The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division

Abstract

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome, however, the exact role of molecular chaperones in this critical process remains unclear. In the important pathogenic bacterium Staphylococcus aureus, the ClpX chaperone is essential for growth at 30{degrees}C and microscopic analyses suggested that ClpX plays a temperature-dependent role in cell division. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of cell division by showing that a spontaneous G325V substitution in the ATP-binding domain of the essential FtsA cell division protein rescues growth and septum synthesis in a Staphylococcus aureus clpX mutant. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with E. coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division. IMPORTANCEEssential biological processes, such as cell division, are performed by multiple proteins working together in dynamic functional complexes. In eukaryotic cells, the disassembly of such molecular machines is often assisted by molecular chaperones capable of unfolding proteins. The ClpX unfoldase is conserved from bacteria to humans, however, the roles of ClpX in bacterial cell biology remain relatively unexplored. By combining genetic methods with super-resolution microscopy techniques, we show here that ClpX and a mutant variant of the essential cell division protein FtsA mutually compensate for each other in controlling cell division of the pathogenic bacterium Staphylococcus aureus. The selected FtsAG325V variant has diminished self-interactions and restored the aberrant FtsA dynamics in clpX cells suggesting that ClpX promotes cell division by antagonizing FtsA protein interactions. This study, for the first time, illuminates the important role of protein unfoldases in the functioning of the divisome, a multienzyme complex fundamental to bacterial reproduction.