SMART MODE:

NORMAL GENOMIC

• Possoz C, Newmark J, Sorto N, Sherratt DJ, Tolmasky ME
• Sublethal concentrations of the aminoglycoside amikacin interfere with cell division without affecting chromosome dynamics.
• Antimicrob Agents Chemother. 2007; 51: 252-6
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• Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.

• Reddy M
• Role of FtsEX in cell division of Escherichia coli: viability of ftsEX mutants is dependent on functional SufI or high osmotic strength.
• J Bacteriol. 2007; 189: 98-108
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• 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.

• Paradis-Bleau C, Beaumont M, Sanschagrin F, Voyer N, Levesque RC
• Parallel solid synthesis of inhibitors of the essential cell division FtsZ enzyme as a new potential class of antibacterials.
• Bioorg Med Chem. 2007; 15: 1330-40
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• As a model system for designing new inhibitors of bacterial cell division, we studied the essential and highly conserved FtsZ GTPase from Pseudomonas aeruginosa. A collection of GTP analogues were prepared using the solid-phase parallel synthesis approach. The synthesized GTP analogues inhibited the GTPase activity of FtsZ with IC(50) values between 450muM and 2.6mM, and 5 compounds inhibited Staphylococcus aureus growth in a biological assay. The FtsZ spectrophotometric assay developed for screening of synthesized compounds is the first step in identification of antibacterials targeting the bacterial cell division essential proteins.

• Goehring NW, Petrovska I, Boyd D, Beckwith J
• Mutants, suppressors, and wrinkled colonies: mutant alleles of the cell division gene ftsQ point to functional domains in FtsQ and a role for domain 1C of FtsA in divisome assembly.
• J Bacteriol. 2007; 189: 633-45
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• Cell division in Escherichia coli requires the concerted action of at least 10 essential proteins. One of these proteins, FtsQ, is physically associated with multiple essential division proteins, including FtsK, FtsL, FtsB, FtsW, and FtsI. In this work we performed a genetic analysis of the ftsQ gene. Our studies identified C-terminal residues essential for FtsQ's interaction with two downstream proteins, FtsL and FtsB. Here we also describe a novel screen for cell division mutants based on a wrinkled-colony morphology, which yielded several new point mutations in ftsQ. Two of these mutations affect localization of FtsQ to midcell and together define a targeting role for FtsQ's alpha domain. Further characterization of one localization-defective mutant protein [FtsQ(V92D)] revealed an unexpected role in localization for the first 49 amino acids of FtsQ. Finally, we found a suppressor of FtsQ(V92D) that was due to a point mutation in domain 1C of FtsA, a domain previously implicated in the recruitment of divisome proteins. However, despite reports of a potential interaction between FtsA and FtsQ, suppression by FtsA(I143L) is not mediated via direct contact with FtsQ. Rather, this mutation acts as a general suppressor of division defects, which include deletions of the normally essential genes zipA and ftsK and mutations in FtsQ that affect both localization and recruitment. Together, these results reveal increasingly complex connections within the bacterial divisome.

• Moreira IS, Fernandes PA, Ramos MJ
• Detailed microscopic study of the full zipA:FtsZ interface.
• Proteins. 2006; 63: 811-21
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• Protein-protein interaction networks are very important for a wide range of biological processes. Crystallographic structures and mutational studies have generated a large number of information that allowed the discovery of energetically important determinants of specificity at intermolecular protein interfaces and the understanding of the structural and energetic characteristics of the binding hot spots. In this study we have used the improved MMPB/SA (molecular mechanics/Poisson-Boltzmann surface area) approach that combining molecular mechanics and continuum solvent permits to calculate the free energy differences upon alanine mutation. For a better understanding of the binding determinants of the complex formed between the FtsZ fragment and ZipA we extended the alanine scanning mutagenesis study to all interfacial residues of this complex. As a result, we present new mutations that allowed the discovery of residues for which the binding free energy differences upon alanine mutation are higher than 2.0 kcal/mol. We also observed the formation of a hydrophobic pocket with a high warm spot spatial complementarity between FtsZ and ZipA. Small molecules could be designed to bind to these amino acid residues hindering the binding of FtsZ to ZipA. Hence, these mutational data can be used to design new drugs to control more efficiently bacterial infections.

• Hamoen LW, Meile JC, de Jong W, Noirot P, Errington J
• SepF, a novel FtsZ-interacting protein required for a late step in cell division.
• Mol Microbiol. 2006; 59: 989-999
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• Summary Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.

