Protein coopted from a phage restriction system dictates orthogonal cell division plane selection in Staphylococcus aureus.

The spherical bacterium Staphylococcus aureus, a leading cause of nosocomial infections, undergoes binary fission by dividing in two alternating orthogonal planes, but the mechanism by which S. aureus correctly selects the next cell division plane is not known. To identify cell division placement factors, we performed a chemical genetic screen that revealed a gene which we termed pcdA. We show that PcdA is a member of the McrB family of AAA+ NTPases that has undergone structural changes and a concomitant functional shift from a restriction enzyme subunit to an early cell division protein. PcdA directly interacts with the tubulin-like central divisome component FtsZ and localizes to future cell division sites before membrane invagination initiates. This parallels the action of another McrB family protein, CTTNBP2, which stabilizes microtubules in animals. We show that PcdA also interacts with the structural protein DivIVA and propose that the DivIVA/PcdA complex recruits unpolymerized FtsZ to assemble along the proper cell division plane. Deletion of pcdA conferred abnormal, non-orthogonal division plane selection, increased sensitivity to cell wall-targeting antibiotics, and reduced virulence in a murine infection model. Targeting PcdA could therefore highlight a treatment strategy for combatting antibiotic-resistant strains of S. aureus.

Nucleotide-dependent activities of FtsA regulate the early establishment of a functional divisome during the Escherichia coli cell cycle.

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. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins, predominantly FtsA in E. coli. 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. Bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and polymerize. Here, we show that several amino acid residues proximal to the nucleotide binding site in FtsA are critical for 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, membrane binding and release, and, most importantly, ATP-dependent PL remodeling, lead to failed Z-ring assembly and division defects in cells.

The Stress-Active Cell Division Protein ZapE Alters FtsZ Filament Architecture to Facilitate Division in Escherichia coli.

During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.

Degradation of MinD oscillator complexes by Escherichia coli ClpXP.

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase-dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.