Carbon-Fixation Potential in Zhuqueibacterota from a Central Indian Ridge Hydrothermal Vent Extends beyond Zhuqueibacterales, Tracking Habitat Rather Than Lineage

Abstract Background Deep-sea hydrothermal vent fields are biological hotspots sustained by chemosynthetic primary production rather than photosynthesis. The Onnuri Vent Field (OVF), discovered in 2018 on the Central Indian Ridge, exhibits an unusual diffuse-flow regime and supports a distinct faunal community, yet its microbial contributors to primary production remain incompletely characterized. Zhuqueibacterota (formerly Ca. KSB1) is an uncultured bacterial phylum long considered strictly heterotrophic, although a recent study proposed that a single order within this phylum, Zhuqueibacterales, can perform carbon fixation via the Calvin-Benson-Bassham (CBB) cycle. Because CBB-competent Zhuqueibacterales had not previously been reported from deep-sea hydrothermal vents, whether Zhuqueibacterota inhabiting the OVF belong to this order, and whether their capacity for carbon fixation extends to this environment, remained unexamined. Results Comparative shotgun metagenomic analysis of sediment from the OVF and two co-located chimney-type vent fields, Edmond and Solitaire, revealed a prokaryotic community at the OVF marked by the near-absence of Epsilonproteobacteria, the typically dominant chemolithoautotrophic primary producers at hydrothermal vents. Metagenome-assembled genome analysis identified Zhuqueibacterota as a lineage with high relative abundance unique to the OVF, encoding the complete gene set for carbon fixation via non-canonical CBB cycle variants. Zhuqueibacterota-derived RuBisCO sequences accounted for 7.2% of total RbcL sequences at the OVF, the second-largest contribution after Gammaproteobacteria. This carbon-fixing genome belonged to Oceanimicrobiales rather than Zhuqueibacterales, the order previously reported as the sole lineage capable of the CBB cycle. Phylogenetic analysis showed that possession of Form I RuBisCO, and thus the potential for active CBB cycle operation, tracked with isolation habitat rather than taxonomic lineage across the phylum, a pattern corroborated by global mapping of Zhuqueibacterota-derived RbcL sequences against environmental metagenomes. Conclusions The identification of a carbon-fixing Zhuqueibacterota genome within Oceanimicrobiales, rather than the previously implicated Zhuqueibacterales, demonstrates that CBB cycle capacity within this phylum cannot be inferred from order-level taxonomy alone. Instead, the association between Form I RuBisCO possession and isolation habitat, corroborated by global metagenomic mapping, indicates that habitat exerts a stronger influence on carbon-fixation potential than phylogenetic lineage in Zhuqueibacterota. These findings identify Zhuqueibacterota as an unrecognized contributor to carbon fixation in deep-sea hydrothermal vent ecosystems.