Genome-resolved insights into the bacterial phylum WOR-3: hydrogenotrophic metabolism and unique carbon fixation via archaeal form III RuBisCO.

The WOR-3 phylum is widely distributed in various environments, including hot springs, marine ecosystems, and hydrothermal vents, yet its ecological roles and metabolic capabilities remain poorly understood. In this study, we analyzed 181 medium- to high-quality metagenome-assembled genomes, including 59 newly reconstructed from environmental samples and 122 retrieved from public databases. Phylogenetic analyses resolved the WOR-3 lineage into four subgroups (subgroup 1-4). Metabolic reconstruction revealed significant divergence of the carbon, sulfur, nitrogen, and hydrogen metabolism pathways among the different subgroups. Subgroup 1 was characterized by fermentative metabolism involving formate and ethanol and uniquely exhibited potential for carbon fixation via the Calvin cycle, as indicated by the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene. Notably, WOR-3 RuBisCO is phylogenetically affiliated with archaeal form III, although the carbon fixation pathway follows the canonical bacterial Calvin cycle-a feature of potential evolutionary significance. Subgroup 3 exhibits metabolic versatility, including genes for dissimilatory sulfate reduction, sulfur oxidation, partial denitrification, and fatty acid degradation. In addition, all subgroups harbored key components of hydrogen metabolism, including widespread NiFe hydrogenases, supporting H2-dependent electron transfer and energy conservation. Within the WOR-3 lineage, the coexistence of two respiratory enzyme systems-the Rnf complex and the oxidative phosphorylation respiratory chain-indicates distinct anaerobic and aerobic metabolic lifestyles, respectively. Collectively, this study expands the genomic framework for the WOR-3 phylum and provides novel insights into the metabolic versatility and ecological functions of this previously uncharacterized lineage in biogeochemical cycles of carbon, nitrogen, and sulfur.IMPORTANCEThe WOR-3 phylum represents a widespread but poorly understood bacterial lineage inhabiting diverse various environments. By integrating 181 metagenome-assembled genomes, including 59 newly reconstructed, this study provides the most comprehensive genomic framework to date for WOR-3. Phylogenomic and metabolic reconstruction revealed four distinct subgroups with divergent capacities for carbon, sulfur, and nitrogen metabolism. Notably, subgroup 1 encodes a complete Calvin-Benson-Bassham cycle featuring an archaeal-type form III ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting an unusual evolutionary trajectory for carbon fixation in this lineage. Subgroup 3 exhibits versatile metabolic potential, including dissimilatory sulfur metabolism, partial denitrification, and fatty acid degradation, highlighting its possible roles in multiple biogeochemical processes. These findings not only expand the taxonomic and functional landscape of the WOR-3 phylum but also offer key insights into its ecological roles in global element cycling.