Whole genome sequencing and molecular characterization of two Bacillus licheniformis strains isolated from hot springs in yellowstone ational park.

Bacillus licheniformis is a Gram-positive, endospore-forming bacterium with broad biotechnological applications. Thermophilic environments such as hot springs may harbor strains with unique biosynthetic capabilities relevant to drug discovery. In this study, we isolated two B. licheniformis strains (S3 and S4) from the Five Sisters hot spring in Yellowstone National Park (68 °C and 65 °C, pH 8) and performed whole-genome sequencing using both the Oxford Nanopore long read and Illumina platforms. Hybrid de novo assembly using Unicycler yielded genome sizes of 4.80 Mbp (S3, 14 contigs) and 4.79 Mbp (S4, 22 contigs); GC contents were 45.12% and 45.10%, and N50 values were 4,546,802 bp and 2,415,736 bp, for S3 and S4, respectively. Both strains were assigned to Multi-Locus Sequence Typing sequence type ST-42. Pangenome comparison with 61 complete B. licheniformis genomes revealed an open pangenome of 10,374 genes, with 3,272 core genes, 430 soft core, 1,250 shell, and 5,422 cloud genes. AMRFinderPlus identified the blaP, encoding a class A beta-lactamase and its regulatory elements (blaI and blaR1); erm(D), encoding a 23S rRNA methyltransferase conferring macrolide-lincosamide-streptogramin B resistance; and catA, encoding a chloramphenicol O-acetyltransferase that inactivates chloramphenicol through acetylation in both strains. A chromosomal arsBC locus was identified in both B. licheniformis S3 and S4, consistent with the arsenic-rich geothermal environment of Five Sisters hot spring. These findings highlight the biosynthetic potential of B. licheniformis strains isolated from extreme environments and provide a genomic foundation for future exploration of novel bioactive compounds with potential applications in drug discovery, agriculture, and biotechnology.