Genetic modification of heavy metal resistant Streptomyces sp. strains

Two heavy metal resistant Streptomyces sp. strains, S. mirabilis P16 B-1 and S. acidiscabies E13, isolated from a former uranium mining site are investigated for genetic metal resistance determinants. After establishing a transformation system for Streptomyces sp. using conjugation with E. coli, the streptomycetes are transformed by random transposon mutagenesis yielding mutants with reduced metal resistance. The integration sites serve as targets for directed deletion via PCR-targeted gene replacement. It is demonstrated that a large linear S. mirabilis plasmid plays a major role in conferring nickel resistance and is transferrable to S. lividans. An NreB-like transporter encoded on this plasmid is identified as a nickel resistance factor, which could also be expressed in E. coli. Additionally, the role of plasmid-borne copper homeostasis system components, i.e. a transcriptional CsoR-type repressor and a CopZ-like chaperone, is surveyed. The tyrosinase-encoding gene melC2, located on a second linear S. mirabilis plasmid, is shown to hamper nickel resistance on complex medium, providing one reason for the strains higher nickel resistance on minimal medium. Furthermore, genes encoding means for coping with different environmental stressors are investigated, e.g. an HAD-like hydrolase, a Na+,Li+/H+ antiporter, a phosphinothricin N-acetyltransferase. Besides, the composition of extracellular matrix around the streptomycete hyphae and cell membrane composition are shown to impact metal resistance, i.e. loss of CslA function increases nickel resistance. Taken together, by demonstrating the applicability of molecular transformation methods for heavy metal resistant Streptomyces sp. strains this study provides the basis for further investigations of the metal resistome. The results indicate that metal resistance of both investigated strains is a result of multiple genetic determinants, several of which are plasmid-encoded.


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