Methane oxidation by extremophilic Verrucomicrobia adapted to geothermal environments

Methane-oxidizing bacteria, or methanotrophs, share the unique ability to use methane, a potent greenhouse gas, as sole carbon and energy source. A novel group of heat- and acid- loving (thermoacidophilic) methanotrophs belonging to the Verrucomicrobia phylum, has been found in acidic volcanic environments. Bacteria belonging to this phylum are widespread in nature but not well understood. In this project, we study these microorganisms special molecular, physiological and genetic characteristics to reveal how methane oxidation takes place in extreme environments and to enhance our knowledge about biological methane oxidation in general. We are using comparative genomics to investigate the phylogenetic relationship between 8-9 different strains isolated from terrestrial hot springs around the world, which shows that they belong to different but still closely related taxa. Furthermore, the analysis revealed large differences in the number of transposable elements and indicates that these have been a major driver for the evolution of the genomic differences between the strains. Finally, we have used genome analysis to predict physiological traits of these organisms, some of which we will verify experimentally in the laboratory. Thermophilic methanotrophic Verrucomicrobia possess three complete sets of the genes encoding the key enzyme complex, particulate methane monooxygenase (Pmo), which catalyzes the first step in the methane oxidation pathway; oxidation of methane to methanol using molecular oxygen. The pmo genes are organized into three distinct but differentially expressed pmoCAB1, 2 and 3 gene clusters. We are working on obtaining a better understanding of the regulation of these clusters with the aim to understand their differential regulation and physiological functions. It has been shown that pmoCAB1 and 2 are differentially expressed under different oxygen concentrations. We have identified a gene encoding a putative transcriptional regulator located close to the genes encoding pmoCAB1 and 2. This putative regulator has been expressed and purified, and we have raised polyclonal antibodies to this protein and are currently performing a ChIP-seq analysis to identify binding sites on the DNA for this regulator. Work is also in progress to map the transcription initiation sites for pmoCAB1 and 2 by using «primer extension», which will enable identification of promoter areas and possible regulatory sequence signals for binding of regulatory proteins. These two approaches will shed light on the regulatory mechanisms(s) for expression of pmoCAB1/2. Recently (June 2021) a Korean group reported that methanotrophic Verrucomicrobia were able to grow on C3 compounds such as 2-propanol, acetone and acetol, and that the pmoCAB3 gene cluster was strongly expressed when growing on these compounds. This indicates that pmoCAB3 is involved in oxidation of C3 compounds rather than methane. When tested, all our isolates also grew on the mentioned C3 compounds. There are still no tools for genetic analysis of methanotrophic Verrucomicrobia. Transformation trials using different plasmid vectors and protocols have been unsuccessful. We collaborate with the Rosenzweig lab at Northwestern University, Illinois, the USA with the aim to solve the crystal structure of the Pmo enzymes. This is crucial for understanding of the biochemical properties and the reaction mechanism, in particular the role of metal ions in the catalytic site. In collaboration with NORCE Stavanger we have produced bacterial biomass that has been shipped to the American partner for purification and crystallization trials of Pmo. This work is very time-consuming and challenging but resolving the crystal structure will give us invaluable information about the differences and similarities of structure/function relationships in proteobacterial and verrucomicrobial Pmos as well as their origin and evolution. We have in hands six new isolates of verrucomicrobial methanotrophs, recently recovered from geothermal regions in Iceland, The Azores, Yellowstone National Park (USA) and The Philippines. Comparison of these strains has revealed that the evolution of these organisms to a large degree is driven by geographic isolation (allopatric evolution), which is rare among prokaryotes. The new isolates include two novel species, both of which are fully genome-sequences using Pacbio technology and are now being compared using bioinformatics. It is interesting that one of these strains lacks a complete pmoCAB1 gene cluster. Strain Kam1 was completely genome sequenced earlier. Results from this project provide novel insights into the evolution, diversity and biochemistry of biological methane oxidation, a presumed ancient metabolic trait and key process in curbing natural greenhouse gas emissions, as well as improve the biotechnological platform for industrial biotransformation of natural gas to added-value products.

Outcomes: Main outcomes are enhanced understanding of the diversity and molecular features of methanotrophic Verrucomicrobia. Competence in large-scale cultivation of gas-utilizing microbes in fermenters and bioinformatics-based genome analyses have been strengthened. A technological platform for use of these microbes for production of high-value products from natural gas has been created. Impacts: The project has contributed to an improved overall understanding of the role of methanotrophs in the carbon cycle with special focus on the greenhouse gas, methane, and how the oxidation of this greenhouse gas is regulated by methanotrophic communities in geothermal environments. It has also strengthened the collaboration between UiB and NORCE on gas fermentation as well as with The Northwestern University, USA. The awareness of the role of methanotrophs as a methane sink in curbing of the methane release to the atmosphere has been strengthened.

Methane-oxidizing bacteria, or methanotrophs, share the unique ability to use methane, a potent greenhouse gas, as sole carbon and energy source. A novel group of thermoacidophilic methanotrophs belonging to the Verrucomicrobia phylum and provisionally given the candidate genus name, Methylacidiphilum, has been found in acidic volcanic environments. These organisms, in particular strain Kam1, which was isolated in Birkelands lab from a hot spring in Kamchatka, will be used for further molecular, physiological and genetic analyses. Methanotrophic Verrucomicrobia possess three complete sets of the genes encoding the complex key enzyme, particulate methane monooxygenase, organized into three distinct but differentially expressed pmoCAB operons. We will further assess the differential expression of these operons with the aim to identify the functions and regulation of the various pmo clusters and the properties of their encoded proteins. In vivo transcription patterns will be further assessed, and transcription initiation sites mapped in search for regulatory elements. We further aim to develop genetic tools to assess the functional differentiation of these gene clusters using a gene inactivation approach. Both native Pmo and recombinant subunits and domains will be analysed structurally and biochemically, in particular to assess the role of metal ions in the catalytic mechanism. Novel isolates, recently recovered from geothermal regions worldwide, will be subjected to complete genome sequence analysis and phenotypic characterisation in order to assess their possible biogeographic structure and evolutionary relatedness. Results from this project will provide novel insights into the evolution, diversity and biochemistry of biological methane oxidation, a presumed ancient metabolic trait and key process in curbing natural greenhouse gas emissions, as well as improve the biotechnological platform for industrial biotransformation of natural gas to added-value products.