# Metabolic Responses, Cell Recoverability, and Protein Signatures of Three Extremophiles: Sustained Life During Long-Term Subzero Incubations

## TL;DR

This study explores how three Arctic bacteria survive and remain active in icy, salty environments over a year, identifying proteins and metabolic responses that could help detect life in extreme conditions.

## Contribution

The study introduces the first use of isotopically labeled leucine and proteomics to track new protein synthesis in subzero conditions for long-term survival of extremophiles.

## Key findings

- All three bacterial strains remained metabolically active at −5 °C after 12 months.
- Newly synthesized proteins in Cp34H at −5 °C were detectable within one hour and included those involved in motility and metabolism.
- Recoverability and metabolic activity varied significantly among strains at −10 and −36 °C.

## Abstract

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.