Single-Cell Genomics of Novel Actinobacteria With the Wood-Ljungdahl Pathway Discovered in a Serpentinizing System

Serpentinite-hosted systems represent modern-day analogs of early Earth environments. In these systems, water-rock interactions generate highly alkaline and reducing fluids that can contain hydrogen, methane, and low-molecular-weight hydrocarbons-potent reductants capable of fueling microbial metabolism. In this study, we investigated the microbiota of Hakuba Happo hot springs (similar to 50 degrees C; pH similar to 10.5-11), located in Nagano (Japan), which are impacted by the serpentinization process. Analysis of the 16S rRNA gene amplicon sequences revealed that the bacterial community comprisesNitrospirae(47%), Parcubacteria (19%),Deinococcus-Thermus(16%), andActinobacteria(9%), among others. Notably, only 57 amplicon sequence variants (ASV) were detected, and fifteen of these accounted for 90% of the amplicons. Among the abundant ASVs, an early-branching, uncultivated actinobacterial clade identified as RBG-16-55-12 in the SILVA database was detected. Ten single-cell genomes (average pairwise nucleotide identity: 0.98-1.00; estimated completeness: 33-93%; estimated genome size: similar to 2.3 Mb) that affiliated with this clade were obtained. Taxonomic classification using single copy genes indicates that the genomes belong to the actinobacterial class-level clade UBA1414 in the Genome Taxonomy Database. Based on metabolic pathway predictions, these actinobacteria are anaerobes, capable of glycolysis, dissimilatory nitrate reduction and CO(2)fixationviathe Wood-Ljungdahl (WL) pathway. Several other genomes within UBA1414 and two related class-level clades also encode the WL pathway, which has not yet been reported for theActinobacteriaphylum. For the Hakuba actinobacterium, the energy metabolism related to the WL pathway is likely supported by a combination of the Rnf complex, group 3b and 3d [NiFe]-hydrogenases, [FeFe]-hydrogenases, and V-type (H+/Na(+)pump) ATPase. The genomes also harbor a form IV ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, also known as a RubisCO-like protein, and contain signatures of interactions with viruses, including clustered regularly interspaced short palindromic repeat (CRISPR) regions and several phage integrases. This is the first report and detailed genome analysis of a bacterium within theActinobacteriaphylum capable of utilizing the WL pathway. The Hakuba actinobacterium is a member of the clade UBA1414/RBG-16-55-12, formerly within the group OPB41. We propose to name this bacterium 'CandidatusHakubanella thermoalkaliphilus.'