Methylosinus

Abstract: 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 (α subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the α and γ 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 101 and 102 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) × 106pmoA molecules g−1 for all methanotrophs. The Methylosinus group was predominant (2.7 × 106 ± 1.1 × 106 target molecules g−1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 × 106 ± 0.9 × 106 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 × 104 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.

Abstract: The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level. Methanotrophs are a group of gram-negative bacteria that can grow on methane as the sole source of carbon and energy. They are widespread in nature and have gotten increased attention in the past two decades due to their potential role in the global methane cycle (11) and their ability to cometabolize a number of environmental contaminants (15). The methanotrophs consist of eight recognized genera (3, 5‐7) that fall into two major phylogenetic groups, the a subgroup of the class Proteobacteria (a-Proteobacteria) (which includes the type II

Abstract: Numerical taxonomic, DNA-DNA hybridization, and phospholipid fatty acid composition analyses were performed on an extensive range of methanotrophic strains, including reference strains and environmental isolates obtained from sites throughout eastern Australia. When the results of these studies were related to the results of a study based on genomic physicochemical properties, they clarified group I and II methanotroph genus and species interrelationships. The group I methanotrophs were found to be made up of three broadly phenotypically and genotypically homologous clusters of species. The first group I methanotroph cluster included the carotenoid-containing species Methylomonas methanica, Methylomonas fodinarum, and Methylomonas aurantiaca. These species represent the true members of the genus Methylomonas. The second group I methanotroph cluster was made up of two subclusters of strains. One subcluster included species not capable of producing resting cells and consisted of the species “Methylomonas agile,” “Methylomonas alba,” and Methylomonas pelagica. The other subcluster included species capable of forming desiccation-resistant cysts and included Methylococcus luteus, marine Methylomonas-like strains, and Methylococcus whittenburyi. Strains designated “Methylococcus ucrainicus” and Methylococcus vinelandii were found to be synonyms of Methylococcus whittenburyi, while Methylococcus bovis was a synonym of Methylococcus luteus. It is proposed that these subclusters represent a new genus, Methylobacter gen. nov. The species in the new genus are type species Methylobacter luteus comb. nov., Methylobacter agilis sp. nov., Methylobacter albus sp. nov., nom. rev., Methylobacter marinus sp. nov., Methylobacter pelagicus comb. nov., and Methylobacter whittenburyi comb. nov. The remaining group I methanotrophs included the moderately thermophilic species Methylococcus capsulatus and Methylococcus thermophilus and a group of unnamed strains closely related to Methylococcus capsulatus. It is proposed that these species represent the true members of the genus Methylococcus. The group II methanotrophs consisted of two closely related groups. The first group included budding, exospore-producing strains, while the second group included nonmotile, cyst-forming strains. These groups represent the genera Methylosinus and Methyocystis, which are revived here. The genus Methylosinus gen. nov., nom. rev. includes the species Methylosinus trichosporium sp. nov., nom. rev. and Methylosinus sporium sp. nov., nom. rev., while the genus Methylocystis gen. nov., nom. rev. includes the species Methylocystis parvus sp. nov., nom. rev. and Methylocystis echinoides sp. nov., nom. rev.

Abstract: Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.