Ecology and genomics of Archaea involved in anaerobic oxidation of ethane

Abstract

In deep sediment layers, geothermal heat degrades organic matter into a complex mix of hydrocarbons. These compounds migrate towards the sediment surface, where a rich microbial community of anaerobic and aerobic microorganisms oxidizes them. Microorganisms involved in anaerobic alkane oxidation have been identified for most compounds. However, the anaerobic oxidation of the second most abundant alkane, ethane, was unexplored. This thesis aimed to cultivate an anaerobic ethane oxidizer and extend our understanding of the anaerobic oxidation of ethane. In this work, an ethane-degrading thermophilic archaeon was cultured and named "Candidatus Ethanoperedens thermophilum" (Chapter 2). Using metagenomic, transcriptomic, and metabolomic data, Ca. E. thermophilum was shown to activate ethane using a methyl-coenzyme M reductase (MCR) homolog. Ethane is completely oxidized to CO2, and electrons are passed to the sulfate-reducing partner bacterium. In a fluorescence in-situ hybridization (CARD-FISH) study, ethanotrophs were detected at various hydrocarbon seepage sites. In chapter 3, a modified mRNA-FISH protocol was developed to analyze activity dynamics in spatially segregated consortia. Tetra-labeled oligonucleotide probes were used to target the mRNA of the metabolic key enzyme of the ethanotroph, ethyl-coenzyme M reductase (ECR). With this method, activity differences were shown and appear to be dependent on the position in the archaeal monospecies cluster and distance from the nearest partner cell. Chapter 4 describes the structural characterization of the ethane-specific MCR homolog from Ca. E. thermophilum. The first structure of a non-canonical MCR showed many sophisticated differences from canonical MCR structures, and the enzyme was named ethyl coenzyme M reductase after its presumed function. The ECR contains a novel dimethylated F430-cofactor and has many amino acid substitutions at the active site building a widened catalytic chamber. Additionally, large insert regions form loops at the enzyme surface, marking the entry to a hydrophobic tunnel that leads ethane to the catalytic chamber. This study forms the base for understanding enzymes involved in the anaerobic oxidation of ethane and a potential future biotechnological application.