A novel mode of bacterial respiration: iron solubilization prior to electron transfer
Fennessey, Christine Michelle
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Microbial iron respiration contributes significantly to the biogeochemical cycling of metals and may be one of the earliest respiratory processes to have evolved on early earth. Metal-respiring microbes also hold great potential for use in microbial fuel cells for the generation of "green" energy and for remediation of radionuclides in contaminated environments. Despite its significance in global metal cycling processes, the molecular mechanism of Fe(III) respiration has yet to be determined. Unlike many other terminal electron acceptors, Fe(III) is a solid at circumneutral pH and, therefore, cannot come into direct contact with the microbial inner membrane: the site of terminal electron transfer in gram-negative bacteria. It is postulated that metal-respiring organisms have developed alternate strategies for the reduction of solid iron. One such strategy involves the production of an Fe(III)-solublizing ligand by the metal-respiring bacteria which solubilizes the Fe(III) prior to respiration, rendering the metal more easily accessible to the Fe(III) reductase complex. In this study, the genes involved in the solubilization of Fe(III) by the gram-negative dissimilatory metal reducing bacteria Shewanella oneidensis MR-1 were determined using random mutagenesis to generate mutations in the wild-type genome and high-throughput square-wave voltammetry to screen for the attenuation of Fe(III) production in the mutants. Two mutants unable to solubilize Fe(III) were identified and designated d29 and d64. After mutation complementation analysis, it was determined that the point mutations were both located in type II secretion genes: gspG and gspE respectively, indicating that the type II secretion system is required for Fe(III) solubilization prior to respiration. It was also hypothesized that the ligand produced for Fe(III) solubilization during dissimilatory Fe(III) respiration was a siderophore: a small Fe(III)-chelating molecule produced by the cells for the assimilation of Fe(III) for growth. A siderophore biosynthesis gene (SO3031) and a siderophore ferric reductase gene (SO3034) were deleted in frame and the resultant mutants screened to determine whether they were capable of Fe(III) solubilization and reduction during anaerobic Fe(III) respiration. Both mutants retained Fe(III) solubilization and reduction activity, indicating that the siderophore Fe(III) assimilatory system is distinct from the Fe(III) solubilization system utilized during Fe(III) respiration. The work presented here is significant in that it describes a rapid screening method for identifying Fe(III) solubilization mutants, reports on the involvement of the type II secretion system in Fe(III) solubilization during iron respiration, and finally demonstrates that a dissimilatory metal reducing bacteria synthesizes and secretes Fe(III)-chelating molecules which are distinct from Fe(III)-siderophores.