Date Published: August 23, 2017
Publisher: Springer Berlin Heidelberg
Author(s): Mohammad Ghashghavi, Mike S. M. Jetten, Claudia Lüke.
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.
Methane is the second most important greenhouse gas contributing to about 20% of global warming (Intergovernmental Panel on Climate Change 2014). The global methane budget is estimated to be around 600 Tg a−1 (Dubey 2001) which is dominated by biogenic sources, where natural wetlands (23%), and rice fields (21%) (Frenzel 2000) account for almost half of the total budget (Chen and Prinn 2005). Methanogenic archaea are assumed to be the sole producers of methane that reside in these environments (Cicerone and Oremland 1988; Conrad et al. 1999; Joulian et al. 1997). These archaea are also present in waste treatment systems, intestines of ruminants and termites and landfills acting as additional CH4 sources. Therefore, microbial methanogenic activity is responsible for nearly 75% of the methane emitted to the atmosphere (Chen and Prinn 2005).
The design of new primers was obtained by using all available pmoCAB operon sequences from MaGe. Interestingly, pmoB contained no conserved sequence stretch as a potential primer target site. Looking at the full operons, the only conserved regions resided within pmoC and pmoA. A new region at the nucleotide position 374 within the PmoC subunit of Methylococcus capsulatus (Bath), as a reference, was found to be highly conserved amongst all the phyla tested in this experiment. The forward primer binding site encodes for a glutamine residue at 126th base within the crystal structure of pmoC anchored to the membrane in Methylococcus capsulatus (Bath) whereas the reverse primer binding site encodes a phenylalanine residue at 107th base within pmoA. Our newly designed forward primer was compared to Holmes’ forward primer and the results are shown in Tables 1 and 2. As the tables illustrate, with zero mismatches, pmoC374 is able to target three out of the seven available sequences from Verrucomicrobia. If a single mismatch is allowed, five out of the seven sequences from Verrucomicrobia are targeted by pmoC374 whereas pmoA189 (Holmes et al. 1995a, b) with one mismatch still does not target any verrucomicrobial pMMO gene. The details of the novel primer set with regards to number of mismatches are listed in Table 2.
In the era of ‘omics’, molecular approaches using either specific or degenerate primers are still of high importance, especially in ecological studies where many samples need to be investigated or screened. They allow for a quick and robust detection of uncultivated microbes and aid in hypothesizing the community structure and the key processes that occur in certain environments, at the molecular level. As our knowledge and understanding of these environments expands, the tools that are used to investigate also need to be updated. More specifically, identification of the diverse organisms responsible for the oxidation of methane within various environments will help to better understand the key players involved in the methane cycle and evaluate their potential effectiveness as a biological methane filter. The currently available pmoA based primers are over 10 years old and since known MOB diversity has since been extended, a novel primer set with broader amplification ability would be highly beneficial in molecular studies. It is also important to distinguish between copper monooxygenases belonging to the AMO superfamily to ensure the detection of MOB and not AOB or the more recently discovered comammox (van Kessel et al. 2015; Pjevac et al. 2016; Pinto et al. 2015).