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: The results show that the influence of predation by protists in anaerobic engineered systems needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.
Abstract: Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates-ciliates frequently found in anoxic ecosystems-on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885-3,190 and 2,387-2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.
21 citations
Cites methods or result from "Methane production from protozoan e..."
...In one UASB reactor, the proliferation of protists was inhibited by the addition of cycloheximide (1 g L–1) at the start of operation (day 0), as described previously (32, 35)....
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...Methane gas production was more prominent in the coculture reactor; this result is consistent with previous findings (8, 32)....
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...Cycloheximide has traditionally been employed to prepare prokaryotic cultures without eukaryotes (32, 33, 35, 58)....
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TL;DR: Results suggest that anode biofilms are an attractive food source for protozoa and that protozoan grazing can be an important factor limiting the current output of sediment microbial fuel cells.
13 citations
Cites background from "Methane production from protozoan e..."
...This concentration of inhibitors was selected because it has previously been shown to inhibit protozoan growth in sediment microcosms (Holmes et al., 2014; Schwarz and Frenzel, 2005)....
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TL;DR: In this article, the authors isolated three anaerobic protists, Cyclidium sp., Trichomitus sp., and Paracercomonas sp., from an up-flow sludge blanket reactor used to treat domestic sewage.
Abstract: Anaerobic protists are major predators of prokaryotes in anaerobic ecosystems. However, little is known about the predation behavior of anaerobic protists because almost none have been cultured. In particular, these characteristics of anaerobic protists in the phyla Metamonada and Cercozoa have not been reported previously. In this study, we isolated three anaerobic protists, Cyclidium sp., Trichomitus sp., and Paracercomonas sp., from anaerobic granular sludge in an up-flow anaerobic sludge blanket reactor used to treat domestic sewage. Ingestion and digestion of food bacteria by anaerobic protists with or without endosymbiotic methanogens were demonstrated using tracer experiments with green fluorescent protein and a stable carbon isotope. These tracer experiments also demonstrated that Cyclidium sp. supplied CO2 and hydrogen to endosymbiotic methanogens. While Cyclidium sp. and Trichomitus sp. ingested both Gram-negative and -positive bacteria, Paracercomonas sp. could only take up Gram-negative bacteria. Archaeal cells such as Methanobacterium beijingense and Methanospirillum hungatei did not support the growth of these protists. Metabolite patterns of all three protists differed and were influenced by food bacterial species. These reported growth rates, ingestion rates, food selectivity, and metabolite patterns provide important insights into the ecological roles of these protists in anaerobic ecosystems.
12 citations
TL;DR: Central OTUs from soil bacterial community networks were also persistent OTUs, thus confirming the existence of a core bacterial community and that these same OTUs could plastically interact according to the perturbation type to quickly stabilize bacterial communities undergoing succession.
10 citations
TL;DR: The current body of knowledge regarding the complex nature of symbioses in anaerobic ciliates, the diversity of these symbionts, their role in the evolution of ciliate anaerobiosis and their significance in ecosystem‐level processes are summarized.
Abstract: Anaerobiosis has independently evolved in multiple lineages of ciliates, allowing them to colonize a variety of anoxic and oxygen‐depleted habitats. Anaerobic ciliates commonly form symbiotic relationships with various prokaryotes, including methanogenic archaea and members of several bacterial groups. The hypothesized functions of these ecto‐ and endosymbionts include the symbiont utilizing the ciliate's fermentative end products to increase the host's anaerobic metabolic efficiency, or the symbiont directly providing the host with energy by denitrification or photosynthesis. The host, in turn, may protect the symbiont from competition, the environment, and predation. Despite rapid advances in sampling, molecular, and microscopy methods, as well as the associated broadening of the known diversity of anaerobic ciliates, many aspects of these ciliate symbioses, including host specificity and coevolution, remain largely unexplored. Nevertheless, with the number of comparative genomic and transcriptomic analyses targeting anaerobic ciliates and their symbionts on the rise, insights into the nature of these symbioses and the evolution of the ciliate transition to obligate anaerobiosis continue to deepen. This review summarizes the current body of knowledge regarding the complex nature of symbioses in anaerobic ciliates, the diversity of these symbionts, their role in the evolution of ciliate anaerobiosis and their significance in ecosystem‐level processes.
8 citations
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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