Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris.
Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.
Dedysh
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Institute of Microbiology, Russian Academy of Sciences, Moscow 117811, Russia.
Derakshani
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Liesack
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D020561:Alphaproteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019068:Bryopsida / Q000382:microbiology*; D015169:Colony Count, Microbial; D006863:Hydrogen-Ion Concentration; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008697:Methane / Q000378:metabolism*; D008772:Methylococcaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012988:Soil Microbiology; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D008697:Methane