The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV.

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.

Role of rare earth elements in methanol oxidation.

For decades rare earth elements (or lanthanides) were considered not to be involved in biological processes, until their discovery in the active site of the XoxF-type methanol dehydrogenase of the methanotrophic bacterium Methylacidiphilum fumariolicum SolV. Follow-up studies revealed the presence of lanthanides in other pyrroloquinoline quinone-containing enzymes involved in alcohol metabolism. This review discusses the biochemistry of the lanthanide-dependent enzymes and the ability of these metals of influencing the gene expression and the type of methanol dehydrogenase used by microorganisms. Furthermore, it highlights novel insights on the uptake mechanism of rare earth elements into bacterial cells.

Current perspectives on the application of N-damo and anammox in wastewater treatment.

The efficient treatment of wastewater for the removal of nitrogen is of key importance to prevent eutrophication and deoxygenation of receiving water bodies. In addition, ineffective wastewater treatment can be a source of greenhouse gasses. The application of newly discovered microbial processes, such as nitrite/nitrate-dependent methane oxidation (N-damo), can make wastewater treatment systems more sustainable; especially when they are combined with anaerobic ammonium oxidation (anammox). A treatment system based on these microbial processes will need oxygen supply for the production of nitrite. This oxygen may inhibit N-damo and anammox and careful regulation of the oxygen supply is of key importance for the success of the application of N-damo in wastewater treatment.

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere.

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

Methylacidiphilum fumariolicum SolV, a thermoacidophilic 'Knallgas' methanotroph with both an oxygen-sensitive and -insensitive hydrogenase.

Methanotrophs play a key role in balancing the atmospheric methane concentration. Recently, the microbial methanotrophic diversity was extended by the discovery of thermoacidophilic methanotrophs belonging to the Verrucomicrobia phylum in geothermal areas. Here we show that a representative of this new group, Methylacidiphilum fumariolicum SolV, is able to grow as a real 'Knallgas' bacterium on hydrogen/carbon dioxide, without addition of methane. The full genome of strain SolV revealed the presence of two hydrogen uptake hydrogenases genes, encoding an oxygen-sensitive (hup-type) and an oxygen-insensitive enzyme (hhy-type). The hhy-type hydrogenase was constitutively expressed and active and supported growth on hydrogen alone up to a growth rate of 0.03 h-1, at O2 concentrations below 1.5%. The oxygen-sensitive hup-type hydrogenase was expressed when oxygen was reduced to below 0.2%. This resulted in an increase of the growth rate to a maximum of 0.047 h-1, that is 60% of the rate on methane. The results indicate that under natural conditions where both hydrogen and methane might be limiting strain SolV may operate primarily as a methanotrophic 'Knallgas' bacterium. These findings argue for a revision of the role of hydrogen in methanotrophic ecosystems, especially in soil and related to consumption of atmospheric methane.

Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.).

The microbes in the gastrointestinal (GI) tract are of high importance for the health of the host. In this study, Roche 454 pyrosequencing was applied to a pooled set of different 16S rRNA gene amplicons obtained from GI content of common carp (Cyprinus carpio) to make an inventory of the diversity of the microbiota in the GI tract. Compared to other studies, our culture-independent investigation reveals an impressive diversity of the microbial flora of the carp GI tract. The major group of obtained sequences belonged to the phylum Fusobacteria. Bacteroidetes, Planctomycetes and Gammaproteobacteria were other well represented groups of micro-organisms. Verrucomicrobiae, Clostridia and Bacilli (the latter two belonging to the phylum Firmicutes) had fewer representatives among the analyzed sequences. Many of these bacteria might be of high physiological relevance for carp as these groups have been implicated in vitamin production, nitrogen cycling and (cellulose) fermentation.

Differences in venom toxicity and antigenicity between females and males Tityus nororientalis (Buthidae) scorpions.

Venom from male and female specimens of the medically important Venezuelan scorpion Tityus nororientalis have been compared. Males showed a significantly higher venom yield (2.39mg/individual) compared to female scorpions (0.98mg/individual). Female venom was significantly more toxic than that of males, with a median lethal dose (LD(50)) in C57BL/6 mice of 9.46 µg venom protein/gm body weight [95% confidence interval (8.91-9.94)] whereas LD(50) for males was 13.36(12.58-14.03) µg/gm. Mass spectral analyses by MALDI-TOF revealed differences in venom composition between males and females. From a clinical standpoint, the time course of toxicity course indicated a tendency, in the case of the female venom, to elicit the earlier occurrence of severe signs such as sialorrhea, dyspnea (bradypnea/apnea) and exophthalmus particularly in the late toxicity phase. Female venom was significantly less efficient than male venom to inhibit the binding of anti-T. discrepans antibodies to immobilized T. discrepans venom in ELISA assays, suggesting sex-related differences in the bioactive surfaces of T. nororientalis toxins. These results indicate that males and females of T. nororientalis produce venoms with different composition and activity which may have epidemiological implications.