Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments The GenBank accession numbers for the nearly complete 16S rRNA gene sequences for the isolates are AJ458466 to AJ458510. Partial sequences of the pmoA, mxaF and mmoX genes have been deposited under the accession numbers AJ458994–AJ459052, AJ459053–AJ459100 and AJ458511–AJ458535, respectively. Where multiple strains contained identical sequences, only one has been deposited.

Abstract

Type II methane-oxidizing bacteria (MOB) were isolated from diverse environments, including rice paddies, pristine and polluted freshwaters and sediments, mangrove roots, upland soils, brackish water ecosystems, moors, oil wells, water purification systems and livestock manure. Isolates were identified based on morphological traits as either Methylocystis spp., Methylosinus sporium or Methylosinus trichosporium. Molecular phylogenies were constructed based on nearly complete 16S rRNA gene sequences, and on partial sequences of genes encoding PmoA (a subunit of particulate methane monooxygenase), MxaF (a subunit of methanol dehydrogenase) and MmoX (a subunit of soluble methane monooxygenase). The maximum pairwise 16S rDNA difference between isolates was 42 %, and considerable variability was evident within the Methylocystis (maximum difference 36 %). Due to this variability, some of the published ' specific ' oligonucleotide primers for type II MOB exhibit multiple mismatches with gene sequences from some isolates. The phylogenetic tree constructed from pmoA gene sequences closely mirrored that constructed from 16S rDNA sequences, and both supported the presently accepted taxonomy of type II MOB. Contrary to previously published phylogenetic trees, morphologically distinguishable species were generally monophyletic based on pmoA or 16S rRNA gene sequences. This was not true for phylogenies constructed from mmoX and mxaF gene sequences. The phylogeny of mxaF gene sequences suggested that horizontal transfer of this gene may have occurred across type II MOB species. Soluble methane monooxygenase could not be detected in many Methylocystis strains either by an enzyme activity test (oxidation of naphthalene) or by PCR-based amplification of an mmoX gene.

Two isozymes of particulate methane monooxygenase with different methane oxidation kinetics are found in Methylocystis sp. strain SC2

“…Growth occurred at 100 ppmv CH 4 , and the cell numbers did not decrease during incubation under 10 ppmv CH 4 . The numbers of wild-type cells and those of mutants SC2-P1 and SC2-P2 increased Ϸ15-to 17-fold during incubation at 100 ppmv CH 4 .…”

“…M ethane (CH 4 ) is present in the atmosphere at a mixing ratio of Ϸ1.75 parts per million by volume (ppmv) (1). It is 20-23 times more effective as a greenhouse gas than carbon dioxide, and its atmospheric concentration over the past two centuries has increased at a rate of Ϸ1% per year (2).…”

“…Type II methanotrophs of the genera Methylocystis and Methylosinus form a distinct clade within the Alphaproteobacteria, whereas the 10 genera currently recognized as type I methanotrophs belong to the Gammaproteobacteria. These well known type I and type II methanotrophs typically inhabit the aerobic interfaces of methanogenic environments, such as natural wetlands and rice paddies, and reduce the potential methane flux to the atmosphere (3)(4)(5). Because of the usually high methane supply in these environments, methane is oxidized with low apparent halfsaturation constants (K m(app) Ͼ1 M CH 4 ).…”

“…These well known type I and type II methanotrophs typically inhabit the aerobic interfaces of methanogenic environments, such as natural wetlands and rice paddies, and reduce the potential methane flux to the atmosphere (3)(4)(5). Because of the usually high methane supply in these environments, methane is oxidized with low apparent halfsaturation constants (K m(app) Ͼ1 M CH 4 ). These K m values are similar to those determined for cultured methanotrophs (K m(app) ϭ 2-12 M CH 4 ) (6, 7).…”

“…This copper-dependent enzyme is synthesized even when only a minuscule amount of copper is available (16,17). Under copper-limiting conditions, a subset of the methanotrophs produce a soluble type of MMO (sMMO) (4,16), an enzyme evolutionarily distinct from pMMO (15). However, to initiate synthesis of pMMO under copper-limiting conditions, some methanotrophs may be able to mobilize and acquire copper from mineral and organic solid phases by releasing the fluorescent chromopeptide methanobactin (17,18).…”