9723157 Poindexter The objectives of the proposed studies are to identify cell components of the Gram-negative bacterium Caulobacter crescentus that participate in the entry of spherical ssRNA viruses (Leviviridae bacteriophages) into susceptible cells. The investigation will examine the hypothesis that pilin, in association with a specific outer membrane (OM) protein, provides adsorption sites for infecting virions, that lipopolysaccharide (LPS) or a protein(s) of the OM effect uncoating of the virion, and that the liberated viral RNA penetrates to the cytoplasm through OM-cytoplasmic membrane adhesion zones. To test this hypothesis with respect to adsorption and uncoating, a battery of phage-resistant mutant clones of the bacterium will be isolated and characterized with respect to their respective abilities to adsorb virions, to uncoat them so that viral NA is freed and becomes infectious for spheroplasts of susceptible cells, and themselves to be penetrated by viral RNA when converted to spheroplasts. The mutants will be employed in comparative studies of protein and LPS components of cell envelopes prepared from susceptible and from resistant C. crescentus cells. In a separate type of experiment, the influence on phage infectibility of various levels of plasmolysis, which disrupts adhesion zones, will be determined with phage-susceptible strains. Leviviruses share a greater number of genetic and structural properties with certain plant and animal viruses than do any other bacteriophages. It is anticipated that elucidation of the role of the cell in the entry of this type of bacteriophage will reveal further similarities among ssRNA viruses and thereby enhance both the understanding of infection by such viruses in diverse cell types, and the usefulness of Levivirus infections as prokaryotic experimental models of these infections in eukaryotic systems. The results may also provide evidence of adhesion zones as a path for entry of exogenous nucleic acid into Gram-negative bacte ria, with implications for both transformation and conjugative transfer of genetic material. Because phage receptors appear only transiently in the cell cycle of C. crescentus, identification of participants in phage entry could further eludidate the reorganization of the cell envelope that occurs during this unique, endogenously-regulated prokaryotic cellular differentiation. Lay abstract: This project addresses the question of how bacterial viruses (bacteriophages) infect susceptible host bacterial cells The entry of viruses into Gram-negative bacteria is a formidable biological challenge, since these bacteria are surrounded not only by a plasma membrane but also by a rigid cell wall and an outer membrane. The studies will proceed as projects conducted by undergraduate student-scientists and will provide for them a technically diverse and sophisticated, but intellectually accessible and stimulating introduction to experimental cell biology. ***