• Kawai Y, Ogasawara N
• Bacillus subtilis EzrA and FtsL synergistically regulate FtsZ ring dynamics during cell division.
• Microbiology. 2006; 152: 1129-41
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• Previous work has shown that the Bacillus subtilis EzrA protein directly inhibits FtsZ ring assembly, which is required for normal cell division, and that loss of EzrA results in hyperstabilization of the FtsZ polymer in vivo. Here, it was found that in ezrA-disrupted cells, artificial expression of YneA, which suppresses cell division during the SOS response, and disruption of noc (yyaA), which acts as an effector of nucleoid occlusion, resulted in accumulation of multiple non-constricting FtsZ rings, inhibition of cell division, and synthetic lethality. Overexpression of the essential cell division protein FtsL suppressed the effect of ezrA disruption. FtsL overexpression recovered the delayed FtsZ ring constriction seen in ezrA-disrupted wild-type cells. Conversely, the absence of EzrA caused lethality in cells producing a lower amount of FtsL than wild-type cells. It has previously been reported that FtsL is recruited to the division site during the later stages of cell division, although its exact role is currently unknown. The results of this study suggest that FtsL and EzrA synergistically regulate the FtsZ ring constriction in B. subtilis. Interestingly, FtsL overexpression also suppressed the cell division inhibition due to YneA expression or Noc inactivation in ezrA-disrupted cells.

• Beuria TK, Shah JH, Santra MK, Kumar V, Panda D
• Effects of pH and ionic strength on the assembly and bundling of FtsZ protofilaments: a possible role of electrostatic interactions in the bundling of protofilaments.
• Int J Biol Macromol. 2006; 40: 30-9
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• Assembly, bundling and stability of FtsZ protofilaments are important for the formation and functioning of the cytokinetic Z-ring during bacterial division. We found that the bundling of FtsZ protofilaments decreased strongly with increasing pH from 6.0 to 7.9, while the assembly of FtsZ monomers did not decrease considerably. In addition, the disassembly of FtsZ protofilaments was strongly suppressed at pH 6.0 as compared to the elevated pHs. The far-UV circular dichroism spectra of the native FtsZ and the tryptophan emission spectra of mutated FtsZ (Y371W) did not change by increasing pH from 6 to 7.9 indicating that the structure of FtsZ was not altered significantly. Further, the inhibition of bundling of FtsZ protofilaments predominantly, and the inhibition of assembly to a lesser extent by salt indicated that electrostatic interactions are important for the assembly and bundling of FtsZ protofilaments. These observations are supported by the results of computational docking of Escherichia coli dimer structure. The results suggest that the basic intracellular pH (7.4-7.8) of E. coli may play a role in regulating the assembly dynamics of FtsZ in the Z-ring by reducing protofilament stability and bundling in bacterial cytoplasm.

• Ito H et al.
• A 4-aminofurazan derivative-A189-inhibits assembly of bacterial cell division protein FtsZ in vitro and in vivo.
• Microbiol Immunol. 2006; 50: 759-64
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• Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4-aminofurazan derivative was found to inhibit FtsZ GTPase with an IC(50) of 80 mug/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189-treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA-deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.

• Harry E, Monahan L, Thompson L
• Bacterial cell division: the mechanism and its precison.
• Int Rev Cytol. 2006; 253: 27-94
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• The recent development of cell biology techniques for bacteria to allow visualization of fundamental processes in time and space, and their use in synchronous populations of cells, has resulted in a dramatic increase in our understanding of cell division and its regulation in these tiny cells. The first stage of cell division is the formation of a Z ring, composed of a polymerized tubulin-like protein, FtsZ, at the division site precisely at midcell. Several membrane-associated division proteins are then recruited to this ring to form a complex, the divisome, which causes invagination of the cell envelope layers to form a division septum. The Z ring marks the future division site, and the timing of assembly and positioning of this structure are important in determining where and when division will take place in the cell. Z ring assembly is controlled by many factors including negative regulatory mechanisms such as Min and nucleoid occlusion that influence Z ring positioning and FtsZ accessory proteins that bind to FtsZ directly and modulate its polymerization behavior. The replication status of the cell also influences the positioning of the Z ring, which may allow the tight coordination between DNA replication and cell division required to produce two identical newborn cells.

