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First Genome Data from Uncultured Upland Soil Cluster Alpha Methanotrophs Provide Further Evidence for a Close Phylogenetic Relationship to Methylocapsa acidiphila B2 and for High-Affinity Methanotrophy Involving Particulate Methane Monooxygenase

Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch Strasse, D-35043 Marburg, Germany,¹ Max-Planck-Institut für molekulare Genetik, Ihnestrasse 63-73, D-14195 Berlin, Germany,² Xanagen Inc., 2-7-14 Higashinakano, Nakano-ku, Tokyo 164-0003, Japan³

Received 21 April 2005/ Accepted 11 July 2005

Members of upland soil cluster alpha (USC) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USC members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USC next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USC, a 42-kb genomic contig of a USC representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USC and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USC and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.