PROTEOSTASIS IN ACIDOPHILES: DIFFERENTIAL EXPRESSION OF PROTEASES AND FUNCTIONAL ROLE OF LON1 UNDER STRESS CONDITIONS

Acidithiobacillus ferrooxidans is an acidophilic microorganism that thrives in extreme environments, facing stresses that cause protein damage, requiring rapid proteome remodeling for survival. Proteases play a central role in proteostasis by degrading nonfunctional proteins, recycling components, and regulating selective degradation to adapt cellular responses to stress. This study aimed to characterize At. ferrooxidans protease systems under stress and evaluate Lon1’s function. mRNA levels of genes encoding Lon1, Lon2, Lon3, HtrA1, HtrA2, ClpP, ClpX, ClpA, FtsH, HslU, and HslV were measured by RT-qPCR under thermal (37°C, 2 h), oxidative (1 mM H₂O₂, 1 h), acidic (pH 1.2 and 2.0, 1 h), and desiccation (30 min) stresses. Controls were cells grown at 30°C and pH 1.6. Stress responses were assessed immediately after exposure (shock) and after acclimatization (adapted stress). Lon1 was selected for functional studies via heterologous expression in Escherichia coli. Its role in oxidative stress tolerance was evaluated by determining the MIC of hydrogen peroxide in cultures exposed to serial dilutions of H₂O₂ (0–10 mM). Lon1’s ability to reduce protein aggregation was analyzed by purifying intracellular aggregates after thermal (45 °C), oxidative (H₂O₂ at IC50), and acidic (pH 4.5) stress. Protein quantification was performed using the Bradford assay. Results showed predominant upregulation of protease mRNAs under adapted versus shock stress, indicating a greater role during prolonged exposure. HslUV was strongly upregulated in most stresses except desiccation, where other systems prevailed, supporting stress-specific protease functions. Differential expression of Lon and HtrA copies suggests distinct cellular roles. Lon1 increased the MIC of hydrogen peroxide from 2.2 to 3.4 mM and decreased protein aggregation by 82% under thermal stress and 79% under acidic conditions. Proteases exhibit stress-specific responses, suggesting a differential adaptation of proteolytic systems to respond to various types of stress in extreme environments in order to maintain proteostasis.