Active site mutations in the bacterial actin homolog FtsA impair direct interactions with the divisome in Escherichia coli

Abstract

During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation early in the division pathway. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins. In E. coli, membrane recruitment and tethering of FtsZ are predominantly carried out by FtsA. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Accordingly, bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and adopt a polymerized conformation. Here, we show that a cluster of amino acid residues in the central region of FtsA and proximal to the nucleotide binding site are critical for FtsA function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, ATP-dependent PL vesicle remodeling, and membrane release, lead to failed Z-ring assembly and division defects in cells.