Dissection of novel resistance mechanisms in Cupriavidus metallidurans

Cupriavidus metallidurans strains, which belong to the Burkholderiaceae family, are characterized by multiple metal resistances and have been mostly isolated from industrial sites linked to mining, metallurgic and chemical industries. Studies at SCK•CEN underscored the rapid evolution of C. metallidurans strains towards significantly increased metal resistance. Surprisingly, the canonical mechanisms did not participate in this adaptive evolution. In contrast, a novel and unique resistance mechanism, involving an uncharacterized small periplasmic protein, was discovered.

In this work, we investigated silver resistance. Different C. metallidurans strains including type strain CH34, its plasmidless derivative AE104 and strain NA4, which was isolated form the silver-sanitized drinking water of International Space Station (ISS), were grown in toxic concentration of silver to obtain silver-resistant mutants. Since silver-resistant mutants were even found in the plasmidless AE104 strain, the dominant silver-resistant mechanism does not include the canonical efflux mechanisms encoded by the megaplasmids. Whole-genome expression profiling was used to compare the up- and downregulated genes of the silver-resistant mutants with their respective parents. Only eight genes were commonly upregulated in all silver-resistant mutants and no commonly downregulated genes were observed. Deletion mutants were constructed for these genes and observations confirmed that deletion of the two-component system agrRS and a copQ-like gene coding for a small periplasmic protein render susceptibility to silver. Furthermore, plasmid-based complementation restored resistance to silver. Further investigation is ongoing with the other differentially expressed genes and to validate the DNA-binding properties of AgrR.

Altogether, our data indicate that C. metallidurans is able to adapt rapidly to toxic silver concentrations without mediation of its known silver efflux pumps. Although the mechanism that confers increased silver resistance is still not fully understood, these results indicate differential regulation via a two-component regulatory system and the involvement of a family of small periplasmic proteins.