• Osawa M, Erickson HP
• FtsZ from divergent foreign bacteria can function for cell division in Escherichia coli.
• J Bacteriol. 2006; 188: 7132-40
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• FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10(-3) to 10(-5), suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.

• Thakur M, Chakraborti PK
• GTPase activity of mycobacterial FtsZ is impaired due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA.
• J Biol Chem. 2006; 281: 40107-13
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• FtsZ, a homolog of eukaryotic tubulin, is involved in the process of cell division, particularly in septum formation in bacteria. The primary amino acid sequences of this protein are fairly conserved in prokaryotes. We observed that a eukaryotic-type Ser/Thr protein kinase, PknA from Mycobacterium tuberculosis, when expressed in Escherichia coli exhibited cell elongation due to a defect in septum formation. We found that FtsZ either from Escherichia coli (eFtsZ) or from M. tuberculosis (mFtsZ) was phosphorylated on co-expression with PknA. Consistent with these observations, solid phase binding and in vitro kinase assays revealed the ability of PknA to interact with mFtsZ protein and also to phosphorylate it. We, therefore, ascertained mFtsZ as a substrate of PknA. Furthermore, the phosphorylated mFtsZ exhibited impairment in its GTP hydrolysis and polymerization abilities. Thus, our results highlighted the ability of PknA to phosphorylate as well as to regulate the functionality of FtsZ, the protein central to cell division throughout the bacterial lineage.

• Tsao DH et al.
• Discovery of novel inhibitors of the ZipA/FtsZ complex by NMR fragment screening coupled with structure-based design.
• Bioorg Med Chem. 2006; 14: 7953-61
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• ZipA is a membrane anchored protein in Escherichia coli that interacts with FtsZ, a homolog of eukaryotic tubulins, forming a septal ring structure that mediates bacterial cell division. Thus, the ZipA/FtsZ protein-protein interaction is a potential target for an antibacterial agent. We report here an NMR-based fragment screening approach which identified several hits that bind to the C-terminal region of ZipA. The screen was performed by 1H-15N HSQC experiments on a library of 825 fragments that are small, lead-like, and highly soluble. Seven hits were identified, and the binding mode of the best one was revealed in the X-ray crystal structure. Similar to the ZipA/FtsZ contacts, the driving force in the binding of the small molecule ligands to ZipA is achieved through hydrophobic interactions. Analogs of this hit were also evaluated by NMR and X-ray crystal structures of these analogs with ZipA were obtained, providing structural information to help guide the medicinal chemistry efforts.

• Wang S, Arends SJ, Weiss DS, Newman EB
• A deficiency in S-adenosylmethionine synthetase interrupts assembly of the septal ring in Escherichia coli K-12.
• Mol Microbiol. 2005; 58: 791-9
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• A mutant in which S-adenosylmethionine synthetase is underexpressed makes filaments with no visible septa. Examination with GFP fusions to various septal proteins shows that FtsZ, ZipA and FtsA localize to the septal ring, but FtsQ, FtsW, FtsI or FtsN do not. The requirement for S-adenosylmethionine suggests that some methylation reaction is required before a complete septal ring can be assembled.

• Goehring NW, Gueiros-Filho F, Beckwith J
• Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli.
• Genes Dev. 2005; 19: 127-37
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• Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell. Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway. Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins. Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step. Here we present a method--premature targeting--for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway. We fused FtsQ to the FtsZ-binding protein ZapA such that FtsQ was targeted to FtsZ rings independently of proteins FtsA and FtsK, which are normally required for FtsQ localization. Analysis of the resulting ZapA-FtsQ fusion suggests that FtsQ associates with a large complex of cell division proteins and that premature targeting of FtsQ can restore localization of this complex under conditions in which neither FtsQ nor the associated proteins would normally be localized.

