This is the more recent paper I mentioned https://www.nature.com/articles/s43705-021-00026-y#citeas

Just to update.....

Papers assuming 2 copies of the pmoA per cell (McDonald & Murrell 1997; Kolb et al. 2003; Kolb et al. 2005; Stolyar et al. 1999; Semrau et al. 1995; Chiri et al. 2020). However this is based largely on work by Semrau et al., (1995)and Stolyar et al., (1999), which concentrate on two methanotroph species (mainly Methylococcus capsulatus). The paper by Semrau et al., (1995) suggests at least 1 copy of pmoA, whilst pmoB was found in duplicate.

There are papers stating pmoA is a single copy gene (Deutzmann et al. 2014; Ettwig et al. 2010), nonetheless there are also recent papers claiming more than one copy. Importantly for me, they have accounted for this when calculating cell specific methane oxidation (Nijman et al. 2021; Chiri et al. 2020). Nijman et al., (2021) suggest mean pmoA copies per cell = 1.62 (no reference provided unfortunately), whereas Chiri et al., (2020) assume duplicate copies.

Taking all this research into account, I will consider 1.5 copies per cell, but plot data with 1, 1.5 and 2 copies per cell to discuss.

References

Chiri, E. et al., 2020. Termite mounds contain soil-derived methanotroph communities kinetically adapted to elevated methane concentrations. The ISME Journal, 14(11), pp.2715–2731. Available at: https://doi.org/10.1038/s41396-020-0722-3.

Deutzmann, J.S. et al., 2014. Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake. Proceedings of the National Academy of Sciences, 111(51), p.18273 LP-18278. Available at: http://www.pnas.org/content/111/51/18273.abstract.

Ettwig, K. et al., 2010. Nitrite-driven anaerobic methane oxdation by oxygenic bacteria. Nature, 464, pp.543–548.

Kolb, S. et al., 2005. Abundance and activity of uncultured methanotrophic bacteria involved in the consumption of atmospheric methane in two forest soils. Environmental microbiology, 7 8, pp.1150–1161.

Kolb, S. et al., 2003. Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays. Applied and Environmental Microbiology, 69(5), pp.2423–2429. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154495/.

McDonald, I.R. & Murrell, J.C., 1997. The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs. FEMS Microbiology Letters, 156(2), pp.205–210. Available at: https://doi.org/10.1111/j.1574-6968.1997.tb12728.x.

Nijman, T.P.A. et al., 2021. Warming and eutrophication interactively drive changes in the methane-oxidizing community of shallow lakes. ISME Communications, 1(1), p.32. Available at: https://doi.org/10.1038/s43705-021-00026-y.

Semrau, J.D. et al., 1995. Particulate methane monooxygenase genes in methanotrophs. Journal of bacteriology, 177(11), pp.3071–3079. Available at: https://pubmed.ncbi.nlm.nih.gov/7768803.

Stolyar, S. et al., 1999. Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath. Microbiology, 145(5), pp.1235–1244. Available at: https://www.microbiologyresearch.org/content/journal/micro/10.1099/13500872-145-5-1235.

Hi Kate

If you have a DNA extract of pure culture and a qPCR method for the pmoA gene and another target that you know as a single copy, you can consider the following approach.

The distribution of positive PCR at very log copy numbers is different when you have two copies instead of one. It would help if you considered the Poison distribution of results in microbiology counts.

You have to do a dilution banc and a 10 PCR of the lowest levels, including a dilution where all the PCR are negatives for both targets. In the previous dilution to those with zero results in all replicates, you will find different positive ratios if the copy number is different. This experiment needs to be done several times to show significant results.

best regards