Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. The mechanism of this form of movement has been a mystery for nearly a century. Recently 12 Gld (for "gliding") proteins required for gliding were identified. Cell-surface proteins are expected to be part of the gliding machinery but surprisingly, none of the Gld proteins appear to be exposed on the cell surface. The genetic approaches used to identify the Gld proteins would have missed redundant motility proteins with overlapping functions. A screen for transposon-induced mutants with partial defects in motility identified new mutations, termed sprA, sprB, sprC, and sprD, which appear to encode some of the missing surface components of the gliding machinery. These proteins may be adhesins that are propelled along the cell surface by the Gld proteins. Cell-surface fibrils were observed on wild-type cells but were lacking on cells of a mutant deficient for sprB, sprC, and sprD. This suggests that the fibrils are composed of Spr proteins and are part of the motility machinery. The specific aims of this project are to: (1) identify and characterize the cell-surface proteins of the motility machinery; (2) visualize the gliding motility apparatus; and (3) identify moving components of the gliding machinery. Molecular experiments will be used to characterize SprA, SprB, SprC, and SprD, and novel genetic approaches will be used to identify additional cell-surface components of the motility machinery. Modern electron microscopy (EM) approaches, including cryo-EM tomography and immuno-EM, will be used to visualize the motility apparatus. The roles of the Spr proteins will be probed by determining the effect of specific antibodies on motility, and by fluorescently labeling epitope-tagged Spr proteins to visualize dynamic changes that occur during cell movement. These studies will elucidate the mechanism of a novel yet common form of bacterial motility and will result in new information regarding molecular motors, transporters, and adhesins.
Broader Impacts: Undergraduate and graduate students will conduct much of this research. These students will gain experience in genetics, molecular biology, and modern microscopic approaches to study questions of bacterial cell biology. Research on F. johnsoniae will also be incorporated into several undergraduate laboratory courses. From past experience, participation of students in real research where the outcome is not known beforehand (even by the instructor) ignites scientific curiosity and appreciation of the scientific method in a way that "cookbook" laboratory exercises cannot. Genetic tools developed for F. johnsoniae and related bacteria within the phylum Bacteroidetes will be made available to the scientific community and the results of the research will be reported by students at national and international meetings and published in leading journals.
