Methylacidiphilum kamchatkense gen. nov., sp. nov., an extremely acidophilic and moderately thermophilic methanotroph belonging to the phylum Verrucomicrobiota.

The thermo-acidophilic aerobic methanotrophic Verrucomicrobia bacterium, designated strain Kam1T was isolated from an acidic geothermal mud spring in Kamchatka, Russia. Kam1T is Gram-stain-negative, with non-motile cells and non-spore-forming rods, and a diameter of 0.45-0.65 µm and length of 0.8-1.0 µm. Its growth is optimal at the temperature of 55 °C (range, 37-60 °C) and pH of 2.5 (range, pH 1-6), and its maximal growth rate is ~0.11 h-1 (doubling time ~6.3 h). Its cell wall contains peptidoglycan with meso-diaminopimelic acid. In addition to growing on methane and methanol, strain Kam1T grows on acetone and 2-propanol. Phylogenetically, it forms a distinct group together with other Methylacidiphilum strains and with the candidate genus Methylacidimicrobium as a sister group. These findings support the classification of the strain Kam1T as a representative of a novel species and genus of the phylum Verrucomicrobiota. For this strain, we propose the name Methylacidiphilum kamchatkense sp. nov. as the type species within Methylacidiphilum gen. nov. Strain Kam1T (JCM 30608T=KCTC 4682T) is the type strain.

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph "Candidatus Methylacidiphilum kamchatkense" strain Kam1 and comparison with its closest relatives.

BACKGROUND: The candidate genus "Methylacidiphilum" comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the "Ca. Methylacidiphilum" and the sister genus "Ca. Methylacidimicrobium" represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms. RESULTS: Here we present the closed genome of "Ca. Methylacidiphilum kamchatkense" strain Kam1 and a comparison with the genomes of its two closest relatives "Ca. Methylacidiphilum fumariolicum" strain SolV and "Ca. Methylacidiphilum infernorum" strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of "Ca. Methylacidiphilum" is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between "Ca. M. kamchatkense" strain Kam1 and strains SolV and V4 is ≤95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements. CONCLUSIONS: Comparing three closed genomes of "Ca. Methylacidiphilum" spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

Differential expression of particulate methane monooxygenase genes in the verrucomicrobial methanotroph 'Methylacidiphilum kamchatkense' Kam1.

Methane monooxygenases (MMOs) are oxygen-dependent enzymes that catalyze the oxidation of methane to methanol in the methanotrophic bacteria. The thermoacidophilic verrucomicrobial methanotroph 'Methylacidiphilum kamchatkense' Kam1 contains three complete and phylogenetically distinct copies of the pmoCAB gene cluster apparently organized as operons, each encoding all three subunits of particulate MMO (pMMO), and a truncated pmoCA cluster encoding only two of the subunits. Two of the clusters are present as a tandem array, but the other clusters occur in isolation. Here, the expression of these clusters has been assessed using the four pmoA genes as targets in reverse transcriptase quantitative PCR analysis. One of the pmoA genes, designated pmoA2, is at least 35-fold more strongly transcribed than the other pmoA copies. Growth at suboptimal temperature and pH conditions did not significantly change the transcription pattern, indicating that the pmoCAB2 cluster encodes the functional pMMO under methane-fuelled growth conditions. During growth on methanol, expression of pmoA2 was reduced approximately tenfold as compared to growth on methane, suggesting a role for the alternative carbon substrates in gene regulation.

Global Biogeographic Distribution Patterns of Thermoacidophilic Verrucomicrobia Methanotrophs Suggest Allopatric Evolution.

Thermoacidophilic methane-oxidizing Verrucomicrobia of the candidate genus Methylacidiphilum represent a bacterial taxon adapted to highly acidic (pH 1-4) and moderate temperature (∼65°C) methane-containing geothermal environments. Their apparent ubiquity in acidic terrestrial volcanic areas makes them ideal model organisms to study prokaryotic biogeography. Three Methylacidiphilum species isolated from distantly-separated geothermal regions in Russia, New Zealand, and Italy were previously described. We have explored the intra-taxon phylogenetic patterns of these organisms based on comparative genome analyses and phenotypic comparisons with six new Verrucomicrobia methanotroph isolates from other globally-separated acidic geothermal locations. Comparison of rRNA and particulate methane monooxygenase (pmoCAB) operon sequences indicates a close phylogenetic relationship among the new isolates as well as with the previously characterized strains. All share similar cell morphology including the presence of extensive intracellular inclusion bodies and lack of intracellular membrane systems, which are typical for proteobacterial methanotrophs. However, genome sequence comparisons and concatenated MLST-based phylogenetic analyses separate the new isolates into three distinct species-level groups. Three recently processed isolates from the Azores (each from geographically-separate hot springs within the region) and a single isolate from Iceland are highly similar, sharing more than 88% in silico genome homology with each other as well as with the previous isolate, Methylacidiphilum fumariolicum strain SolV, from Italy. These appear to constitute a distinct European/Atlantic clade. However, two of the new isolates - one from the Yellowstone National Park (United States) and another from The Philippines - constitute separate and novel Methylacidiphilum species. There is no clear correlation between fatty acid profiles and geographic distance between origins, or any phylogenetic relationship. Serological analysis using antiserum raised against M. kamchatkense strain Kam1 revealed large differences in the degree of cross-reactivity with no correlation with other factors. However, the genetic distance between the strains does correlate to the distance between their geographic origins and suggests a global biogeographic pattern shaped by an isolation-by-distance mechanism. These results further confirm terrestrial geothermal springs as isolated islands featuring allopatric prokaryotic speciation.

Draft Genome Sequence of "Candidatus Methylacidiphilum kamchatkense" Strain Kam1, a Thermoacidophilic Methanotrophic Verrucomicrobium.

"Candidatus Methylacidiphilum kamchatkense" strain Kam1 is an aerobic methane-oxidizing thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. It was recovered from an acidic geothermal site in Uzon Caldera, Kamchatka, Russian Federation. Its genome possesses three complete pmoCAB gene clusters encoding particulate methane monooxygenase enzymes and a complete Calvin-Benson-Bassham cycle for carbon assimilation.