Mechanisms of unique chaotropic resistance and biocalcification in a halophilic Virgibacillus isolated from ephemeral microbial mats of the calcium-rich Don Juan pond in Antarctica

Introduction Don Juan Pond (DJP), located in the McMurdo Dry Valleys of Antarctica, is one of the most extreme cold aquatic environments on our planet and most similar to Mars due to its CaCl₂-rich brines, low water activity, exceptional chaotropicity, average annual subzero temperatures and intense solar radiation during the Antarctic summer. Although the central DJP brine is considered anathema to life, the surrounding basin seasonally hosts temporary streams, ponds, and, very rarely, ephemeral microbial mats. Methods In this study, we investigated the diversity of these microbial mats and attempted to cultivate Ca 2+ -tolerant bacteria and analyze the phenotypic and molecular characteristics of the most promising isolates. To understand the molecular mechanisms underlying their adaptive strategy, further analysis of their genomic potential, adaptive epigenetic transformations and differential transcriptomics was conducted. Predicted functions were verified through physiological experiments. Results The most calcium-tolerant isolate, Virgibacillus flavescens DJP025-4, was studied in detail, including its adaptive physiology and, in particular, its remarkable tolerance to varying calcium concentrations, unmatched by other prokaryotes. DJP025-4 cells were able to survive and remain cultivable after 3 months of exposure to 3.0 M Ca 2+ , corresponding to a water activity of approximately 0.63 and total chaotropicity value of +276.6 kJ kg −1 , conditions well below the current limit for life survival equivalent. Transcriptome analysis revealed that the primary metabolic response of strain DJP025-4 to Ca 2+ stress includes enzymatically induced urease-independent CaCO 3 precipitation, enhanced uptake of external compatible solutes, and activation of cell surface structures biosynthesis, including peptidoglycan metabolism, biofilm matrix formation, and cellular appendage assembly, which finally increase the cell surface area and the number of Ca 2 + precipitation centers on it. Whole-genome analysis revealed a high degree of genetic plasticity in this bacterium, involving natural transformation with genetic material acquired from the bacterial community and adaptive epigenetic mechanisms of gene regulation. Discussion Among the key findings, it is worth noting that, in addition to developing remarkable adaptation mechanisms to withstand and thrive in a harsh, calcium-rich environment, strain DJP025-4 appears to remain dependent on the presence of external compatible solutes. We hypothesize that within the DJP stream ecosystem, it is able to survive at high Ca 2+ concentrations by utilizing trace amounts of such substances produced by other organisms, specifically, moderate halophiles inhabiting surface microbial mats in lower-salinity zones, which release dissolved organic matter and osmolytes into the environment.