USCa Cluster

Holmes et al. (1999) characterized atmospheric methane-oxidizing bacteria using a dual approach of retrieving pmoA genes from soil and radiolabelling microbial phospholipid fatty acids (PLFAs) by incubation of soil under 14CH4. The pmoA gene codes for the active site-containing subunit of pMMO, which is universal to all known methanotrophs except Methylocella spp. Phylogenies constructed based on pmoA gene sequences closely reflect phylogenies based on 16S rRNA sequences, and therefore retrieval and sequencing of pmoA from environmental DNA extracts allows identification of the methanotrophs present (Holmes et al., 1995; Heyer et al., 2002).

In the study of Holmes et al. (1999), a novel group of pmoA sequences was predominantly recovered from several forest

Forest Nitrogen Deposition

Fig. 10.3. Phylogenetic tree based on sequences of derived amino acid sequences of partial pmoA or amoA genes of methanotrophs and ammonia oxidizers. Groups that are believed to be involved in the consumption of atmospheric methane (cluster I, Methylocystis, USCa, USCg) are indicated. (Modified from Knief et al., 2005b. With permission of Blackwell Publishing.) Tree construction with the tree-puzzle algorithm and the Jones-Taylor-Thornton evolutionary model was based on 147 derived amino acid residues. Black circles indicate branches that were recovered in 90% of 25,000 reconstructed tree-puzzle trees; white circles indicate branches with 80% recovery. The AmoA sequences of nitrifiers were set as the outgroup. The bar represents 0.10 change per amino acid position.

Fig. 10.3. Phylogenetic tree based on sequences of derived amino acid sequences of partial pmoA or amoA genes of methanotrophs and ammonia oxidizers. Groups that are believed to be involved in the consumption of atmospheric methane (cluster I, Methylocystis, USCa, USCg) are indicated. (Modified from Knief et al., 2005b. With permission of Blackwell Publishing.) Tree construction with the tree-puzzle algorithm and the Jones-Taylor-Thornton evolutionary model was based on 147 derived amino acid residues. Black circles indicate branches that were recovered in 90% of 25,000 reconstructed tree-puzzle trees; white circles indicate branches with 80% recovery. The AmoA sequences of nitrifiers were set as the outgroup. The bar represents 0.10 change per amino acid position.

soils that oxidized atmospheric methane. These sequences were not closely related to the pmoA of any known methanotroph. However, they were more similar to pmoA genes of type II (Alphaproteobacteria) than type I (Gammaproteobacteria) methanotrophs.

The most closely related pmoA sequence from a cultivated methanotroph is that of Methylocapsa acidiphila, an alphaproteo-bacterium isolated from acidic peat (Dedysh et a!., 2002). The pmoA data were supported by incubating soils under a low mixing ratio

(<50 ppmv) of 14C-labelled CH4. The resulting 14C-labelled PFLA profiles were somewhat related to profiles of known type II metha-notrophs, especially Methylocapsa (Holmes et a!., 1999; Roslev and Iversen, 1999).

This unknown group of pmoA sequences, called unidentified soil cluster Alphaproteobacteria (USCa) after Knief et al. (2003), has been recovered from many different upland soils in subsequent studies (Henckel et al., 2000; Jensen et al., 2000; Bourne et al., 2001; Knief et al., 2003). Labelling of PLFAs and bacteriohopanoids by incubation with 13C-labelled methane has supported the theory that unknown species of type II methanotrophs are responsible for atmospheric methane uptake in many forest soils (Bull et al., 2000; Knief et al., 2003; Crossman et al., 2005).

Recent quantification of pmoA genes in upland soils has provided a clue that the USCa cluster represents a high-specific-affinity methane oxidizer. Kolb et al. (2005) used quantitative real-time polymerase chain reaction (PCR) assays targeting specific phylogenetic groups of pmoA genes, including USCa, to estimate methanotroph population densities in soils. In one of two upland soils tested, USCa was the most abundant pmoA sequence recovered, with a population density of ~2 x 106 cells/g soil. According to this population, and the measured methane oxidation rates in the soil, the per-cell oxidation rates of USCa metha-notrophs at 1.7 ppmv CH4 (i.e. the specific affinities) were estimated to be much higher than rates in known methanotrophs. This estimate assumed that all abundant metha-notrophic species were detectable, and that populations were not underestimated due to methodological limitations. These are problematic assumptions, but if they are correct, USCa methanotrophs might be the high-affinity species originally postulated by Bender and Conrad (1992).

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