Genome-resolved characterization of candidate thermotolerance traits and predicted protein conformational behavior in Calditerricola during hyperthermophilic composting of organic wastes.
Hyperthermophilic composting (HC) can generate temperatures above 80 °C without external heating, thereby accelerating organic-waste stabilization; however, how dominant heat-adapted microorganisms maintain cellular function under such extreme conditions remains unclear. Here, we integrated metagenomics, metagenome-assembled genome reconstruction, Calditerricola-resolved functional profiling, partial least squares path modeling, and molecular dynamics simulations to investigate candidate thermotolerance-related traits associated with Calditerricola enrichment during HC. The pile temperature reached 82.6 °C on day 2 and peaked at 86.6 °C on day 4, accompanied by progressive humification, with humic substances increasing from 40.45 to 51.28 mg/g and HA/FA reaching 3.45. Microbial communities differed significantly among composting phases (R2 = 0.975, P = 0.004), and Calditerricola increased from 0.02% in the initial phase to 6.1% in the thermophilic phase before declining to 0.7% in maturation. Community-level profiles showed comparatively modest variation in selected thermotolerance-related pathways, whereas the independently normalized Calditerricola profile displayed clearer phase-associated increases in functions linked to polyamine metabolism, membrane/envelope homeostasis, proteostasis, and DNA maintenance. Path modeling revealed consistent positive associations between Calditerricola enrichment and polyamine synthesis, membrane stability, and proteostasis. qPCR further revealed phase-associated increases in the community-level copy numbers of representative target genes, particularly polA and speE. During 100-ns simulations at 360 K, the predicted apo structures of PolA, AtpD, SpeE, and FabH retained their overall folds and comparatively stable catalytic-residue geometries. Together, these results define an association-based multi-module framework of candidate traits linked to Calditerricola persistence during HC, providing a genome-resolved basis for prioritizing testable thermotolerance targets in engineered high-temperature waste-treatment systems.