Project description:BACKGROUND: Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture. RESULTS: In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5'-m6ACN4GT-3' and 5'-CCm6AN5CTC-3' methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif. CONCLUSIONS: Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

Project description: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.

Project description:Industrial methanol production converts methane from natural gas into methanol through a multistep chemical process. Biological methane-to-methanol conversion under moderate conditions and using biogas would be more environmentally friendly. Methanotrophs, bacteria that use methane as an energy source, convert methane into methanol in a single step catalyzed by the enzyme methane monooxygenase, but inhibition of methanol dehydrogenase, which catalyzes the subsequent conversion of methanol into formaldehyde, is a major challenge. In this study, we used the thermoacidophilic methanotroph "Methylacidiphilum fumariolicum" SolV for biological methanol production. This bacterium possesses a XoxF-type methanol dehydrogenase that is dependent on rare earth elements for activity. By using a cultivation medium nearly devoid of lanthanides, we reduced methanol dehydrogenase activity and obtained a continuous methanol-producing microbial culture. The methanol production rate and conversion efficiency were growth-rate dependent. A maximal conversion efficiency of 63% mol methanol produced per mol methane consumed was obtained at a relatively high growth rate, with a methanol production rate of 0.88?mmol/g (dry weight)/h. This study demonstrates that methanotrophs can be used for continuous methanol production. Full-scale application will require additional increases in the titer, production rate, and efficiency, which can be achieved by further decreasing the lanthanide concentration through the use of increased biomass concentrations and novel reactor designs to supply sufficient gases, including methane, oxygen, and hydrogen.IMPORTANCE The production of methanol, an important chemical, is completely dependent on natural gas. The current multistep chemical process uses high temperature and pressure to convert methane in natural gas to methanol. In this study, we used the methanotroph "Methylacidiphilum fumariolicum" SolV to achieve continuous methanol production from methane as the substrate. The production rate was highly dependent on the growth rate of this microorganism, and high conversion efficiencies were obtained. Using microorganisms for the production of methanol might enable the use of more sustainable sources of methane, such as biogas, rather than natural gas.

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Methylacidiphilum kamchatkense

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