The diversity of the methanogenic archaea associated with the six segments of the horse and donkey hindgut (caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and rectum) was analyzed using 16S rDNA gene clone library. A total of 641 positive clones, 321 from the horse and 320 from the donkey hindgut, were examined by the RFLP, revealing 9 different ribotypes, 8 in the horse and 5 in the donkey hindgut. In both the animals Methanobacteriales (Methanobrevibacter-like sequences) and Methanomicrobiales (Methanocorpusculum-like sequences) were detected as the dominant orders followed by the uncultured Methanomassiliicoccales. The composition of the equine archaeal community was found to be dependent on the gut region. In both the two animals no Methanobrevibacter-like clones were detected in the caeca, which were instead inhabited by the Methanocorpusculum-like archeons. The Methanosarcinales were found only in distal regions of the horse hindgut.
- MeSH
- Archaea klasifikace genetika izolace a purifikace metabolismus MeSH
- biodiverzita MeSH
- DNA archebakterií genetika MeSH
- Equidae mikrobiologie MeSH
- fylogeneze MeSH
- koně mikrobiologie MeSH
- methan metabolismus MeSH
- ribozomální DNA genetika MeSH
- tlusté střevo mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Studies on methanogenesis from freshwater sediments have so far primarily focused on lake sediments. To expand our knowledge on the community composition of methanogenic archaea in river sediments, we studied the abundance and diversity of methanogenic archaea at two localities along a vertical profile (top 50 cm) obtained from sediment samples from Sitka stream (the Czech Republic). In this study, we compare two sites which previously have been shown to have a 10-fold different methane emission. Archaeal and methanogen abundance were analyzed by real-time PCR and T-RFLP. Our results show that the absolute numbers for the methanogenic community (qPCR) are relatively stable along a vertical profile as well as for both study sites. This was also true for the archaeal community and for the three major methanogenic orders in our samples (Methanosarcinales, Methanomicrobiales, and Methanobacteriales). However, the underlying community structure (T-RFLP) reveals different community compositions of the methanogens for both locations as well as for different depth layers and over different sampling times. In general, our data confirm that Methanosarcinales together with Methanomicrobiales are the two dominant methanogenic orders in river sediments, while members of Methanobacteriales contribute a smaller community and Methanocellales are only rarely present in this sediment. Our results show that the previously observed 10-fold difference in methane emission of the two sites could not be explained by molecular methods alone.
- MeSH
- Archaea klasifikace genetika metabolismus MeSH
- biodiverzita * MeSH
- DNA archebakterií genetika MeSH
- geologické sedimenty mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- methan metabolismus MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- řeky mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake.
- MeSH
- amoniak metabolismus MeSH
- Archaea klasifikace genetika metabolismus MeSH
- biodiverzita * MeSH
- DNA archebakterií chemie genetika MeSH
- fylogeneze MeSH
- geologické sedimenty mikrobiologie MeSH
- hybridizace in situ fluorescenční MeSH
- jezera mikrobiologie MeSH
- molekulární sekvence - údaje MeSH
- oxidace-redukce MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.
- MeSH
- Archaea genetika izolace a purifikace MeSH
- Bacteria genetika izolace a purifikace MeSH
- chemické techniky analytické metody MeSH
- DNA archebakterií genetika izolace a purifikace MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- DNA fungální genetika izolace a purifikace MeSH
- houby genetika izolace a purifikace MeSH
- mikrobiologie životního prostředí MeSH
- polymerázová řetězová reakce MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5-6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.
- MeSH
- denaturační gradientová gelová elektroforéza MeSH
- DNA archebakterií chemie genetika MeSH
- fylogeneze MeSH
- geologické sedimenty mikrobiologie MeSH
- Methanosarcinales izolace a purifikace MeSH
- molekulární sekvence - údaje MeSH
- řeky MeSH
- ribozomální DNA chemie genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- shluková analýza MeSH
- společenstvo * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.
- MeSH
- anaerobióza MeSH
- bachor mikrobiologie MeSH
- DNA archebakterií genetika MeSH
- DNA fungální genetika MeSH
- feces mikrobiologie MeSH
- fermentace MeSH
- fylogeneze MeSH
- konfokální mikroskopie MeSH
- kozy mikrobiologie MeSH
- methan biosyntéza MeSH
- Methanobacteriales klasifikace genetika izolace a purifikace metabolismus MeSH
- mezerníky ribozomální DNA genetika MeSH
- Neocallimastigomycota klasifikace genetika izolace a purifikace metabolismus MeSH
- polymerázová řetězová reakce MeSH
- sekvenční analýza DNA MeSH
- symbióza fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5-3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.
- MeSH
- Archaea genetika izolace a purifikace klasifikace MeSH
- biodiverzita MeSH
- Chiroptera mikrobiologie MeSH
- DNA archebakterií genetika MeSH
- ekosystém MeSH
- feces chemie mikrobiologie MeSH
- financování organizované MeSH
- fylogeneze MeSH
- molekulární sekvence - údaje MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 16S genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Geografické názvy
- Slovenská republika MeSH
The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.
- MeSH
- anaerobióza MeSH
- Archaea genetika izolace a purifikace klasifikace růst a vývoj MeSH
- bachor mikrobiologie parazitologie MeSH
- Ciliophora genetika izolace a purifikace růst a vývoj MeSH
- DNA archebakterií analýza izolace a purifikace MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- kultivační média MeSH
- molekulární sekvence - údaje MeSH
- ovce MeSH
- protozoální DNA analýza izolace a purifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- symbióza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
