The long-term goals of the proposed research are to elucidate the role of bacteria in modifying metal ion (particularly copper) speciation in aquatic environments. Specifically, this study will further document the occurrence of the bacterial response to copper ion stress involving production of extracellular, proteinaceous metal chelators and their physiological role(s). Environmental health-related benefits of the research include an improved understanding of the response of aquatic environments to heavy metal contamination, influence of aquatic biota in """"""""buffering"""""""" toxicity of heavy metals, and the influence of metals on lower members of the human food chain. In general, the research will contribute to our understanding of homeostatic mechanisms involved in trace metal metabolism and detoxification. The first phase of the study will focus on pinpointing the role of the copper binding proteins (CuBP) of Vibrio alginolyticus in metal detoxification. Tn5-l32 transductants will be screened for sensitivity to copper, and copper sensitive mutants will be screened for CuBP production. A copper sensitive Vibrio alginolyticus deficient in CuBP expression will provide a direct link between CuBP and copper detoxification. In addition, cultures will be screened for copper resistant mutants which overproduce CuBP. A Chesapeake Bay isolate of Vibrio alginolyticus will be tested for production of compounds similar to CuBP, as will Escherichia coli and Pseudomonasputida. The possibility that CuBP is induced by other environmental stresses, such as iron and carbon limitation, will also be examined. Specific methods employed in the study are polyacrylamide and agarose gel electrophoresis, immobilized metal ion affinity chromatography (IMAC) for characterization and quantitation of copper complexing supernatant proteins, and transduction by the transposable element Tn5-l32. Tangential flow filtration, IMAC, gel permeation chromatography and anion exchange chromatography will be used to concentrate and purify CuBP. Human health implications of the research regard the environmental fate of potentially toxic heavy metals, and, therefore, mechanisms by which they may be incorporated into the food chain.
| Gordon, A S; Howell, L D; Harwood, V (1994) Responses of diverse heterotrophic bacteria to elevated copper concentrations. Can J Microbiol 40:408-11 |
| Harwood, V J; Gordon, A S (1994) Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus. Appl Environ Microbiol 60:1749-53 |
| Gordon, A S; Harwood, V J; Sayyar, S (1993) Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus. Appl Environ Microbiol 59:60-6 |
