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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.

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.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after completion of successful antibiotic treatment. Many clinical isolates, including the well characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex IHF and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two hybrid assays. We report direct interactions between succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions likely enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility.

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.