• Ramos A, Letek M, Campelo AB, Vaquera J, Mateos LM, Gil JA
• Altered morphology produced by ftsZ expression in Corynebacterium glutamicum ATCC 13869.
• Microbiology. 2005; 151: 2563-72
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• Corynebacterium glutamicum is a Gram-positive bacterium that lacks the cell division FtsA protein and actin-like MreB proteins responsible for determining cylindrical cell shape. When the cell division ftsZ gene from C. glutamicum (ftsZ(Cg)) was cloned in different multicopy plasmids, the resulting constructions could not be introduced into C. glutamicum; it was assumed that elevated levels of FtsZ(Cg) result in lethality. The presence of a truncated ftsZ(Cg) and a complete ftsZ(Cg) under the control of Plac led to a fourfold reduction in the intracellular levels of FtsZ, generating aberrant cells displaying buds, branches and knots, but no filaments. A 20-fold reduction of the FtsZ level by transformation with a plasmid carrying the Escherichia coli lacI gene dramatically reduced the growth rate of C. glutamicum, and the cells were larger and club-shaped. Immunofluorescence microscopy of FtsZ(Cg) or visualization of FtsZ(Cg)-GFP in C. glutamicum revealed that most cells showed one fluorescent band, most likely a ring, at the mid-cell, and some cells showed two fluorescent bands (septa of future daughter cells). When FtsZ(Cg)-GFP was expressed from Plac, FtsZ rings at mid-cell, or spirals, were also clearly visible in the aberrant cells; however, this morphology was not entirely due to GFP but also to the reduced levels of FtsZ expressed from Plac. Localization of FtsZ at the septum is not negatively regulated by the nucleoid, and therefore the well-known occlusion mechanism seems not to operate in C. glutamicum.

• Aarsman ME, Piette A, Fraipont C, Vinkenvleugel TM, Nguyen-Disteche M, den Blaauwen T
• Maturation of the Escherichia coli divisome occurs in two steps.
• Mol Microbiol. 2005; 55: 1631-45
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• Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14-21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles.

• Beuria TK, Santra MK, Panda D
• Sanguinarine Blocks Cytokinesis in Bacteria by Inhibiting FtsZ Assembly and Bundling.
• Biochemistry. 2005; 44: 16584-93
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• Bacterial diseases are among the leading causes of human death. The development of antibiotic resistance greatly contributes to the high mortality rate, and thus, the discovery of antibacterial drugs with novel mechanisms of action is needed. In this study, we found that sanguinarine, a benzophenanthridine alkaloid, strongly induced filamentation in both Gram-positive and Gram-negative bacteria and prevented bacterial cell division by inhibiting cytokinesis. Sanguinarine did not perturb the membrane structure in Escherichia coli. However, it perturbed the cytokinetic Z-ring formation in E. coli. In addition, sanguinarine strongly reduced the frequency of the occurrence of Z rings/micrometer of Bacillus subtilis length but did not alter the number of nucleoids/micrometer of cell length. The results suggested that sanguinarine inhibited cytokinesis in B. subtilis by inhibiting Z-ring formation without affecting nucleoid segregation. Sanguinarine inhibited the assembly of purified FtsZ and reduced the bundling of FtsZ protofilaments in vitro. Further, the interaction of sanguinarine to FtsZ was investigated using size-exclusion chromatography, an extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, and tryptophan fluorescence of mutated FtsZ (Y371W). Sanguinarine was found to bind to FtsZ with a dissociation constant of 18-30 muM. The results together show that sanguinarine inhibits bacterial division by perturbing FtsZ assembly dynamics in the Z ring and provide evidence in support of the hypothesis that the assembly and bundling of FtsZ play a critical role in bacterial cytokinesis. The results suggest that sanguinarine may be used as a lead compound to develop FtsZ-targeted antibacterial agents.

• Weart RB, Nakano S, Lane BE, Zuber P, Levin PA
• The ClpX chaperone modulates assembly of the tubulin-like protein FtsZ.
• Mol Microbiol. 2005; 57: 238-49
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• Summary Assembly of the tubulin-like cytoskeletal protein FtsZ into a ring structure establishes the location of the nascent division site in prokaryotes. Factors that modulate FtsZ assembly are essential for ensuring the precise spatial and temporal regulation of cytokinesis. We have identified ClpX, the substrate recognition subunit of the ClpXP protease, as an inhibitor of FtsZ assembly in Bacillus subtilis. Genetic data indicate that ClpX but not ClpP inhibits FtsZ-ring formation in vivo. In vitro, ClpX inhibits FtsZ assembly in a ClpP-independent manner through a mechanism that does not require ATP hydrolysis. Together our data support a model in which ClpX helps maintain the cytoplasmic pool of unassembled FtsZ that is required for the dynamic nature of the cytokinetic ring. ClpX is conserved throughout bacteria and has been shown to interact directly with FtsZ in Escherichia coli. Thus, we speculate that ClpX functions as a general regulator of FtsZ assembly and cell division in a wide variety of bacteria.

