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Survey of methanotrophic diversity in various ecosystems by degenerate methane monooxygenase gene primers.

ABSTRACT: Methane is the second most important greenhouse gas contributing to about 20% of global warming. Its mitigation is conducted by methane oxidizing bacteria that act as a biofilter using methane as their energy and carbon source. Since their first discovery in 1906, methanotrophs have been studied using a complementary array of methods. One of the most used molecular methods involves PCR amplification of the functional gene marker for the diagnostic of copper and iron containing particulate methane monooxygenase. To investigate the diversity of methanotrophs and to extend their possible molecular detection, we designed a new set of degenerate methane monooxygenase primers to target an 850 nucleotide long sequence stretch from pmoC to pmoA. The primers were based on all available full genomic pmoCAB operons. The newly designed primers were tested on various pure cultures, enrichment cultures and environmental samples using PCR. The results demonstrated that this primer set has the ability to correctly amplify the about 850 nucleotide long pmoCA product from Alphaproteobacteria, Gammaproteobacteria, Verrucomicrobia and the NC10 phyla methanotrophs. The new primer set will thus be a valuable tool to screen ecosystems and can be applied in conjunction with previously used pmoA primers to extend the diversity of currently known methane-oxidizing bacteria.

SUBMITTER: Ghashghavi M

PROVIDER: S-EPMC5567572 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Survey of methanotrophic diversity in various ecosystems by degenerate methane monooxygenase gene primers.

Ghashghavi Mohammad M Jetten Mike S M MSM Lüke Claudia C

AMB Express 20170823 1

Methane is the second most important greenhouse gas contributing to about 20% of global warming. Its mitigation is conducted by methane oxidizing bacteria that act as a biofilter using methane as their energy and carbon source. Since their first discovery in 1906, methanotrophs have been studied using a complementary array of methods. One of the most used molecular methods involves PCR amplification of the functional gene marker for the diagnostic of copper and iron containing particulate methan ...[more]

PMID: 28831762

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Project description:The anaerobic oxidation of methane (AOM) is a key biogeochemical process regulating methane emission from marine sediments into the hydrosphere. AOM is largely mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), and has mainly been investigated in deep-sea sediments. Here we studied methane seepage at four spots located at 12 m water depth in coastal, organic carbon depleted permeable sands off the Island of Elba (Italy). We combined biogeochemical measurements, sequencing-based community analyses and in situ hybridization to investigate the microbial communities of this environment. Increased alkalinity, formation of free sulfide and nearly stoichiometric methane oxidation and sulfate reduction rates up to 200 nmol g(-1) day(-1) indicated the predominance of sulfate-coupled AOM. With up to 40 cm thickness the zones of AOM activity were unusually large and occurred in deeper sediment horizons (20-50 cm below seafloor) as compared to diffusion-dominated deep-sea seeps, which is likely caused by advective flow of pore water due to the shallow water depth and permeability of the sands. Hydrodynamic forces also may be responsible for the substantial phylogenetic and unprecedented morphological diversity of AOM consortia inhabiting these sands, including the clades ANME-1a/b, ANME-2a/b/c, ANME-3, and their partner bacteria SEEP-SRB1a and SEEP-SRB2. High microbial dispersal, the availability of diverse energy sources and high habitat heterogeneity might explain that the emission spots shared few microbial taxa, despite their physical proximity. Although the biogeochemistry of this shallow methane seep was very different to that of deep-sea seeps, their key functional taxa were very closely related, which supports the global dispersal of key taxa and underlines strong selection by methane as the predominant energy source. Mesophilic, methane-fueled ecosystems in shallow-water permeable sediments may comprise distinct microbial habitats due to their unique biogeochemical and physical characteristics. To link AOM phylotypes with seep habitats and to enable future meta-analyses we thus propose that seep environment ontology needs to be further specified.

Project description:Deep-sea mussels of the genus Bathymodiolus are numerically dominant macrofauna in many cold seep and hydrothermal vent ecosystems worldwide, and they depend on organic carbon produced by symbionts present in the epithelial cells of the gills. Although Bathymodiolus platifrons represents typical methanotrophic endosymbiosis, our understanding of molecular mechanisms of methane oxidization and carbon fixation is still in its infancy. Moreover, the laboratory maintenance of B. platifrons and the symbiont abundance dynamics during maintenance has not been reported. In the present study, we report the first systematic identification and phylogenetic analysis of three subunits of methane monooxygenase (pmoA, pmoB, and pmoC) obtained from the endosymbiotic bacteria found in B. platifrons. The coding sequences (CDS) of the three genes in the B. platifrons endosymbiont were 750, 1,245, and 753 bp, encoding 249, 414, and 250 amino acids, respectively. Sequence alignment and phylogenetic analysis revealed that the symbiont of B. platifrons belongs to the type I methanotrophs. In order to clarify the impact of environmental methane on symbiont abundance, a 34-day laboratory maintenance experiment was conducted in which B. platifrons individuals were acclimatized to methane-present and methane-absent environments. Symbiont abundance was evaluated by calculating the relative DNA content of the methane monooxygenase gene using quantitative real-time PCR. We found that symbiont quantity immediately decreased from its initial level, then continued to gradually decline during maintenance. At 24 and 34 days of maintenance, symbiont abundance in the methane-absent environment had significantly decreased compared to that in the methane-present environment, indicating that the maintenance of symbionts relies on a continuous supply of methane. Our electron microscopy results validated the qPCR analysis. This study enriches our knowledge of the molecular basis and the dynamic changes of the methanotrophic endosymbiosis in B. platifrons, and provides a feasible model biosystem for further investigation of methane oxidization, the carbon fixation process, and environmental adaptations of deep-sea mussels.

Dataset Information

Survey of methanotrophic diversity in various ecosystems by degenerate methane monooxygenase gene primers.

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Survey of methanotrophic diversity in various ecosystems by degenerate methane monooxygenase gene primers.

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