To Mid-cell and Beyond: Characterizing the Roles of GpsB and YpsA in Cell Division Regulation in Gram-positive Bacteria

The bacterial cell division protein FtsZ is a tubulin homolog that forms a ring-like structure at the site of cell division in most bacterial species. There it acts as a scaffold, aiding in the recruitment of other divisome proteins to the site of cell division. Furthermore, studies focusing on the role of FtsZ treadmilling and septal peptidoglycan synthesis implicates that FtsZ plays a direct role in the ultimate closure of the division septum. Thus, many studies in the field of bacterial cell division have focused on FtsZ in terms of its spatial and temporal regulation as well as its ability to interact with other division proteins. The finding that cells lacking well- studied regulators of FtsZ activity still divide at wild type like capacities suggests the existence of other yet to be discovered factors involved in the cell division process. Work in our lab has focused on the identification and characterization of novel regulators of cell division in the rod- shaped model organism, Bacillus subtilis, and in the spherical bacterium, Staphylococcus aureus. Here, we describe the function of YpsA, previously annotated as a protein of unknown function, and GpsB in B. subtilis and S. aureus respectively. Our results indicate that YpsA provides oxidative stress protection in B. subtilis cells. Furthermore, YpsA appears to be functioning as a growth rate-dependent cell division inhibitor, though the mechanism by which YpsA elicits this function has not yet been identified. It is interesting to note that this proposed function of YpsA, as well as the growth rate dependency, is also conserved in S. aureus. Sequence and structural analysis of YpsA allowed for the identification of several amino acid residues critical for the ability of YpsA to elicit filamentation. We have also found that disruption of a conserved substrate binding groove, through site directed mutagenesis, resulted in an impairment of YpsA-GFP foci formation in addition to cell division inhibition. That had indicated a link between foci formation and the ability of YpsA to inhibit cell division. A follow up study utilizing a screen to identify intragenic and extragenic suppressors of YpsA-mediated filamentation allowed for the identification of additional amino acid residues important for the function of YpsA, many of which also lined the predicted DNA binding groove. Using this screen, we were also able to isolate an extragenic suppressor mutation in yfhS, which was annotated as a sporulation gene based on transcriptomics data. Our results indicate that YfhS may also play a role in cell size regulation during vegetative growth, as cells harboring yfhS null mutations appeared smaller in both length and width when compared to a wild type control. Collectively, these results constituted the first reports on the role of YpsA in cell division in B. subtilis. Our study focusing on GpsB in S. aureus indicated a role for GpsB in regulating the dynamics of FtsZ during the cell cycle. Our results indicate that production of S. aureus GpsB in B. subtilis is lethal and results in severe filamentation indicative of cell division inhibition. Furthermore, overexpression of gpsB in S. aureus also resulted in cell division inhibition, and depletion of intracellular GpsB levels resulted in a lethal phenotype thereby confirming its essentiality in S. aureus. Fluorescence microscopy revealed that GpsB localization was dynamic during the cell cycle as GpsB-GFP was observed at the leading edge of the invaginating membrane. In combination with in vitro analysis of GpsB suggesting a direct interaction with FtsZ, we propose a model where GpsB in S. aureus forms lateral interactions between FtsZ protofilaments. This increases the local concentrations of FtsZ at the division site and subsequently triggers FtsZ protofilament disassembly via GpsB activity, and ultimately contributes to FtsZ treadmilling facilitating septum closure