Anna Cristina Garza

STRUCTURE/FUNCTION ANALYSIS OF A CELL DIVISION PROTEIN IN BACILLUS SUBTILIS. Anna Cristina Garza¹, Daniel P. Haeusser¹, Amy Zoch Buscher¹, Petra Anne Levin¹, Department of Biology, Washington University, St. Louis, MO¹.

During bacterial cell division the tubulin-homolog FtsZ localizes to midcell and assembles into a membrane-associated ring, establishing the site of septation. FtsZ assembly is precisely regulated to ensure that cell division occurs at the right time and in the right place.[1] EzrA is an important regulator of FtsZ assembly in the low G-C family of Gram-positive bacteria. EzrA is a membrane protein that inhibits FtsZ assembly at cell poles. Paradoxically, EzrA also localizes to the FtsZ ring at midcell; although its function there is not known, genetic data suggests that it might stabilize preformed polymers in the FtsZ ring.[2]^,[3]

To identify residues of EzrA that are important for one or both of these activities, I initiated a structure/function analysis of EzrA in Bacillus subtilis. Sequence analysis predicts that EzrA consists of an N-terminal membrane anchor followed by four cytoplasmic coiled coil domains. While coiled coil domains are known to mediate protein interactions, their specific role in EzrA function is uncertain. In addition to the coiled coils, the C-terminus of EzrA contains a stretch of seven highly conserved amino acid residues.

I began my analysis with a functional fusion of EzrA to the green fluorescent protein (GFP). Using PCR-based site-directed mutagenesis, I created a series of mutant EzrA-GFP fusions, including a deletion of and a set of point mutations within the conserved C-terminal domain as well as constructs missing the third and fourth coiled coils, respectively. I verified mutant protein expression by Western blot and assayed the ability of each fusion to localize to the FtsZ ring via live cell fluorescence microscopy.

Most notably, I found that a single point mutation within the conserved sequence, R510D, resulted in an EzrA-GFP fusion that was unable to localize to the midcell FtsZ ring. Preliminary immunofluorescence microscopy (IFM) indicated that the EzrA-GFP fusion missing all seven conserved residues still inhibited polar ring formation, although it was unable to localize to midcell. These data suggest that the conserved seven residues play an essential role in EzrA’s ability to localize to FtsZ at midcell but may not be necessary for preventing polar FtsZ ring formation. In addition, EzrA-GFP with deletions of the third or the fourth coiled coils did not localize to midcell.

In future work I will create mutant EzrA-GFP fusions containing more subtle disruptions of the third and fourth coiled coils; mutagenesis of the first and second coiled coils is also pending. In collaboration with Dr. Amy Buscher and Daniel Haeusser I will examine the stability of each mutant protein without the GFP tag, as well as the ability of each to inhibit FtsZ assembly in vitro.

[1] Romberg, L. & Levin, P.A. (2003) Annu. Rev. Microbiol. 57, 125-157.

[2] Haeusser, D.P., Schwartz, R.L., Smith, A.M., Oates, M.E., & Levin, P.A. (2004) Mol. Microbiol. 52, 801-814.

[3] Levin, P.A., Kurtser, I.G., & Grossman, A.D. (1999) Proc. Natl. Acad. Sci. USA 96, 9642-9647.