Print Email Facebook Twitter McrA primers for the detection and quantification of the anaerobic archaeal methanotroph ‘Candidatus Methanoperedens nitroreducens’ Title McrA primers for the detection and quantification of the anaerobic archaeal methanotroph ‘Candidatus Methanoperedens nitroreducens’ Author Vaksmaa, Annika (Radboud Universiteit Nijmegen) Jetten, M.S.M. (TU Delft BT/Environmental Biotechnology; Radboud Universiteit Nijmegen; Soehngen Institute of Anaerobic Microbiology) Ettwig, Katharina F. (Radboud Universiteit Nijmegen) Lüke, Claudia (Radboud Universiteit Nijmegen) Date 2017-02-01 Abstract The nitrogen and methane cycles are important biogeochemical processes. Recently, ‘Candidatus Methanoperedens nitroreducens,’ archaea that catalyze nitrate-dependent anaerobic oxidation of methane (AOM), were enriched, and their genomes were analyzed. Diagnostic molecular tools for the sensitive detection of ‘Candidatus M. nitroreducens’ are not yet available. Here, we report the design of two novel mcrA primer combinations that specifically target the alpha sub-unit of the methyl-coenzyme M reductase (mcrA) gene of ‘Candidatus M. nitroreducens’. The first primer pair produces a fragment of 186-bp that can be used to quantify ‘Candidatus M. nitroreducens’ cells, whereas the second primer pair yields an 1191-bp amplicon that is with sufficient length and well suited for more detailed phylogenetic analyses. Six different environmental samples were evaluated with the new qPCR primer pair, and the abundances were compared with those determined using primers for the 16S rRNA gene. The qPCR results indicated that the number of copies of the ‘Candidatus M. nitroreducens’ mcrA gene was highest in rice field soil, with 5.6 ± 0.8 × 106 copies g−1 wet weight, whereas Indonesian river sediment had only 4.6 ± 2.7 × 102 copies g−1 wet weight. In addition to freshwater environments, sequences were also detected in marine sediment of the North Sea, which contained approximately 2.5 ± 0.7 × 104 copies g−1 wet weight. Phylogenetic analysis revealed that the amplified 1191-bp mcrA gene sequences from the different environments all clustered together with available genome sequences of mcrA from known ‘Candidatus M. nitroreducens’ archaea. Taken together, these results demonstrate the validity and utility of the new primers for the quantitative and sensitive detection of the mcrA gene sequences of these important nitrate-dependent AOM archaea. Furthermore, the newly obtained mcrA sequences will contribute to greater phylogenetic resolution of ‘Candidatus M. nitroreducens’ sequences, which have been only poorly captured by general methanogenic mcrA primers. Subject Anaerobic oxidation of methaneANMEmcrA‘Candidatus Methanoperedens nitroreducens’ To reference this document use: http://resolver.tudelft.nl/uuid:491127c5-2c9c-44da-a7b1-bc491292e1ed DOI https://doi.org/10.1007/s00253-016-8065-8 ISSN 0175-7598 Source Applied Microbiology and Biotechnology, 101 (4), 1631-1641 Part of collection Institutional Repository Document type journal article Rights © 2017 Annika Vaksmaa, M.S.M. Jetten, Katharina F. Ettwig, Claudia Lüke Files PDF 10.1007_2Fs00253_016_8065_8.pdf 714.74 KB Close viewer /islandora/object/uuid:491127c5-2c9c-44da-a7b1-bc491292e1ed/datastream/OBJ/view