Dissecting the Role of a Protease Subunit in Bacterial Cell Division

David Robert Erickson¹, Petra Anne Levin, Ph.D.,Biology Department, Washington University, St. Louis, MO¹

FtsZ, a homolog of the eukaryotic protein tubulin, is essential to bacterial cell division. As bacteria prepare for cell division, FtsZ assembles into a ring at the future division site. This ring serves as a scaffold for other cell division proteins to assemble. FtsZ exists in a balance between monomer and polymer states. Prior to the initiation of cell division, the equilibrium is pushed towards the monomer state by various factors, thereby preventing inappropriate FtsZ ring formation. However, as the cell approaches division, the equilibrium shifts towards the polymer state. This shift promotes FtsZ ring formation, thus permitting cell division to proceed.

ClpX, isolated in a screen for negative regulators of FtsZ in Bacillus subtilis, pushes the FtsZ equilibrium towards the monomer state and therefore antagonizes FtsZ ring formation. ClpX is an ATPase chaperone protein that works with ClpP to form the ClpXP protease. ClpXP is involved in the degradation of many proteins within the cell. In in B. subtilis ClpXP is also involved in the regulation of many cellular processes including genetic competence and sporulation. In addition to its role in guiding the activities of the ClpXP protease, ClpX has been shown to have ClpP independent activities in E. coli. These include the remodeling and disassembly of protein complexes.

Past experimentation has shown that the overexpression of ClpX in a temperature sensitive ftsZ (ftsZts) background is lethal to B. subtilis cells because it blocks FtsZ ring formation. It is not known, however, whether ClpX blocks FtsZ ring formation by utilizing the ClpXP proteolytic pathway or the ClpP independent, protein remodeling pathway. In order to establish the pathway by which ClpX regulates FtsZ ring assembly, my goal was to overexpress ClpX in a ClpP null mutant bearing the ftsZts allele. If ClpX regulates FtsZ through the ClpP proteolytic pathway, then I would expect to find normal cell growth and division. If, however, ClpX regulates FtsZ ring formation independently of ClpP, then I would expect that B. subtilis cells will filament and die.

To test the effect of overexpressing ClpX in the clpP deficient background, I first had to construct the clpP null allele. The cloning strategy involved amplifying sequences flanking clpP by PCR and inserting them into the plasmid pUC18erm. The resulting plasmid would then be used to delete clpP in B. subtilis via insertional deletion in which ClpP was replaced by an erythromycin resistance gene. Unfortunately, my research stalled at this point due to problems with the cloning reactions, and much of my effort was spent troubleshooting. However, preliminary data suggests that the first of the two inserts has been successfully cloned into pUC18erm thus allowing the future continuation of this project.