Biololgical methane oxidation by methanotrophic Verrucomicrobia under hot and acidic conditions; evolution of an ancient metabolic trait

Methylacidiphilum represents a novel lineage of extremophilic methane-oxidizing bacteria belonging to the phylum Verrucomicrobia. These bacteria are extremely acidophilic (acid loving) and grow in geothermal environments with pH down to 1 and below. They are moderately thermophilic (heat-loving). The group is distantly related to the "classic" methanotrophic Proteobacteria that are prevalent in many natural environments. The discovery of methanotrophic bacteria within the Verrucomicroba phylum a few years ago has thrown new light on the evolution and diversity of biological methane oxidation. These bacteria have 3 different but conserved gene complexes that each encodes the key enzyme particulate methane monooxygenase (pMMO). This enzyme consists of three different subunits, PmoC, A and B, which together form a multi-component enzyme complex. One of the objectives of the project is to understand the role of the three different gene complexes using the bacterium Methylacidiphilum kamchatkense strain Kam1 as a model organism. We have shown that one of these complexes, the pmoCAB2 operon is much more strongly expressed than the other two under standard culture conditions, indicating that these gene complexes probably have different functions and are expressed under different growth conditions. We have worked on this problem and tested the expression of the other two operons, but so far we have no clear results that can provide information about the conditions under which the other two operons are expressed. Proteomics aimed at identification of pMMO subunits under different growth conditions has been difficult to perform, which may be due to technical problems with trypsin treatment of integral membrane proteins, but preliminary results support the transcription assays, i.e. that the pmoCAB2 operon is the functional gene complex under normal growth conditions. The corresponding enzyme (pMMO) of methanotrophic Proteobacteria uses copper ions as an essential metal in its active center for the oxidation of methane to methanol. High copper concentrations are also required for the expression of the pmo genes. Methanotrophic Verrucomicrobia are isolated from geothermal areas with relatively little copper, and copper appears to play less of a role for these organisms than for the Proteobacteria. We have succeeded in getting strain Kam1 to grow without copper addition to the medium. This reinforces our earlier suggestion that methane oxidation within this group of organisms can be copper-independent and may use a different metal than copper, which we must examine carefully by means of biochemical analyzes of the methane oxidation activity in the presence of various metal ions. This is also supported by the fact that copper-binding motifs identified in pMMO from Proteobacteria cannot be identified in pMMO from Verrucomicrobia. Homology protein modelling also indicates that copper is not bound by pMMO from verrucomicrobia. A domain of the pmoB2 subunit from Kam1 believed to contain the active site for methane oxidation (based on studies of methanotrophic proteobacteria) has been cloned and overexpressed in E. coli. The domain is however insoluble, but we have at last managed to get it partially solubilized by creating a fusion protein. Attempts to use this fusion protein in enzymatic assays are in progress. We have made rabbit antibodies against the recombinant pmoB2 protein using insoluble protein antigen and we now have a strong and specific antibody against this domain. The antibody is tested in immunogold-labeling experiments to determine the sub-cellular localization of pMMO enzyme in Kam1, and preliminary analyzes indicate an association with the cell membrane. Another subgoal of the project was to isolate additional methane-oxidizing bacteria belonging to phylum Verrucomicrobium in order to perform biogeographical and evolutionary analyses. We now have several new isolates from a number of geographically separate geothermal areas; The Azores, Yellowstone National Park (USA), Iceland and The Philippines. The isolates are very similar, both physiologically and morphologically, as well as regarding phylogenetic marker genes. The results indicate that there exists a biogeographic structure where the phylogenetic difference may be correlated with the geographic distance. The genome of strain Kam1 is sequenced to draft level and compared to the other isolates. This analysis mainly confirms a high degree of specialization for one-carbon metabolism and assimilation of carbon using the Calvin-Benson-Bassham cycle such as in plants, algae and most other autotrophic organisms. These organisms have thus a carbon metabolism that can be described as a kind of "autotrophic" metanothrofy as they assimilate carbon from the carbon dioxide formed by oxidation of methane.

Recent isolation of thermoacidophilic methane-oxidizing bacteria belonging to the Verrucomicrobia lineage of evolution has expanded our understanding of the diversity of biological methane oxidation. These microorganisms share the unique ability to use me thane, a potent greenhouse gas, as a sole carbon and energy source. Methylacidiphilum kamchatkense, strain Kam1, which my lab isolated from an acidic hot spring in Kamchatka, Russia, will be used as a model for further molecular and physiological analyses of methane oxidation in these organisms, which possess 3-4 conserved operons each encoding 3 particulate methane monooxygenase (Pmo) protein subunits. Preliminary analyses indicate that only one is functionally expressed in Kam1 under standard growth con ditions. Through further transcriptional and proteomics analyses, the effect of environmental factors, such as substrate limitation and available copper, on the expression of pmo operons will be assessed as well as the mechanisms for operon regulation. Th e intracellular polyhedral bodies in these organisms are of particular interest; they may represent a novel subcellular micro-compartment for methane oxidation, compensating for the lack of the typical Pmo-associated intracellular membrane system found in other methanotrophs. These unique intracellular structures may also play a role in detoxification and/or carbon assimilation. The organelles will be purified from Kam1 and their functional role will be assessed. The diversity and activity of methanotroph ic Verrucomicrobia populations from other geothermal regions will also be explored, in part, through international collaboration. Results from this project will provide novel insights into the evolution and diversity of biological methane oxidation, a pre sumed "ancient" metabolic trait and key process in curbing natural greenhouse gas emissions.