Since the discovery of perchlorates in martian soils, astrobiologists have been curious if and how life could survive in these low-water, high-salt environments. Perchlorates induce chaotropic and oxidative stress but can also confer increased cold tolerance in some extremophiles. Though bacterial survival has been demonstrated at subzero temperatures and in perchlorate solution, proteomic analysis of cells growing in an environment like martian regolith brines–perchlorate with subzero temperatures–has yet to be demonstrated. By defining biosignatures of survival and growth in perchlorate-amended media at subzero conditions, we move closer to understanding the mechanisms that underlie the feasibility of life on Mars. Colwellia psychrerythraea str. 34H (Cp34H), a marine psychrophile, was exposed to perchlorate ions in the form of a diluted Phoenix Mars Lander Wet Chemistry Laboratory solution at –1°C and –5°C. At both temperatures in perchlorate-amended media, Cp34H grew at reduced rates. Mass spectrometry-based proteomics analyses revealed that proteins responsible for mitigating effects of oxidative and chaotropic stress increased, while cellular transport proteins decreased. Cumulative protein signatures suggested modifications to cell–cell or cell–surface adhesion properties. These physical and biochemical traits could serve as putative identifiable biosignatures for life detection in martian environments.

Few halophilic strains have been examined in detail for their culturability and metabolic activity at subzero temperatures, within the ice matrix, over the longer term. Here, we examine three Arctic strains with varied salinity tolerances: Colwellia psychrerythraea str. 34H (Cp34H), Psychrobacter sp. str. 7E (P7E), and Halomonas sp. str. 3E (H3E). As a proxy for biosignatures, we examine observable cells, metabolic activity, and recoverability on 12-month incubations at –5, –10 and –36°C. To further develop life-detection strategies, we also study the short-term tracking of new protein synthesis on Cp34H at –5°C for the first time, using isotopically labeled ¹³C₆-leucine and mass spectrometry-based proteomics. All three bacterial species remained metabolically active after 12 months at –5°C, while recoverability varied greatly among strains. At –10 and –36°C, metabolic activity was drastically reduced and recoverability patterns were strain-specific. Cells were observable at high numbers in all treatments, validating their potential as biosignatures. Newly synthesized proteins were detectable and identifiable after one hour of incubation. Proteins prioritized for synthesis with the provided substrate are involved in motility, protein synthesis, and in nitrogen and carbohydrate metabolism, with an emphasis on structural proteins, enzymatic activities in central metabolic pathways, and regulatory functions.