Genomic and physiological characterization of two heavy metal resistant bacteria isolated from a long-term metalliferous ecosystem.

Remediation of mixed heavy metal contamination in U.S. nuclear legacy sites remains a persistent challenge. This study examines the genomic and functional traits of two newly isolated bacterial strains-Stenotrophomonas strain 3 and Pseudomonas strain 8-from the metal-contaminated D-Area Ash Plume at the Savannah River Site (SRS). Growth assays showed strain 8 tolerated cobalt (Co) (400 ppm) and copper (Cu) (1000 ppm), while strain 3 thrived in zinc (Zn) (20 000 ppm). Both exhibited limited growth under high nickel exposure, indicating distinct metal-specific resistance profiles. Whole-genome sequencing revealed distinct genomic adaptations: strain 8 carried copA, cnrA, NiCoT, and zitB, supporting its Co and Cu tolerance, while strain 3 harbored czcA and zitB, consistent with Zn resistance. Strain 3's genome comprised 61 contigs (4.41 Mb, 66.6% GC), and strain 8's included 62 contigs (6.93 Mb, 63.48% GC). Comparative genomic analysis of strains 3 and 8 with several previously reported SRS isolates revealed that Burkholderia spp. (SRS-25, SRS-46, SRS-W-2-2016) possessed the most extensive resistance gene repertoire, followed by Stenotrophomonas and Pseudomonas. Co-localized metal resistance genes and antibiotic resistance genes suggest shared stress response pathways and horizontal gene transfer potential, underscoring the bioremediation promise of native SRS bacteria for both metal and antibiotic contaminants.