Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (␣ subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the ␣ and ␥ subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10 1 and 10 2 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ؎ 1.4) ؋ 10 6 pmoA molecules g ؊1 for all methanotrophs. The Methylosinus group was predominant (2.7 ؋ 10 6 ؎ 1.1 ؋ 10 6 target molecules g ؊1 ). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 ؋ 10 6 ؎ 0.9 ؋ 10 6 target molecules g of soil ؊1 ). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 ؋ 10 4 target molecules g of soil ؊1 . Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.In soils, methane is oxidized and consumed by methaneoxidizing bacteria (MOB) (6). Based on their main substrates, MOB can be divided into two groups, comprising the autotrophic ammonium-oxidizing bacteria (AAOB) and the methaneassimilating bacteria (MAB), the so-called methanotrophs (25). MAB are affiliated with the ␣ (Methylocystaceae; type II) and ␥ (Methylococcaceae; type I) subclasses of Proteobacteria, whereas most of the AAOB belong to the ␤ subclass (2, 14). The first step and key reaction of methane oxidation is the introduction of a hydroxyl group by methane monooxygenase (MMO). Two different forms of this enzyme exist: a membrane-bound particulate MMO (pMMO), which is present in almost all methanotrophs isolated so far, and the soluble form (sMMO), which has been found in only some methanotrophs (34). The pMMO is homologous to the ammonium monooxygenase (AMO), the key enzyme of the AAOB (23). AMO catalyzes not only the oxidation of ammonia but also that of CH 4 , albeit at a much lower activity (2). In the following report,...

Aerobic methanotrophs in forest soils are the largest biological sink for atmospheric methane (CH4 ). Community structures in 53 soils from Europe, Russia, North and South America, Asia and New Zealand located in boreal, temperate and tropical forests were analysed and maximal abundances of 2.1 × 10(7) methanotrophs g(-1)   DW were measured. In acidic soils, the most frequently detected pmoA genotypes were Upland Soil Cluster α (USCα) and Methylocystis spp. Phospholipid fatty acids that were labelled by consumption of (14/13) CH4 suggested the activity of type II methanotrophs. Cluster 1 (Methylocystaceae), USCγ and Methylocystis spp. were frequently detected genotypes in pH-neutral soils. Genotypes with ambiguous functional affiliation were co-detected (Clusters MR1, RA21, 2) and may represent aerobic methanotrophs, ammonia oxidizers or enzymes with an unknown function. The physiological traits of atmospheric CH4 oxidizers are largely unknown because organisms possessing the key forest soil pmoA genotypes (USCα, USCγ, Cluster 1) have not been cultivated. Some methanotrophic strains belonging to the family Methylocystaceae have been shown to oxidize CH4 at atmospheric mixing ratios. Methylocystis strain SC2 was found to have an alternative particulate CH4 monooxygenase responsible for CH4 oxidation at atmospheric mixing ratios. pH, forest type and temperature might be environmental factors that shape methanotrophic communities in forest soils. However, specific effects on individual species are largely unknown, and only a limited number of studies have addressed environmental controls of methanotrophic diversity, pointing to the need for future research in this area.

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.

Norway spruce (Picea abies) forests exhibit lower annual atmospheric methane consumption rates than do European beech (Fagus sylvatica) forests. In the current study, pmoA (encoding a subunit of membranebound CH 4 monooxygenase) genes from three temperate forest ecosystems with both beech and spruce stands were analyzed to assess the potential effect of tree species on methanotrophic communities. A pmoA sequence difference of 7% at the derived protein level correlated with the species-level distance cutoff value of 3% based on the 16S rRNA gene. Applying this distance cutoff, higher numbers of species-level pmoA genotypes were detected in beech than in spruce soil samples, all affiliating with upland soil cluster ␣ (USC␣). Additionally, two deep-branching genotypes (named 6 and 7) were present in various soil samples not affiliating with pmoA or amoA. Abundance of USC␣ pmoA genes was higher in beech soils and reached up to (1.2 ؎ 0.2) ؋ 10 8 pmoA genes per g of dry weight. Calculated atmospheric methane oxidation rates per cell yielded the same trend. However, these values were below the theoretical threshold necessary for facilitating cell maintenance, suggesting that USC␣ species might require alternative carbon or energy sources to thrive in forest soils. These collective results indicate that the methanotrophic diversity and abundance in spruce soils are lower than those of beech soils, suggesting that tree species-related factors might influence the in situ activity of methanotrophs.

Methanotrophic communities were studied in several periodically water-saturated gleyic soils. When sampled, each soil had an oxic upper layer and consumed methane from the atmosphere (at 1.75 ppmv). In most gleyic soils the K(m(app)) values for methane were between 70 and 800 ppmv. These are higher than most values observed in dry upland soils, but lower than those measured in wetlands. Based on cultivation-independent retrieval of the pmoA-gene and quantification of partial pmoA gene sequences, type II (Alphaproteobacteria) methanotrophs of the genus Methylocystis spp. were abundant (> 10(7) pmoA target molecules per gram of dry soil). Type I (Gammaproteobacteria) methanotrophs related to the genera Methylobacter and Methylocaldum/Methylococcus were detected in some soils. Six pmoA sequence types not closely related to sequences from cultivated methanotrophs were detected as well, indicating that diverse uncultivated methanotrophs were present. Three Gleysols were incubated under different mixing ratios of (13)C-labelled methane to examine (13)C incorporation into phospholipid fatty acids (PLFAs). Phospholipid fatty acids typical of type II methanotrophs, 16:0 and 18:1omega7c, were labelled with (13)C in all soils after incubation under an atmosphere containing 30 ppmv of methane. Incubation under 500 ppmv of methane resulted in labelling of additional PLFAs besides 16:0 and 18:1omega7c, suggesting that the composition of the active methanotrophic community changed in response to increased methane supply. In two soils, 16:1 PLFAs typical of type I methanotrophs were strongly labelled after incubation under the high methane mixing ratio only. Type II methanotrophs are most likely responsible for atmospheric methane uptake in these soils, while type I methanotrophs become active when methane is produced in the soil.