Methane oxidation by extremophilic Verrucomicrobia adapted to geothermal environments

Informations

Funding country

Norway

Acronym

-

URL

-

Start date

1/1/2017

End date

12/31/2022

Budget

817,212 EUR

Fundings

Abstract

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.