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 (400 ppm) and copper (1000 ppm), while strain 3 thrived in zinc (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 cobalt and copper tolerance, while strain 3 harbored czcA and zitB, consistent with zinc 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 os 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 MRGs and ARGs suggest shared stress response pathways and horizontal gene transfer potential, underscoring the bioremediation promise of native SRS bacteria for both metal and antibiotic contaminants.