Cold and Salt Tolerant Hydrolase Producing Bacteria from the High Altitude Cold Desert of Ladakh, India: Characterization and Comparative In Silico Analysis

Abstract

The high-altitude cold desert of Ladakh represents a polyextreme environment where low temperature, repeated freeze-thaw cycles, desiccation, intense solar radiation, and salinity fluctuations collectively shape microbial survival. In the present study, water samples from Pangong Lake and the Indus River were used to isolate culturable bacteria across NaCl gradients (0.9-12%, w/v) at 20 and 37 °C. A total of 112 bacterial isolates were recovered and screened for extracellular amylase, protease, cellulase, and xylanase production, revealing broad hydrolytic potential along with notable pigmentation diversity. Six representative enzyme-hyperproducing isolates were selected for phenotypic, biochemical, and 16S rRNA gene-based characterization and showed closest affiliation to Halobacillus trueperi, Alkalibacillus flavidus, Glutamicibacter nicotianae, Brachybacterium rhamnosum, and Pantoea vagans. To extend the culture-based observations, comparative in silico analyses were performed using publicly available reference proteomes corresponding to the closest identified taxa. These analyses revealed trends associated with adaptation to low temperature and osmotic stress, including variation in glycine-to-proline ratio, shifts in mean isoelectric point, and reduced salt-bridge networks in modeled cold shock proteins relative to the thermophilic reference Thermus thermophilus. Comparative gene mining also indicated the presence of UV-repair and oxidative-stress functions relevant to persistence in high-altitude environments. Together, these findings highlight the biotechnological potential of culturable Ladakh bacteria as sources of hydrolases active under combined cold and saline conditions, while providing preliminary comparative insights into molecular traits associated with survival in high-altitude cold-desert habitats.