Q-301. Strategies for Living with High Concentrations of Copper; Kinetics and Equilibrium of Copper Sequestration by Ralstonia pickettii

F. Yang, S-H. Kim, M. Fujimoto, T. L. Marsh;
Ctr. for Microbial Ecology, Michigan State Univ., East Lansing, MI.

We have previously described the isolation of Ralstonia pickettii strains from a copper-contaminated lake sediment. These strains are resistant to high concentrations of copper (1200 µg/ml) and sequester large amounts of the metal. We have determined that the isolates fall into two genomovar groups that differ significantly in genome structure, carriage of a filamentous phage and resistance to other metals besides copper. Here we present our genomic analysis of representatives from each genomovar focusing on genes that provide resistance to metals and copper in particular. In both genomovars we find multiple copies of copper resistance proteins, copper efflux proteins, copper binding proteins and copper oxidases. Multiple copies of the copABCD operon are also present in these strains, frequently close to transposases or insertion sequences. Scanning and transmittance electron microscopy coupled with energy dispersive spectroscopy revealed that cells grown in the presence of high copper had an accumulation of copper in the outer cell envelope. Using a rapid filtration protocol coupled to a modified bicinchoninic acid assay, we determined the kinetics of copper sequestration. The sequestration by live cells reached equilibrium (35 mg Cu/g dry wt.) within 24 hours. Dead cells sequestered less copper (17 mg Cu/g dry wt.) but reached equilibrium in 1 hour. A positive cooperative association during copper binding was observed in live R. pickettii but not in dead cells. Finally, active cultures exported a putative polysaccharide that formed a colloidal complex with copper when copper concentration was greater than 1200 µg/ml. Up to 55% of the copper in solution could be precipitated by this extracellular factor derived from a stationary phase culture. These multiple strategies for survival in high concentrations of copper may provide exploitable technologies for bioremediation.