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1 CB - 10 ( B ) RI SURF 2019 This
CB - 10 ( B ) RI SURF 2019 This work was supported by RI INBRE ZapE is a Novel Cell Division A ssociated ATPase i n Escherichia coli Rebecca Dickinson , Eric DiBiasio & Jodi Camberg Cell & Molecular Biology , University of Rhode Island , Kingston, RI During early septal phase of cell division in bacteria, a large protein structure called the Z - ring assembles at the division site. The major protein that comprises the Z - ring is FtsZ, a tubulin homolog that hydrolyzes GTP and assembles into polymers. In E scherichia coli , many cell division proteins interact with FtsZ and direct Z - ring assembly, while others may modulate constriction or direct cell wall insertion and remodeling. Several accessory proteins that interact with FtsZ are called Z - ring associated proteins (Zaps). The Zaps (ZapA, ZapB, ZapC, ZapD, and ZapE) are recruited to the division site and are predicted to influence Z - ring assembly and/or stability. ZapE was recently identified to be an ATPase that accumulates during late constriction in E. c oli and is important for bacterial growth under low - oxygen conditions and high temperatures (Marteyn, et al. , 2014). In vitro , ZapE destabilizes FtsZ polymers suggesting that it may promote Z - ring disassembly in vivo (Marteyn, et al. , 2014). To evaluate Za pE function and interactions we cloned ZapE with a N - terminal hexahistidine tag (H6 - ZapE) into a high - copy inducible expression vector and purified via metal affinity chromatography. We determined that H6 - ZapE hydrolyzes ATP with an average rate of 1.25 Pi /min/pmol. ZapE is predicted to contain an ATP binding pocket with putative Walker A (GGVGRGK84T) and Walker B nucleotide - binding sites. We performed site directed mutagenesis on the Walker A nucleotide - binding site at position K84. H6 - ZapE(K84A) was defe ctive for ATP hydrolysis. Since ZapE has been shown to interact with FtsZ we decided to further probe this interaction using a colorimetric assay detecting free phosphate released by H6 - ZapE in the presence of ATP and FtsZ and later GTP and FtsZ. Under the conditions tested, H6 - ZapE had no impact on ATP or GTP hydrolysis in the presence of FtsZ. We also tested the oligomeric status of H6 - ZapE using size exclusion chromatography. Under these conditions, H6 - ZapE eluted as a monomer with and without the presen ce of ATP. Overall, these experiments will help to further understand the role of ZapE during prokaryotic cell division.