Comparison of pmoA PCR Primer Sets as Tools for Investigating Methanotroph Diversity in Three Danish Soils

Abstract

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.

Diversity of Aerobic Methanotrophic Bacteria in a Permafrost Active Layer Soil of the Lena Delta, Siberia

“…Our methodological approach is based on primer combinations that were designed to detect both 16S rRNA and pmoA gene sequences of known and unknown type I as well as type II MOB [3,8,59]. These primer combinations were used in several studies where type I and type II MOB were detected either based on the 16S rRNA gene [57], based on the pmoA gene [29,42], or based on both genes [28,44].…”

One millimetre makes the difference: high-resolution analysis of methane-oxidizing bacteria and their specific activity at the oxic–anoxic interface in a flooded paddy soil

“…Compared with the 682r reverse primer (Holmes et al, 1995), the reverse primer mb661r (Costello and Lidstrom, 1999;Bourne et al, 2001) covers methanotroph diversity, but not the homologous amoA gene encoding for a subunit of ammonium monooxygenase. Furthermore, primer mb661r seems to be superior for resolving type I diversity (Bourne et al, 2001;Lü ke et al, 2010). Table 1 Average number of pmoA gene copies and transcripts in the three different depth zones defined in Figure 4 Depth zone (mm)…”

Response of methanotrophic communities to afforestation and reforestation in New Zealand

“…Response of methanotrophic communities L Nazaries et al using terminal-restriction fragment length polymorphism of particulate methane monooxygenase (pmoA) genes using the primer pair A189-mb650 (Bourne et al, 2001). Cloning and sequencing of pmoA genes from different samples of Turangi shrublands and Puruki-Native confirmed that terminal-restriction fragment (T-RF) 245 belonged to type-I methanotrophs and a close relative of Methylococcus capsulatus, while T-RF 33 and T-RF 129 were distant relatives of the uncultured clade USCa (a distant relative of cultured Methylocapsa sp., a type-II methanotroph) Table 2).…”