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Supplementary Information
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Environmental distribution and abundance of the facultative
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methanotroph Methylocella
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Md.Tanvir Rahman, Andrew Crombie, Yin Chen, Nancy Stralis-Pavese, Levente Bodrossy,
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Patrick Meir, Niall P. McNamara, and J. Colin Murrell
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Materials and methods
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PCR protocol for amplification of Methylocella 16S rRNA and mmoX genes
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DNA was extracted from soils as described by Chen et al. (2007). Around 30 ng of the total
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extracted DNA was used as the template for PCR to amplify 16S rRNA and mmoX genes.
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16S rRNA genes related to Methylocella were amplified using nested PCR. The first round
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(30 cycles), using primers 27f/1492r (Lane 1991), was followed by 30 cycles using primers
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Type IIF (5´-GGGAMGATAATGACGGTACCWGGA-3´) and Mcell-1445 (5´-
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CCTCTCTCCTTGCGGTT-3´), with 1 μl of the first round product as template. In some
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cases one round was sufficient, and it was possible to dispense with the first (non-specific)
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round of PCR. Initially the presence of substances that inhibit PCR, such as humic acids, was
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ruled out by amplifying bacterial 16S rRNA genes from DNA (30 ng) extracted from all the
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samples tested here using primers 27f/1492r. The cycling conditions were 94° C for 5 min,
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followed by 30 cycles of 94° C for 1 min, 55° C (primers 27f/1492r) or 63° C (primers Type
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IIF/Mcell-1445) for 1 min, 72° C for 1 min, with a final extension at 72° C for 10 min.
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Methylocella genus-specific mmoX forward primer mmoXLF (5´-GAAGATTGG
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GGCGGCATCTG -3´) and reverse primer mmoXLR (5´-
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CCCAATCATCGCTGAAGGAGT -3´) were designed to amplify mmoX from Methylocella
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spp. Initially all the available mmoX gene sequences covering both type I and type II
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methanotrophs were downloaded from the GenBank database and analysed using the ARB
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software package (Ludwig et al., 2004). The sequence alignment was manually verified for
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alignment accuracy. Potential primers were identified and their specificity tested using the
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Probe Match function of ARB. The primers were analysed for hairpin structures and potential
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duplex formation using the OLIGO 6 program (http://www.oligo.net/oligo.htm). For the
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amplification of Methylocella mmoX by conventional PCR, two consecutive rounds of PCR
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were adopted using primers mmoXLF and mmoXLR, where 1 µl of PCR product from the
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first round was used as template DNA for a second round of PCR (2 30 cycles). The PCR
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cycling conditions were 94° C for 5 min, followed by 30 cycles of 94° C for 1 min, 68° C for
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1 min, 72° C for 1 min, with a final extension at 72° C for 10 min. Both Methylocella mmoX
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and 16S rRNA gene-targeting PCR conditions were optimized with DNA from pure cultures
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of Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis
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parvus, Methylomonas agile, Methylomonas rubra, Methylomonas methanica, Methylosinus
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trichosporium and Methylosinus sporium. Specificity of these primers to detect Methylocella
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mmoX and 16S rRNA genes in environmental DNA was verified by clone library analysis
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(Table 1).
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Real-time quantitative PCR
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Methylocella mmoX-specific primers mmoXLF and mmoXLR were applied in a SYBR-
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Green based real-time quantitative PCR (qPCR) assay to quantify the Methylocella
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population present in the environment. qPCR assays were conducted in polypropylene 96-
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well plates on an ABI PRISM ® 7000 Sequence Detection System (Applied Biosystems). All
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assays were carried out in triplicate in a 25 µl volume containing 12.5 µl Power SYBR®
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Master Mix (Applied Biosystems, UK), 1 µl (10 µM; Invitrogen, UK) of primer mmoXLF
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and moXLR, 0.5 µl of 3.2% (w/v) BSA (Roche, Switzerland), 2 µl template DNA (1/10 to
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1/100 dilution of the extracted DNA) and water 8 µl. Non-template controls (NTCs) were
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also run in triplicate in each assay. A two-step qPCR protocol was adopted, consisting of an
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initial denaturation at 95 º C for 5 minutes followed by 45 cycles of denaturation at 95 ° C for
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15 seconds and combined annealing and elongation at 68 ° C for 1 min. Absolute
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quantification of mmoX copies was achieved by comparing the reaction Ct (threshold cycle)
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value with a standard curve, made from a dilution series of M. silvestris BL2 genomic DNA
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ranging from 102 copies to 106 copies per reaction (Supplementary Figure S1). The
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concentration of genomic DNA was determined by measuring the absorbance at 260 nm
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using a NanoDrop spectrophotometer (ND-1000; NanoDrop™, USA). Gene copies were
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calculated according to the method described by Fogel et al. (1999) using the mass of the M.
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silvestris BL2 genome (approximately 4.3 Mb). Only one copy of mmoX is present in the
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genome of M. silvestris BL2 (accession number CP001280). Therefore we assume that the
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number of mmoX copies present in a particular sample represents the number of Methylocella
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spp. cells in that sample. A fluorescence amplification plot of 10-fold serial dilutions of M.
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silvestris BL2 genomic DNA and NTCs is shown in Supplementary Figure S2.
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Amplification of specific single amplicons and the absence of primer-dimer formation were
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confirmed by melting-curve analysis (Supplementary Figure S3). During melting-curve
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analysis, the temperature was increased from 60° C to 95° C at approximately 2° C min-1.
