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Applied and Environmental Microbiology, October 2001, p. 4850-4857, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4850-4857.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Svetlana N. Dedysh,1,2 Manigee Derakshani,2 and Werner Liesack2,*

Institute of Microbiology, Russian Academy of Sciences, Moscow 117811, Russia,1 and Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany2

Received 16 March 2001/Accepted 9 July 2001

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 106 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 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 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) × 106 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.

* Corresponding author. Mailing address: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany. Phone: 49 (6421) 178 720. Fax: 49 (6421) 178 809. E-mail address: Liesack{at}mailer.uni-marburg.de.

Applied and Environmental Microbiology, October 2001, p. 4850-4857, Vol. 67, No. 10
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.10.4850-4857.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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