Intellectual merit: Shewanella oneidensis MR-1 is a bacterium that utilizes a sigma-28 regulated transcriptional network to express coordinately regulated genes. A computer-based (in silico) analysis predicted that the sigma-28 regulatory network contained several genes necessary for flagellar motility. However, several additional genes, mostly annotated as encoding hypothetical proteins, were also linked to these motility genes. The purpose of this project is to analyze the products of transcriptional regulation of these genes using reverse transcriptase Polymerase Chain Reaction Assays. Then, the biological roles of the proteins will be assessed by constructing in-frame deletion mutants and screening these mutants for motility defects using chemotaxis assays and video-microscopy. Using these approaches it will be possible to evaluate the in silico method used to construct the sigma-28-regulated transcriptional network and potentially identify roles for a number of currently hypothetical proteins. This research will lay the foundations for a better understanding of the diversity of bacterial motility mechanisms (a topic about which very little is known), provide information on the role of bacterial motility in the biogeochemical cycling of metals (because MR-1 is a dissimilatory metal-reducing microbe), and facilitate modeling for bioremediation studies that involve microbial metal reduction.
Broader impacts: The PI will enhance educational infrastructure by producing an open-access, web-based, series of lectures: Modules in Microbiology. Each module will encourage interdisciplinary thinking by emphasizing how different disciplines have impacted the field of microbiology. These modules will be advertised on the Society for Microbiology web sites of different countries and will provide an e-text for students and teachers unable to afford regular textbooks.
Shewanella oneidensis is a bacterium that can survive in the absence of oxygen by respiring minerals, including insoluble manganese (IV) oxides. Mn(III) is a soluble form of manganese that is found in certain environments. During studies aimed at determining if Shewanella can use soluble Mn(III) for respiration and growth we observed the formation of crystals in the cultures (Fig.1). These crystals were most likely a manganese phosphate (Fig.2) and the cells were shown to preferrentially attach to them. Confocal microscopy showed many of the cells to be respiring when attached to the crystals (Fig.3). Our preliminary studies suggested that Shewanella can respire Mn(III) in addition to Mn(IV) and that this respiration can occur even in the presence of oxygen. However, mutants that are unable to respire Mn(IV) could still respire Mn(III), suggested that the cellular components required for Mn(III) respiration are distinct from those used for Mn(IV) respiration. Understanding the mechanism associated with Mn(III) respiration proved difficult because the process killed a large proportion of the cells. Therefore, although we obtained evidence that Shewanella can respire Mn(III), we obtained no evidence that the cells could link this respiratory process with growth. Instead most cells were shown to be killed by the process, although attachment to the crystals formed in the assays did allow the formation of living biofilms. Because manganese is normally found in either the Mn(IV) or Mn(II) oxidation states, it is possible that microbes that can respire insoluble Mn(IV) oxides [a process that generates soluble Mn(II)] are not similarly adapted to respire soluble Mn(III). These studies suggest that the solubility state of metals may influence the ability of bacteria to use them for respiration and growth.
