Grace P. Lee

GENETIC SCREEN FOR NEGATIVE REGULATORS OF BACILLUS SUBTILIS CELL DIVISION. Grace P. Lee¹, Amy Z. Buscher¹, Petra A. Levin¹. Department of Biology, Washington University, St. Louis, MO¹.

FtsZ is an essential bacterial cell division protein that polymerizes into a ring (the Z-ring) at midcell as the cell initiates division. Due to the importance of cell division for survival, the temporal and spatial regulation of Z-ring formation has to be precise. Positive and negative regulators carefully control this process so that only one Z-ring forms at mid-cell during each round of cell division. Using a genetic screen for negative regulators of FtsZ polymerization, the Levin Lab has previously discovered three inhibitors, EzrA, YhxB and ClpX. My goal is to check for additional inhibitors by taking the genetic screen to saturation, and to characterize any previously unidentified regulators that I find.

To identify inhibitors of FtsZ assembly, I mutagenized B. subtilis cells carrying an ftsZts allele with a transposon called Tn10. FtsZts is a temperature sensitive allele of wild-type ftsZ. B. subtilis cells harboring ftsZts cannot form Z-rings at high temperatures, fail to divide, and ultimately die. I grew the cells at 45˚C to select for mutants that survived at the restrictive temperature. Mutants that grow at 45˚C have overcome the ftsZts cell division defect, perhaps through a Tn10 insertion in a gene that normally inhibits FtsZ polymerization. To ensure that the ftsZts suppression was linked to the Tn10 insertion, I transformed chromosomal DNA from those strains into a fresh ftsZts background, selecting for both the transposon and heat resistance. Approximately 2 x 10⁷ cells were screened, and 42 were confirmed to have viability linked to Tn10 at 45˚C.

Next, I determined if the Tn10 insertion was linked to known inhibitors of FtsZ polymerization by performing linkage analysis on the possible ftsZts suppressors. Of the 42 strains screened, 4 were linked to EzrA, 1 to YhxB and 1 to ClpX. The remaining 36 unlinked mutants represent possible unknown negative regulators of ftsZts polymerization. As a first step in characterizing these potential new factors, I quantified the level of ftsZts suppression by performing plating efficiencies, and comparing the number of colony forming units at 45°C versus 30°C. To date, percentage viability at 45°C of several possible ftsZts suppressors ranged from 88% to 130%, which is comparable the suppression levels of EzrA, YhxB and ClpX. To identify the Tn10 insertion site, I PCR amplified B. subtilis DNA adjacent to the transposon, and sequenced PCR products. These studies are ongoing.

To characterize the potential regulators of FtsZ assembly that I found in the genetic screen, I will examine the effect of the Tn10 insertion on wild-type ftsZ by examining the ability of the Tn10 insertion to suppress the lethality induced by overexpressing another bacterial cell division inhibitor, MinCD. Subsequently, I will obtain sequences of all the potential regulators that suppress ftsZts and MinCD overexpression. In addition, I will carry out fluorescence microscopy to learn how the Tn10 insertion affects FtsZ localization, cell size, morphology, or DNA segregation. This will shed light on the possible function of the gene of interest.