Twenty-two novel glacier Pseudomonas species expand cultivated representation: nitrogen-cycling potential and temperature-responsive proteomes.

Accelerating glacier retreat exports cryospheric microbiomes into downstream ecosystems, raising questions about their functional roles. However, a persistent gap between genomic predictions and physiologically validated functions exists, largely due to the scarcity of cultivated and taxonomically resolved model organisms from these environments. Here we characterize 23 Pseudomonas strains isolated from ice, cryoconite, and supraglacial meltwater collected across ten glaciers in China. Phylogenomic inference places them within the Pseudomonas mandelii subgroup, and genome metrics delineate 22 strains as distinct novel species (ANI ≤95.71%; dDDH ≤64.6% to their closest type strains). Phenotypic analyses showed broad environmental tolerance, including growth at -2°C, strain-dependent upper growth limits of 30°C-37°C, and growth over an approximate pH range of 5.0-11.0. Genome-based reconstruction predicted heterogeneous nitrogen-transforming potential, including DNRA-related genes in all strains, nitrate-reduction genes in 14 strains, complete denitrification marker sets in 12 strains, and nitrogen-fixation genes in one strain. Phenotypic assays showed broader nitrate-reduction activity than predicted, with all 23 strains reducing nitrate to nitrite and seven showing reduction beyond nitrite, underscoring the value of cultivation-based functional validation in non-model cryospheric bacteria. Comparative proteomics of four representative species at 0°C versus 28°C reveals shared temperature-responsive pathways and a common network backbone involving translation, proteostasis, and carbon economy. Together, these cultured genomes provide valuable models for linking cryospheric Pseudomonas diversity to temperature-responsive physiology and biogeochemical potential.