Investigating the mechanism of action of cell division proteins FtsZ and FtsA of cell wall-less bacteria

Abstract

FtsZ and FtsA are the two primary components of the Z-ring involved in bacterial cell division. Despite extensive research, their roles in driving cell constriction are not well defined. In this context, FtsZ and FtsA from mollicutes or the cell wall-less bacteria can serve as a model system to investigate the role of Z-ring in the division process in the absence of a cell wall synthesis machinery. In this work, we have employed an in vitro approach to study FtsZ and FtsA proteins from Mycoplasma and Spiroplasma, the two most studied genera under mollicutes. In Mycoplasma, we discovered a phylogenetically separate FtsZ group containing putative membrane-binding amphipathic helix at the N-terminal or C-terminal extensions. We showed the proposed Cterminal amphipathic helix (CTAH) in Mycoplasma genitalium FtsZ can bind membrane in vitro. Furthermore, a cholesterol recognition motif was identified within the CTAH region of M. genitalium FtsZ. Bacterial FtsZs are not so far known to bind membrane or cholesterol, which makes these observations quite interesting and novel as well. We also characterized FtsA from Spiroplama melliferum (SmFtsA) and its interaction with FtsZ. Here, we found SmFtsA to be inactive for ATP and GTP hydrolysis unlike many other reported homologs. We observed stimulation in GTPase activity of SmFtsZ along with higher order lateral assembly of SmFtsZ protofilaments in the presence of FtsA (a property generally shown by FtsZ stabilizers in cell walled bacteria). Based on the observations, we proposed a plausible mechanism for GTPase stimulation and higher order assembly which will be validated further in future studies. Finally, we determined the structure of SmFtsZ filament in a GTP bound state using cryo-EM at 3.5 Å resolution. Our preliminary analysis suggests that the filament is composed of T state monomers with an inter-subunit distance of 45 Å. We observed an intrinsic curvature of the filament with the N-terminal domain facing the concave side. Additionally, the ordered T3 loop indicates a possible correlation with the presence of gamma phosphate in the nucleotide pocket and interface stabilization. Currently, more structural analyses are underway to gain structural insights into the native state conformational changes of the two domains that drive polymerization dynamics. Altogether, our results give an overview of the cell division genes in mollicutes and a new perspective of the diverse functions of FtsZ and FtsA in Mycoplasma and Spiroplasma.