Dual Screen for Metal-Tolerant Metallophore Producers Evaluated with Soil from the Carpenter Snow Creek Site, a Heavy-Metal-Toxified Site in Montana

Abstract

Bacteria have evolved numerous mechanisms to resist metal toxicity, including small-molecule metal chelators (metallophores). This study presents a dual screening methodology to isolate metallophore-producing bacteria from the Carpenter Snow Creek Mining District for potential use in heavy-metal bioremediation. Soil samples were screened on metal-supplemented plates from which colonies were picked onto chrome azurol S (CAS)-dyed plates. Copper or cerium toxicity was used as the primary selection step, while the CAS assay revealed the excretion of metal-binding compounds. From the pool of bacteria encompassed in the native soil microbiome, fifty-one isolates were picked from metal-toxified media by colony morphology. Out of these colonies, 17 exhibited positive results in the CAS assay. 16S rRNA sequencing identified eight unique species within these CAS-positive hits, the nearest BLAST hits of which were from the genera: Rhodanobacter, Dyella, Bradyrhizobium, Luteibacter, Cupriavidus, Arthrobacter, and Paraburkholderia. To validate our workflow, we profiled our Cupriavidus isolate by LCMS metabolomics and genome mining and purified its metabolites. These efforts led to the reisolation of the known metallophore taiwachelin. In efforts to identify lead strains for heavy-metal bioremediation applications, the present work suggests the utility of our screening method in rapidly targeting the metallophore producers from the soil microbiome.

Figure 1

Dual screening method successfully targets metallophore-producing bacteria from the soil microbiome by substantially reducing the number of bacterial candidates for the desired application in heavy-metal bioremediation.

Figure 2

Metabolomic analysis of C. basilensis BL-MT-10 revealed a cluster of putative taiwachelins (highlighted section [M + H]⁺ of m/z 963 and 991 for taiwachelin (1) and its analogue (2), respectively). Nodes are colored by the samples in which they were observed: blue = supernatants from C. basilenesis BL-MT-10, red = blank runs, and yellow = both. Thicker lines indicate higher cosine scores.