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Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in Escherichia coli.

ABSTRACT: During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317-383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics.

SUBMITTER: Viola MG

PROVIDER: S-EPMC5256927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:When deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.ImportanceOur results suggest a role for FtsZ, in addition to its already known effect in the constriction of E. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated by E. coli, an observation that may help in the design of effective antimicrobial compounds.

Project description:The Min system in Escherichia coli, consisting of MinC, MinD, and MinE proteins, regulates division site selection by preventing assembly of the FtsZ-ring (Z-ring) and exhibits polar oscillation in vivo MinC antagonizes FtsZ polymerization, and in vivo, the cellular location of MinC is controlled by a direct association with MinD at the membrane. To further understand the interactions of MinC with FtsZ and MinD, we performed a mutagenesis screen to identify substitutions in minC that are associated with defects in cell division. We identified amino acids in both the N- and C-domains of MinC that are important for direct interactions with FtsZ and MinD in vitro, as well as mutations that modify the observed in vivo oscillation of green fluorescent protein (GFP)-MinC. Our results indicate that there are two distinct surface-exposed sites on MinC that are important for direct interactions with FtsZ, one at a cleft on the surface of the N-domain and a second on the C-domain that is adjacent to the MinD interaction site. Mutation of either of these sites leads to slower oscillation of GFP-MinC in vivo, although the MinC mutant proteins are still capable of a direct interaction with MinD in phospholipid recruitment assays. Furthermore, we demonstrate that interactions between FtsZ and both sites of MinC identified here are important for assembly of FtsZ-MinC-MinD complexes and that the conserved C-terminal end of FtsZ is not required for MinC-MinD complex formation with GTP-dependent FtsZ polymers.IMPORTANCE Bacterial cell division proceeds through the coordinated assembly of the FtsZ-ring, or Z-ring, at the site of division. Assembly of the Z-ring requires polymerization of FtsZ, which is regulated by several proteins in the cell. In Escherichia coli, the Min system, which contains MinC, MinD, and MinE proteins, exhibits polar oscillation and inhibits the assembly of FtsZ at nonseptal locations. Here, we identify regions on the surface of MinC that are important for contacting FtsZ and destabilizing FtsZ polymers.

Project description: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.

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Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in Escherichia coli.

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