Genome-wide analysis reveals structured ecological and functional divergence within Geobacillus stearothermophilus.

Geobacillus stearothermophilus is a thermophilic bacterium widely used in food sterilization and industrial processes. Although it has long been treated as a single, well-defined species, its internal genomic diversity has not been systematically evaluated. Here, we analyzed 36 strains using comparative genomics to clarify the structure of diversity within this species. Phylogenetic analyses consistently revealed two major genomic groups. Genome similarity measurements showed that most strains met current species-level criteria, yet clear internal differentiation was present. The two groups differed in ecological origin and genome composition. Strains associated with food-related environments tended to have smaller genomes and fewer metabolic genes, whereas strains from natural thermal habitats possessed larger genomes and broader metabolic capabilities, including genes for carbohydrate and fatty acid utilization. A small number of strains displayed intermediate positions, suggesting gradual diversification rather than sharp separation. Despite pronounced internal structuring, the strains remain within accepted species boundaries. These findings demonstrate that substantial ecological and functional divergence can accumulate within a single bacterial species. Our results provide a genomic framework for understanding intraspecific diversity in thermophilic bacteria and illustrate the importance of interpreting genome similarity thresholds in the context of population structure.