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Abstract
Candidatus Dormibacterota is a soil bacterial phylum identified in unusually high relative abundances within East Antarctic desert soil. Despite being widely distributed, albeit at low relative abundances (< 1%), within soils across the globe, Ca. Dormibacterota lacks a cultured representative. Given its ‘unculturable’ status there is little functional information available on this enigmatic phylum. Metabolic reconstructions from metagenome-assembled genomes (MAGs) of Ca. Dormibacterota from East Antarctic deserts implicated members of Ca. Dormibacterota as potentially capable of a novel metabolic process called ‘atmospheric chemosynthesis’. During atmospheric chemosynthesis, H2, CO2 and CO from the atmosphere are oxidised by bacteria containing high-affinity enzymes as a source of energy and carbon. The proposed enzymes include Type 1h/5 hydrogenases, Type 1E RuBisCO and carbon monoxide dehydrogenases.
Firstly, we established a protocol for the extraction of bacterial cells from cold and temperate soil samples. The optimised protocol resulted in improved cell yields and decreased carry-over of detritus from soil, which is known to hamper downstream microscopy and DNA extraction processes. This allowed for the successful isolation of microbial cells from Antarctic soil, for subsequent analysis via confocal microscopy using newly designed order-level fluorescence in situ hybridisation (FISH) probes (DORM1164-Cy3 and AEOL1170-Cy3). Cells were revealed to be small (312 nm-1.4 µm in diameter) and consistently coccoid in shape. Confocal microscopy showed that some members of Ca. Dormibacterota form interspecific aggregations with another bacterial species, indicating a potentially symbiotic relationship.
Next, we produced six new high-quality MAGs and combined them with three publicly available MAGs to perform a phylogenetic analysis of Ca. Dormibacterota present in Antarctic soils. We found that Ca. Dormibacterota consists of a single class, Ca. Dormibacteria, that contains two order level divisions; Ca. Dormibacterales and Ca. Aeolococcales, with a total of four genera and five species identified. Through reconstructions of metabolic pathways within each MAG, we describe the genetic capabilities of Ca. Dormibacterota and propose metabolic strategies by which they thrive in Antarctic desert soils. We found that Ca. Dormibacterota are a metabolically diverse phylum with a wide range of mechanisms to provide protection against the harsh Antarctic environment. Primary amongst these was the potential capacity of all species examined to oxidise trace gases to below atmospheric levels. Through the proposed use of Type 1h/5 hydrogenases, atmospheric hydrogen can be oxidised as a source of energy to drive CO2 fixation via the Calvin-Bassham-Benson cycle using a Type 1E RuBisCO. We also assessed the environmental determinants of Ca. Dormibacterales and Ca. Aeolococcales through the analysis of 41 soil physicochemical properties which showed that both orders were negatively correlated with total environmental phosphorous. The primary environmental determinant of Ca. Dormibacterales relative abundance was titanium dioxide and the primary determinant of Ca. Aeolococcales relative abundance was sodium, both displaying a negative correlation with the relevant order.
Finally, a novel culturing method was developed that attempted to enrich for the presumably slow growing Ca. Dormibacterota. The method was designed to exploit the phylum’s genetic capacity to use hydrogen as an energy source and its oligotrophic nature by culturing in nutrient-limited conditions combined with ~60 ppmv hydrogen gas. While attempts were not successful in isolating Ca. Dormibacterota, the study demonstrated enrichment of a single strain from Ca. Dormibacterota and provided invaluable insight for future cultivation strategies. The method did, surprisingly, prove invaluable for the enrichment of uncultured bacterial species and for the isolation of a suite of novel Antarctic fungi.
