Genetic Analysis of Flavobacterium johnsoniae (Cytophaga johnsonae) Gliding Motility

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McBride 9727825 Gliding motility (the active movement of cells over surfaces without the aid of flagella) is a trait shared by many unrelated bacteria, yet the mechanism of gliding motility has remained a mystery for over 100 years. Newly developed genetic techniques will be used to analyze Flavobacterium johnsoniae (formerly Cytophaga johnsonae) gliding motility. Transposon mutagenesis and cosmid complementation will be used to identify F. johnsoniae gliding motility genes (gld genes). The gld genes will be cloned in E. coli, sequenced, and analyzed. The sequences will be compared to sequence databases to determine possible functions for the individual components of the gliding machinery. Mutations will created in individual gld genes and the effects on cell motility will be assessed. gld genes will also be cloned onto expression vectors in E. coli to allow overproduction of the Gld proteins. These proteins will be used for biochemical experiments aimed at determining their functions. They will also be used to raise antibodies. The antibodies against the individual GLD proteins will be used to determine the amounts of each GLD protein in the motile cells. The antibodies will also be used to localize the proteins within the cells by cell fractionation experiments and by immunoelectron microscopy. These studies will lead to the development of a model to explain the mechanism of bacterial gliding motility. In addition to expanding our knowledge on this common but little understood means of bacterial locomotion, these studies could have more practical value since gliding bacteria are important as pathogens of plants, animals, and humans, as agents of biodegredation, and as producers of enzymes, antibodies and other secondary metabolites.