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

ABSTRACT: 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.

SUBMITTER: Kruse T

PROVIDER: S-EPMC6688271 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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

Kruse Thomas T Ratnadevi Chandini Murarilal CM Erikstad Helge-André HA Birkeland Nils-Kåre NK

BMC genomics 20190809 1

<h4>Background</h4>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 evoluti ...[more]

PMID: 31399023

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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:The Solfatara volcano near Naples (Italy), the origin of the recently discovered verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was shown to contain ammonium ([Formula: see text]) at concentrations ranging from 1 to 28 mM. Ammonia (NH3) can be converted to toxic hydroxylamine (NH2OH) by the particulate methane monooxygenase (pMMO), the first enzyme of the methane (CH4) oxidation pathway. Methanotrophs rapidly detoxify the intermediate NH2OH. Here, we show that strain SolV performs ammonium oxidation to nitrite at a rate of 48.2 nmol [Formula: see text].h-1.mg DW-1 under O2 limitation in a continuous culture grown on hydrogen (H2) as an electron donor. In addition, strain SolV carries out nitrite reduction at a rate of 74.4 nmol [Formula: see text].h-1.mg DW-1 under anoxic condition at pH 5-6. This range of pH was selected to minimize the chemical conversion of nitrite ([Formula: see text]) potentially occurring at more acidic pH values. Furthermore, at pH 6, we showed that the affinity constants (K s ) of the cells for NH3 vary from 5 to 270 μM in the batch incubations with 0.5-8% (v/v) CH4, respectively. Detailed kinetic analysis showed competitive substrate inhibition between CH4 and NH3. Using transcriptome analysis, we showed up-regulation of the gene encoding hydroxylamine dehydrogenase (haoA) cells grown on H2/[Formula: see text] compared to the cells grown on CH4/[Formula: see text] which do not have to cope with reactive N-compounds. The denitrifying genes nirk and norC showed high expression in H2/[Formula: see text] and CH4/[Formula: see text] grown cells compared to cells growing at μmax (with no limitation) while the norB gene showed downregulation in CH4/[Formula: see text] grown cells. These cells showed a strong upregulation of the genes in nitrate/nitrite assimilation. Our results demonstrate that strain SolV can perform ammonium oxidation producing nitrite. At high concentrations of ammonium this may results in toxic effects. However, at low oxygen concentrations strain SolV is able to reduce nitrite to N2O to cope with this toxicity.

Project description:Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5-3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h-1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

Project description:BACKGROUND:The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. RESULTS:We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins. CONCLUSION:The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria. REVIEWERS:This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.

Dataset Information

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

Publications

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

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