Orthology-based comparative transcriptomic analysis reveals conserved osmoadaptive responses in Bacillus velezensis and Bacillus paralicheniformis.

Salinity imposes severe osmotic and ionic challenges that require coordinated adaptive responses in microorganisms. Although numerous studies have characterized salt-stress responses in individual Bacillus species, the extent to which these responses are conserved across phylogenetically distinct taxa remains poorly understood. Here, we performed an orthology-based comparative transcriptomic analysis of Bacillus velezensis and Bacillus paralicheniformis using publicly available RNA-seq datasets comparing low- and high-salinity conditions within each species. Publicly available RNA-seq datasets were analyzed using Salmon for transcript quantification and DESeq2 for differential expression analysis, followed by reciprocal best-hit orthology inference and functional annotation through KEGG Orthology assignments. Differential expression analysis identified 1,893 and 1,879 genes with significant expression changes (padj < 0.05, |log2FC|≥ 1) in B. velezensis and B. paralicheniformis, respectively. Reciprocal best-hit analysis recovered 2,963 orthologous gene pairs, of which 725 orthologous pairs met the differential-expression criteria in both species and were retained for comparative response classification. Among these, 234 orthologs were consistently upregulated and 230 were consistently downregulated under salt stress. Conserved induction was dominated by genes associated with compatible-solute metabolism, transport systems, and sodium homeostasis, with additional contributions from oxidative-stress-related functions. Notably, proline biosynthesis genes (proB and proC), glycine betaine-related genes (betB and gbsB), compatible-solute transporters (opuAA and opuAB), and the sodium/proton antiporter nhaC exhibited coordinated activation in both species. Conversely, conserved repression primarily affected biofilm-associated functions and extracellular matrix-related genes. Although KEGG Orthology enrichment analyses did not retain significance after false-discovery-rate correction, functional annotations consistently supported osmoadaptation-related processes as the dominant shared response. These results support the presence of conserved osmoadaptive transcriptional responses shared between Bacillus velezensis and Bacillus paralicheniformis and highlight orthology-guided comparative transcriptomics as an effective approach for identifying candidate conserved stress-response mechanisms.