Cross and unique stress adaptation strategies of the polyextremophile Natranaerobius thermophilus to individual high salt, alkaline pH, and elevated temperature.

N. thermophilus is the first true anaerobic halophilic alkalithermophile. It employs a unique dual mechanism for hypersaline adaptation, utilizing both "compatible solutes" and "salt in" strategies. However, the molecular mechanisms underlying its responses to alkaline pH and thermal stress remain poorly characterized. An iTRAQ-based quantitative proteomics analysis revealed that N. thermophilus used a cross and unique adaptation strategies to three individual extreme stresses. This study fills gaps by elucidating previously unexplored alkaline-specific regulatory processes. It also provides the first comprehensive analysis of its thermal adaptation mechanisms. In response to high-salt and alkaline stress, the organism shifts its metabolism toward glycolysis and pyruvate-derived acetate synthesis, helping to meet increased ATP demands. Heat shock proteins are up-regulated during both alkaline and thermal adaptations, reflecting the "No free lunch" principle. Alkaline pH uniquely induces DNA repair proteins and S-adenosylmethionine biosynthesis enzymes, promoting genomic stability in proton-deficient environments. Besides, the compact genome and the positive correlation between GC content with growth temperature may be also a lineage-specific thermal adaptation of the halophilic and alkalithermophilic order Natranaerobiales. These findings illuminate the layered adaptation strategies that help address cross-stress challenges. Meanwhile, stress-specific reconfigurations enhance flexibility for survival in individual extremes. This work provides novel insights into the survival mechanisms of polyextremophiles, as well as advancing their potential biotechnological applications. SIGNIFICANCE: Halophilic alkalithermophile N. thermophilus exemplify life's capacity to thrive in environments where multiple physicochemical extremes intersect. However, the mechanisms underlying alkaline adaptation remain inadequately characterized, and our understanding of thermal adaptation is limited to genomic analyses. This study addresses critical gaps by disentangling the responses to hypersaline, alkalinity, and thermal stress, thereby elucidating how N. thermophilus organizes its survival strategies. This research reveals that N. thermophilus employs a strategy that combines conserved cross-stress mechanisms with unique stress adaptations to cope with the three distinct extreme stresses of high salinity, alkalinity, and temperature. By identifying the molecular modules through which these mechanisms operate, this research sets the stage for future applications in synthetic biology, particularly in the design of extremophile chassis for bioprocessing under multi-extreme conditions. These insights not only enhance our understanding of polyextremophiles but also pave the way for innovative biotechnological solutions.