Prevalence of the Calvin-Benson-Bassham cycle in chemolithoautotrophic psychrophiles and the potential for cold-adapted Rubisco

The act of fixing inorganic carbon into the biosphere is largely facilitated by one enzyme, Rubisco. Beyond well-studied plants and cyanobacteria, many bacteria use Rubisco for chemolithoautotrophy in extreme environments on Earth. Here, we characterized the diversity of chemolithoautotrophic Rubiscos in subzero environments. First, we surveyed subzero environments and found that the Calvin-Benson-Bassham cycle was the most prevalent chemolithoautotrophic pathway. Second, we uncovered potential for chemolithoautotrophy in metagenomes from two distinct subzero, hypersaline Arctic environments: 40-kyr relic marine brines encased within permafrost (cryopeg brines) and first-year sea ice. Again, the Calvin-Benson-Bassham cycle was the dominant chemolithoautotrophic pathway in both environments, though with different Rubisco forms. From cryopeg brine, we reconstructed four metagenome-assembled genomes with the potential for chemolithoautotrophy, of which the sulfur-oxidizing genus Thiomicrorhabdus was most abundant. A broader survey of Thiomicrorhabdus genomes from diverse environments identified a core complement of three Rubisco forms (II, IAc, IAq) with distinct patterns of gain and loss. We developed a model framework and compared these different Rubisco forms across [CO ₂ ], [O ₂ ], and temperature. We found that form II outcompetes form I at low O ₂ , but cold temperatures minimize this advantage. However, further inspection of form II from cold environments uncovered signals of thermal adaptation of key amino acids which resulted in a more exposed active site. These modifications suggest that these form II Rubisco proteins may have unique kinetics or thermal stability. This work can help address the limits of autotrophic functionality in extreme environments on Earth and other planetary bodies.