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The qPCR assay was validated by a spiking study with the Ufton landfill cover soil (UK).
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Five g soil was spiked in triplicate with known amounts of M. silvestris BL2 cells ranging
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from 103 to 105 cells g-1 soil. M. silvestris BL2 cells in pure culture were quantified by
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microscopy (Axiophot; Zeiss) using a Neubauer cell counting chamber (Glaswarenfabrik Karl
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Hecht KG, Sondheim, Germany). Detection of Methylocella-specific mmoX sequences in the
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amplified qPCR product was verified by cloning and sequencing (data not shown).
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Quantification of pmoA copy number in DNA extracted from selected environmental
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samples was carried out using primers A189F and Mb661R at an annealing temperature of
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52° C according to the method described by Kolb et al., (2003). Standards were generated
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using Methylosinus trichosporium genomic DNA ranging from 102 to 107 copies of pmoA
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per reaction.
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Construction of mmoX and 16S rRNA gene clone libraries, restriction fragment length
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polymorphism analysis and sequencing
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Before cloning, PCR products were run on a 1% (w/v) agarose gel to check for size and PCR
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specificity. PCR products of the correct size were excised from the gel and purified using the
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Qiagen gel purification kit (Qiagen, USA) according to the manufacturer's instructions.
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Purified PCR products were ligated into plasmid pCR2.1 (Invitrogen, San Diego, CA, USA)
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according to the manufacturer's instructions, cloned and inserts were amplified using
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M13F/M13R primers. PCR products were subjected to restriction fragment length
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polymorphism analysis by digesting with RsaI (for mmoX) and MspI (for 16S rRNA genes).
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Digested DNA fragments were resolved by electrophoresis in a 2.5% (w/v) agarose gel.
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Clone inserts displaying identical restriction patterns were grouped into operational
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taxonomic units (OTUs). One to two clones were sequenced per OTU. DNA sequencing was
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performed at the University of Warwick Molecular Biology Facility by cycle sequencing with
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a BigDye Dideoxy Terminator Ready Reaction kit (Applied Biosystems, Warrington, UK)
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and ABI3100 capillary DNA sequencers. The identities of the cloned sequences were
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determined by BLASTn searches of the GenBank database (Altschul et al., 1990), and the
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phylogenetic affiliations of 16S rRNA gene sequences were determined using RDP classifier
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(Cole et al., 2008).
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References
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Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990). Basic local alignment search
tool. J Mol Biol 215: 403-410.
Chen Y, Dumont MG, Cébron A, Murrell JC. (2007). Identification of active methanotrophs
in a landfill cover soil through detection of expression of 16S rRNA and functional genes.
Environ Microbiol 9: 2855-2869.
Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ et al. (2009). The Ribosomal
Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res
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Fogel GB, Collins CR, Li J, Brunk CF. (1999). Prokaryotic genome size and SSU rDNA
copy number: estimation of microbial relative abundance from a mixed population. Microbial
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Heid CA, Stevens J, Livak KJ, Williams PM. (1996). Real time quantitative PCR. Genome
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Kolb S, Knief C, Stubner S, Conrad R. (2003). Quantitative detection of methanotrophs in
soil by novel pmoA-targeted real-time PCR assays. Appl Environ Microbiol 69: 2423-2429.
Lane DJ. (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds).
Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons: New York, NY, USA.
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Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004). ARB: a
software environment for sequence data. Nucleic Acids Res 32: 1363-1371.
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Supplementary Information
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Figures
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Supplementary Figure S1
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Standard curve Ct value against template DNA mass. The Ct value is defined as the number
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of cycles at which the accumulation of amplicons, as measured by an increase in fluorescence,
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reaches a predetermined level significantly above the background (Heid et al., 1996).
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Supplementary Figure S2
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Fluorescence amplification plot of 10-fold serial dilutions (102 to 106 copies) of M. silvestris
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BL2 genomic DNA (standards) and the non-template control (NTC).
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Supplementary Figure S3
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Melting-curve analysis for the mmoX amplicons generated from DNA extracted from
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environmental samples using Methylocella-specific mmoX primers.
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Supplementary Figure S4
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Alignment of mmoX gene sequences of Methylocella spp. and mmoX from closely related
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methanotrophs. Conserved regions selected to design the Methylocella genus-specific
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forward primer (mmoXLF) and reverse primer (mmoXLR) are indicated by boxes. Numbers
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represent the position of the primers with respect to the mmoX nucleotide sequence of
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Methylocella silvestris BL2.
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Supplementary Table 1.
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Total number of mmoX and pmoA gene copies per gram of soil or sediment
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Source of samples
mmoX gene copies
pmoA gene copies
Hornavan (Sweden)
3.3 (± 0.6)  106
3.8 (± 0.5)  109
Uddjaure (Sweden)
2.1 (± 0.5)  106
2.9 (± 0.1)  109
Moor House peat (UK)
2.3 (± 0.6)  106
2.5 (± 0.3)  108
Colne Estuary sediment, Essex (UK)
0.9 (± 0.2)  106
1.8 (± 0.1)  107
Cloud forest, San Pedro (Peru)
1.2 (± 0.6)  106
1.6 (± 0.2)  108
Rain forest, Tono (Peru)
2.7 (± 0.9)  106
1.2 (± 0.1)  108
Lonar lake sediment (India)
Below detection limit
Not done
Svalbard (Arctic)
Below detection limit
Not done
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