ≥ 10 mg/L for squalamine and its parent derivatives S2-S12. Under the same experimental conditions, chlorhexidine and peracetic acid lead to a MAC of 0.2 and 1.5 mg/L, respectively against all tested archaea. CONCLUSIONS/SIGNIFICANCE: Squalamine derivative S1 exhibited a 10-200 higher archaeacidal activity than other tested squalamine derivatives, on the majority of human-associated archaea. As previously reported and due to their week corrosivity and their wide spectrum of antibacterial and antifungal properties, squalamine and more precisely derivative S1 appear as promising compounds to be further tested for the decontamination of medical devices contaminated by human-associated archaea.
Asunto(s)
Colestanoles/farmacología , Desinfectantes/farmacología , Methanobacteriaceae/efectos de los fármacos , Clorhexidina/química , Clorhexidina/farmacología , Colestanoles/síntesis química , Colestanoles/química , Medios de Cultivo , Desinfectantes/química , Humanos , Methanobacteriaceae/crecimiento & desarrollo , Methanobacteriaceae/ultraestructura , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Estructura Molecular , Ácido Peracético/química , Ácido Peracético/farmacología , Relación Estructura-ActividadRESUMEN
Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI:http://dx.doi.org/10.7554/eLife.00218.001.
Asunto(s)
Proteínas Arqueales/química , Microscopía por Crioelectrón , Hidrogenasas/química , Methanobacteriaceae/enzimología , Riboflavina/análogos & derivados , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Proteínas Arqueales/ultraestructura , Sitios de Unión , Hidrogenasas/metabolismo , Hidrogenasas/ultraestructura , Methanobacteriaceae/clasificación , Methanobacteriaceae/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Unión Proteica , Estructura Cuaternaria de Proteína , Riboflavina/química , Riboflavina/metabolismoRESUMEN
A morphologically distinct, filamentous methanogen was isolated from hindguts of the subterranean termite, Reticulitermes flavipes (Kollar) (Rhinotermitidae), wherein it was part of the microbiota colonizing the hindgut wall. Individual filaments of strain RFM-3 were 0.23-0.28 micron in diameter and usually > 50 micron in length and aggregated into flocs that were often >/= 0.1 mm in diameter. Optimal growth of strain RFM-3 was obtained at pH 7.0-7.2 and 30 degrees C with a yeast-extract-supplemented, dithiothreitol-reduced medium in which cells produced stoichiometric amounts of methane from H2 + CO2. The morphology and gram-positive staining reaction of strain RFM-3, as well as its resistance to cell lysis by various chemical agents and its restriction to H2 + CO2 as an energy source, suggested that it was a member of the Methanobacteriaceae. The nucleotide sequence of the SSU-rRNA-encoding gene of strain RFM-3 confirmed this affiliation and also supported its recognition as a new species of Methanobrevibacter, for which the epithet filiformis is herewith proposed. Although M. filiformis was one of the dominant methanogens in R. flavipes collected from Woods Hole (Mass., USA), cells of similar morphology were not consistently observed in R. flavipes collected from different geographical locations.
Asunto(s)
Euryarchaeota/aislamiento & purificación , Insectos/microbiología , Intestinos/microbiología , Methanobacteriaceae/aislamiento & purificación , Animales , Euryarchaeota/genética , Euryarchaeota/ultraestructura , Methanobacteriaceae/genética , Methanobacteriaceae/ultraestructura , Filogenia , ARN de Archaea/análisis , ARN de Archaea/genética , ARN Ribosómico/análisis , ARN Ribosómico/genéticaRESUMEN
The flagellins of Methanospirillum hungatei strains JF1 and GP1, Methanococcus deltae, and Methanothermus fervidus are glycosylated. Isolated flagellar filaments from these organisms are dissociated by low concentrations (0.5% (v/v)) of Triton X-100. Flagellar filaments from other methanogens (Methanococcus voltae, Methanococcus vannielii and Methanoculleus marisnigri) composed of non-glycosylated flagellins are resistant to Triton X-100 treatment. Consequently, the isolation techniques (employing Triton X-100) used to isolate basal body-hook-filament complexes in eubacteria may not be applicable to many methanogens.