9604298 Saffarini Anaerobic metal reduction is a major respiratory process in aquatic environments and in some instances it can account for 80-90% of the total organic carbon that is oxidized. Although this process is widespread in aquatic environments, very little is known about the mechanisms of iron reduction. Iron oxides and oxyhydroxides are solids and highly insoluble. Contact with the iron particles is required for reduction to occur. This raises the question as to the mechanisms used for solid Fe(III) reduction. Is the iron reduced outside the cell, on the outer membrane as has been suggested previously, or is it solubilized first and then transported to the inner membrane where it is reduced? Shewanella putrefaciens is an iron reducer that can use amazingly large number of terminal electron acceptors, both soluble and solid. The organization and localization of the electron transport chain components, and the mechanisms for dealing with soluble vs. solid terminal electron acceptors are intriguing problems. S. putrefaciens serves as an excellent model system to study iron reduction. It is a facultative anaerobe, and therefore amenable to genetic manipulations. Other metal reducers identified so far are strict anaerobes. The goal of the project is to elucidate the mechanisms of iron transport and reduction and to identify the genes involved. Transposon mutants deficient in iron reduction have been isolated. These mutants will be complemented and the genes sequenced. DNA sequences will be analyzed to determine similarities to other reductase systems. The genes encoding the ferric reductase will be expressed in E. coli and antibodies will be generated for immunolocalization of the enzymes in the cell. The factors involved in regulating the expression of the iron reductase will also be investigated. Anaerobic metal reduction is a major respiratory process in aquatic environments and can account for 80-90% of the total organic carbon that is oxidized. Furthermore, an increasingly large number of bacteria are being identified that are capable or iron reduction. One such organism is Shewanella putrefaciens which has been isolated from a variety of different environments. Although iron reduction is widespread in aquatic environments, very little is known about the mechanisms by which bacteria use this metal for respiration. This project will shed light on the mechanisms of iron reduction and its regulation using molecular biological techniques. In addition to providing a basis for understanding of a novel enzyme, a detailed study of the iron reductase will provide insights into the physiology and genetics of an environmentally significant organism. Iron reducing bacteria are being considered as agents of bioremediation because of their ability to anaerobically oxidize aromatic compounds coupled to the reduction of iron. These bacteria cannot be used or manipulated unless the processes by which iron is reduced and regulated are understood.
