Title

Assembly of the FtsZ cell division ring and the action of cell division inhibitors of E. coli

Date of Completion

January 2001

Keywords

Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Intermediates in the assembly of the machinery required for bacterial septation have not been defined. The earliest known step in the assembly of the septation machinery is the formation of an FtsZ ring at midcell. While it is believed that this ring is formed from polymerization of FtsZ into microtubule-like structures, nothing is known about the mechanism of FtsZ assembly at midcell. This study describes a new system to evaluate FtsZ polymerization in the presence of cytoplasmic proteins. Size exclusion chromatography was used to determine that the polymerization of endogenous FtsZ within cytoplasmic extracts proceeds by cooperative incorporation of an FtsZ dimer into the growing oligomer. These oligomers were stable in the absence of GTP, suggesting that cytoplasmic molecules may aid in the polymerization of FtsZ or may alter the GTPase activity of FtsZ. This hypothesis was supported by the observation that purified FtsZ displayed GTP-independent oligomerization when added to FtsZ-depleted cytoplasmic extracts. This assay can be used to identify and characterize molecules which regulate the assembly of the FtsZ ring at midcell. ^ SulA and MinCD are specific inhibitors of cell division in Eschehchia coli. In this study, size exclusion chromatography was used to determine the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented formation of high molecular weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in cell extracts nor the formation of FtsZ and ZipA ring-structures in vivo. However, MinCD did prevent formation of FtsA rings. Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring. ^