Methane production from protozoan endosymbionts following stimulation of microbial metabolism within subsurface sediments
TL;DR: Results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
Abstract: Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and s-tubulin sequences from this phase of an in situ uranium bioremediation field experiment were analyzed. Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus s-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus s-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
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TL;DR: This study sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River and reconstructed the first complete genomes for Archaea using cultivation-independent methods, which dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum.
Abstract: Summary Background Archaea represent a significant fraction of Earth's biodiversity, yet they remain much less well understood than Bacteria. Gene surveys, a few metagenomic studies, and some single-cell sequencing projects have revealed numerous little-studied archaeal phyla. Certain lineages appear to branch deeply and may be part of a major phylum radiation. The structure of this radiation and the physiology of the organisms remain almost unknown. Results We used genome-resolved metagenomic analyses to investigate the diversity, genomes sizes, metabolic capacities, and potential roles of Archaea in terrestrial subsurface biogeochemical cycles. We sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River (USA) and reconstructed the first complete genomes for Archaea using cultivation-independent methods. To provide taxonomic context, we analyzed an additional 151 newly sampled archaeal sequences. We resolved two new phyla within a major, apparently deep-branching group of phyla (a superphylum). The organisms have small genomes, and metabolic predictions indicate that their primary contributions to Earth's biogeochemical cycles involve carbon and hydrogen metabolism, probably associated with symbiotic and/or fermentation-based lifestyles. Conclusions The results dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum. This study, in combination with recently published work on bacterial phyla lacking cultivated representatives, reveals a fascinating phenomenon of major radiations of organisms with small genomes, novel proteome composition, and strong interdependence in both domains.
463 citations
Cites background from "Methane production from protozoan e..."
...Although evidence for their activity has been detected during in situ acetate amendment [27], methanogenic Archaea did not appear to be abundant in the filtrate....
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TL;DR: The results suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp.
Abstract: The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.
229 citations
Cites methods from "Methane production from protozoan e..."
...Sulfate reduction was monitored with an ion chromatograph (ICS-2100; Dionex, CA) equipped with an AS18 column under isocratic elution with 32 mM KOH as the eluent (57)....
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TL;DR: The current understanding of the archaeome in humans, the specific adaptations involved in interaction with the resident microbial community as well as with the host, and the roles of the archaeological community in both health and disease are presented.
Abstract: Host-associated microbial communities have an important role in shaping the health and fitness of plants and animals. Most studies have focused on the bacterial, fungal or viral communities, but often the archaeal component has been neglected. The archaeal community, the so-called archaeome, is now increasingly recognized as an important component of host-associated microbiomes. It is composed of various lineages, including mainly Methanobacteriales and Methanomassiliicoccales (Euryarchaeota), as well as representatives of the Thaumarchaeota. Host–archaeome interactions have mostly been delineated from methanogenic archaea in the gastrointestinal tract, where they contribute to substantial methane production and are potentially also involved in disease-relevant processes. In this Review, we discuss the diversity and potential roles of the archaea associated with protists, plants and animals. We also present the current understanding of the archaeome in humans, the specific adaptations involved in interaction with the resident microbial community as well as with the host, and the roles of the archaeome in both health and disease. The archaeal community, the archaeome, is now increasingly recognized as an important component of host-associated microbiomes. In this Review, Moissl-Eichinger and colleagues discuss the diversity and potential roles of the archaea associated with protists, plants and animals, highlighting the potential roles of archaea in human health and disease.
106 citations
12 Feb 2019
TL;DR: Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production.
Abstract: Recent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH4) production from beef cattle by up to ~ 99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH4 without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US. Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH4 in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed. The methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted.
86 citations
Cites background from "Methane production from protozoan e..."
...It is well known that there is a mutualistic relationship between protozoa and methanogens [33, 34], and it has been shown before that the removal of rumen protozoa results in a reduction of the methanogen population and methanogenesis during enteric fermentation [35, 36]....
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TL;DR: These studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea, and suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the ext racellular environment.
Abstract: Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea. IMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.
63 citations
Cites methods from "Methane production from protozoan e..."
...Methane in the headspace was measured by gas chromatography with a flame ionization detector (Shimadzu, GC-8A) as previously described (85)....
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References
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TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
70,111 citations
TL;DR: ClUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W, providing an integrated system for performing multiple sequence and profile alignments and analysing the results.
Abstract: CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
38,522 citations
"Methane production from protozoan e..." refers methods in this paper
...Alignments were made in ClustalX (Thompson et al., 1997) and corrected with ProSeq v2....
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TL;DR: An advanced version of the Molecular Evolutionary Genetics Analysis software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, is released, which enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
37,956 citations
"Methane production from protozoan e..." refers methods in this paper
...9 (Filatov, 2002) before phylogenetic trees were constructed with Mega v6 (Tamura et al., 2013)....
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01 Jan 2013
TL;DR: The Molecular Evolutionary Genetics Analysis (MEGA) software as discussed by the authors provides facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, including the inference of timetrees.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www. megasoftware.net free of charge.
30,478 citations
TL;DR: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.
Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.
Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement.
Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
12,469 citations
"Methane production from protozoan e..." refers methods in this paper
...Transcript abundances and qPCR efficiencies (90–99%) were calculated from appropriate standard curves and all qPCR experiments followed MIQE guidelines (Bustin et al., 2009)....
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...%) were calculated from appropriate standard curves and all qPCR experiments followed MIQE guidelines (Bustin et al., 2009)....
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