scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals The C-Terminal Domain of MinC Inhibits Assembly of the Z Ring in Escherichia coli

2006 ◽  
Vol 189 (1) ◽  
pp. 236-243 ◽  
Author(s):  
Daisuke Shiomi ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Additional Function ◽  
Ring Assembly ◽  
C Terminus ◽  
Min Proteins ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC122-231) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC122-231, the C terminus of full-length MinC, or the C terminus of MinC122-231 perturbed MinC function, which may explain why cell division inhibition by MinC122-231 was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

scholarly journals Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling

2016 ◽  
Vol 198 (11) ◽  
pp. 1683-1693 ◽  
Author(s):  
Elyse J. Roach ◽  
Charles Wroblewski ◽  
Laura Seidel ◽  
Alison M. Berezuk ◽  
Dyanne Brewer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Bacterial Cell ◽  
Site Directed Mutagenesis ◽  
Bacterial Cell Division ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Z Ring

ABSTRACTBacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associatedproteins (Zap) for stability throughout the process of division. InEscherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure ofEscherichia coliZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by β-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Usingin vitroFtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments.IMPORTANCEZ-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins inE. colithat associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure ofE. coliZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2021 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Multiple Alternative ◽  
Z Ring ◽  
Cell Division Inhibitor

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals DivIVA is essential in Deinococcus radiodurans and its C terminal domain regulates new septum orientation during cell division

2020 ◽  
Author(s):  
Reema Chaudhary ◽  
Swathi Kota ◽  
Hari S Misra
Keyword(s):  
Cell Division ◽  
Deinococcus Radiodurans ◽  
Wild Type ◽  
Orthogonal Axis ◽  
Septum Formation ◽  
Terminal Domain ◽  
Cell Position ◽  
Type Pattern ◽  
In Trans ◽  
Ftsz Assembly

AbstractFtsZ assembly at mid cell position in rod shaped bacteria is regulated by gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles and the next plane of cell division is perpendicular to previous plane, the determination of site for FtsZ assembly is intriguing. Deinococcus radiodurans a coccus shaped bacterium, is characterized for its extraordinary resistance to DNA damage. Here we report that DivIVA a putative component of Min system in this bacterium (drDivIVA) interacts with cognate cell division and genome segregation proteins. The deletion of full length drDivIVA was found to be indispensable while its C-terminal deletion (ΔdivIVAC) was dispensable but produced distinguishable phenotypes like slow growth, altered plane for new septum formation and angular septum. Both wild type and mutant showed FtsZ foci formation and their gamma radiation responses were nearly identical. But unlike in wild type, the FtsZ localization in mutant cells was found to be away from orthogonal axis with respect to plane of previous septum. Notably, DivIVA-RFP localizes to membrane during cell division and then perpendicular to previous plane of cell division. In trans expression of drDivIVA in ΔdivIVAC background could restore the wild type pattern of septum formation perpendicular to previous septum. These results suggested that DivIVA is an essential protein in D. radiodurans and the C-terminal domain that contributes to its interaction with MinC determines the plane of new septum formation, possibly by controlling MinC oscillation through orthogonal axis in the cells.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2020 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Z Ring ◽  
Response To Stress ◽  
Cell Division Inhibitor

AbstractRod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC+ phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple division inhibition pathways exist during the stress-induced SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.ImportanceFilamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

2001 ◽  
Vol 183 (22) ◽  
pp. 6630-6635 ◽  
Author(s):  
Sebastien Pichoff ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Ring Formation ◽  
Ring Assembly ◽  
Z Ring ◽  
Alternative Fixation ◽  
Green Fluorescent

ABSTRACT The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles offtsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

scholarly journals Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division

2001 ◽  
Vol 183 (24) ◽  
pp. 7190-7197 ◽  
Author(s):  
Amit Mukherjee ◽  
Cristian Saez ◽  
Joe Lutkenhaus
Keyword(s):  
Cell Division ◽  
Critical Concentration ◽  
Gtpase Activity ◽  
Support Cell ◽  
Z Ring ◽  
Ftsz Assembly ◽  
The Stability

ABSTRACT FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.

scholarly journals Role of FtsEX in Cell Division of Escherichia coli: Viability of ftsEX Mutants Is Dependent on Functional SufI or High Osmotic Strength

2006 ◽  
Vol 189 (1) ◽  
pp. 98-108 ◽  
Author(s):  
Manjula Reddy
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Salt Transport ◽  
High Osmolarity ◽  
Growth Defects ◽  
Ring Assembly ◽  
Multiple Copies ◽  
Z Ring ◽  
Twin Arginine Translocase ◽  
The Stability

ABSTRACT In Escherichia coli, at least 12 proteins, FtsZ, ZipA, FtsA, FtsE/X, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC, are known to localize to the septal ring in an interdependent and sequential pathway to coordinate the septum formation at the midcell. The FtsEX complex is the latest recruit of this pathway, and unlike other division proteins, it is shown to be essential only on low-salt media. In this study, it is shown that ftsEX null mutations are not only salt remedial but also osmoremedial, which suggests that FtsEX may not be involved in salt transport as previously thought. Increased coexpression of cell division proteins FtsQ-FtsA-FtsZ or FtsN alone restored the growth defects of ftsEX mutants. ftsEX deletion exacerbated the defects of most of the mutants affected in Z ring localization and septal assembly; however, the ftsZ84 allele was a weak suppressor of ftsEX. The viability of ftsEX mutants in high-osmolarity conditions was shown to be dependent on the presence of a periplasmic protein, SufI, a substrate of twin-arginine translocase. In addition, SufI in multiple copies could substitute for the functions of FtsEX. Taken together, these results suggest that FtsE and FtsX are absolutely required for the process of cell division in conditions of low osmotic strength for the stability of the septal ring assembly and that, during high-osmolarity conditions, the FtsEX and SufI functions are redundant for this essential process.

scholarly journals Assembly Dynamics of FtsZ Rings in Bacillus subtilis and Escherichia coli and Effects of FtsZ-Regulating Proteins

2004 ◽  
Vol 186 (17) ◽  
pp. 5775-5781 ◽  
Author(s):  
David E. Anderson ◽  
Frederico J. Gueiros-Filho ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescence Recovery ◽  
Half Time ◽  
Experimental Conditions ◽  
Z Ring

ABSTRACT FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (∼9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an ∼3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, ∼8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

scholarly journals New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

2005 ◽  
Vol 187 (1) ◽  
pp. 358-365 ◽  
Author(s):  
Stephen G. Addinall ◽  
Elaine Small ◽  
Duncan Whitaker ◽  
Shane Sturrock ◽  
William D. Donachie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Point Mutations ◽  
Ring Formation ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Z Ring

ABSTRACT We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109→Ser109 for ftsZ6460, Ala129→Thr129 for ftsZ972, Val157→Met157 for ftsZ2066, Pro203→Leu203 for ftsZ9124, and Ala239→Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

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