Intellectual merit: Cells of Flavobacterium johnsoniae crawl over surfaces by a process known as gliding motility. These cells do not have well-studied motility organelles such as flagella or Type IV pili. Instead they rely on a novel motility machine composed of proteins that are unique to the phylum Bacteroidetes. Twenty-four proteins involved in motility have been identified. These include 1) Gld proteins that are components of the gliding 'motor', 2) mobile cell surface adhesins (SprB, RemA, and others) that appear to be propelled by the 'motor' and may function somewhat like a tank tread, and 3) components of a novel protein secretion system (PorSS) that is required for assembly of SprB on the cell surface. Electron microscopic observations suggest that SprB may form long filaments extending from the cell. SprB and RemA allow movement of cells on different surfaces, and they have lectin-like carbohydrate-binding domains that may facilitate binding to these surfaces. Cells with mutations in genes involved in polysaccharide synthesis and export also result in motility defects. These results suggest a model for gliding in which the gliding 'motors' propel adhesive filaments along the cell surface. Cells express different adhesins (SprB, RemA and others) to allow movement on different surfaces, and exopolysaccharides might enhance motility by coating the surfaces and interacting with the adhesins. Genetic, cell biology, biochemical, and electron microscopic approaches will be used to address 3 specific aims related to the cell-surface components of the motility machinery. Specific Aim 1. Characterize the mobile cell-surface adhesins involved in gliding. Antibodies against RemA will be used to test the hypothesis that RemA is a mobile cell-surface protein and to determine if the PorSS is required for secretion of RemA. Fluorescently labeled epitope-tagged RemA will also be used to visualize dynamic changes that occur during cell movement. The effect of soluble sugars on RemA function will be analyzed to determine the binding specificity of the lectin domain. Recombinant versions of RemA will be generated to determine the functions of individual domains of the protein, and cryo-electron microscopy tomography will be used to test the hypothesis that RemA and SprB are components of different cell-surface filaments. Specific Aim 2. Determine the role of extracellular polysaccharides in gliding. Wza and Wzc are components of the putative exopolysaccharide secretion system linked to gliding. Exopolysaccharides will be isolated and characterized. Cells with mutations in wza and wzc will be analyzed to test the hypothesis that they are deficient in exopolysaccharides. Purified polysaccharides will be used to determine if they interact with the lectin domain of RemA and extracellular complementation will determine if exogenously supplied polysaccharide overcomes the motility defects of wza and wzc mutants. Specific Aim 3. Characterize the protein secretion system involved in assembly of the cell-surface adhesins. Genetic approaches will be used to identify additional components of the PorSS involved in protein secretion and to determine the features of secreted proteins that target them to this system. Protein-protein interactions will be studied to address questions related to structure and function of the PorSS.
Broader Impacts: Undergraduate students, graduate students and postdoctoral scientists will conduct the research. Students will gain experience in genetics, molecular biology, and modern light and electron microscopic approaches to study questions of bacterial cell biology. The projects are well suited to expose students to a broad array of techniques. Students and postdoctoral scientists will receive extensive training in scientific writing and in presentation techniques. Research on F. johnsoniae will be incorporated into several undergraduate laboratory courses. Undergraduate students will have primary responsibility for constructing and characterizing mutations in potential components of the PorSS. Research results will be reported by students at national meetings and published in leading journals, and genetic tools developed for F. johnsoniae and related bacteria will be made available to the scientific community. Similar gliding motility and protein secretion machines are found in many members of the phylum Bacteroidetes, including bacteria of environmental and agricultural importance. Some of these bacteria are important pathogens of fish that impact aquaculture systems, and others have novel mechanisms for digesting cellulose and may aid conversion of plant material into biofuels. An improved understanding of F. johnsoniae motility and protein secretion may thus have practical applications.