• Lara B et al.
• Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein.
• Mol Microbiol. 2005; 55: 699-711
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• We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide-dependent manner, forming long corkscrew-like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.

• Geissler B, Margolin W
• Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK.
• Mol Microbiol. 2005; 58: 596-612
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• In Escherichia coli, at least 12 proteins colocalize to the cell midpoint, assembling into a membrane-associated protein machine that forms the division septum. Many of these proteins, including FtsK, are essential for viability but their functions in cell division are unknown. Here we show that the essential function of FtsK in cell division can be partially bypassed. Cells containing either the ftsA R286W mutation or a plasmid carrying the ftsQAZ genes suppressed a ftsK44(ts) allele efficiently. Moreover, ftsA R286W or multicopy ftsQAZ, which can largely bypass the requirement for the essential cell division gene zipA, allowed cells with a complete deletion of ftsK to survive and divide, although many of these ftsK null cells formed multiseptate chains. Green fluorescent protein (GFP) fusions to FtsI and FtsN, which normally depend on FtsK to localize to division sites, localized to division sites in the absence of FtsK, indicating that FtsK is not directly involved in their recruitment. Cells expressing additional ftsQ, and to a lesser extent ftsB and ftsN, were able to survive and divide in the absence of ftsK, although cell chains were often formed. Surprisingly, the cytoplasmic and transmembrane domains of FtsQ, while not sufficient to complement an ftsQ null mutant, conferred viability and septum formation in the absence of ftsK. These findings suggest that the N-terminal domain of FtsK is normally involved in stability of the division protein machine and shares functional overlap with FtsQ, FtsB, FtsA, ZipA and FtsN.

• Pichoff S, Lutkenhaus J
• Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA.
• Mol Microbiol. 2005; 55: 1722-34
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• The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.

• Bernhardt TG, de Boer PA
• SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over Chromosomes in E. coli.
• Mol Cell. 2005; 18: 555-64
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• Cell division in Escherichia coli begins with assembly of the tubulin-like FtsZ protein into a ring structure just underneath the cell membrane. Spatial control over Z ring assembly is achieved by two partially redundant negative regulatory systems, the Min system and nucleoid occlusion (NO), which cooperate to position the division site at midcell. In contrast to the well-studied Min system, almost nothing is known about how Z ring assembly is blocked in the vicinity of nucleoids to effect NO. Reasoning that Min function might become essential in cells impaired for NO, we screened for mutations synthetically lethal with a defective Min system (slm mutants). By using this approach, we identified SlmA (Ttk) as the first NO factor in E. coli. Our combined genetic, cytological, and biochemical results suggest that SlmA is a DNA-associated division inhibitor that is directly involved in preventing Z ring assembly on portions of the membrane surrounding the nucleoid.

• Jensen SO, Thompson LS, Harry EJ
• Cell division in Bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-Ring assembly.
• J Bacteriol. 2005; 187: 6536-44
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• The earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA in Bacillus subtilis cells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in a B. subtilis strain containing an ftsA deletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.

• Rajagopalan M et al.
• Genetic evidence that mycobacterial FtsZ and FtsW proteins interact, and colocalize to the division site in Mycobacterium smegmatis.
• FEMS Microbiol Lett. 2005; 250: 9-17
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• We provide genetic evidence to show that the Mycobacterium tuberculosis FtsZ and FtsW proteins interact, and that these interactions are biologically relevant. Furthermore, we show by fluorescence microscopy that Mycobacterium smegmatis FtsW is part of its septasomal complex and colocalizes with FtsZ to the midcell sites. Colocalization experiments reveal that approximately 27% of the cells with septal Z-rings contain FtsW whereas 93% of the cells with FtsW bands are associated with FtsZ indicating that FtsW is late recruit to the septum, as in Escherichia coli. Our results suggest that mycobacterial FtsZ can localize to the septum independent of FtsW, and that interactions of FtsW with FtsZ are critical for the formation of productive FtsZ-rings and the cell division process in mycobacteria.

• Goehring NW, Beckwith J
• Diverse paths to midcell: assembly of the bacterial cell division machinery.
• Curr Biol. 2005; 15: 51426-51426
• Display abstract

• At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.

• Corbin BD, Geissler B, Sadasivam M, Margolin W
• Z-ring-independent interaction between a subdomain of FtsA and late septation proteins as revealed by a polar recruitment assay.
• J Bacteriol. 2004; 186: 7736-44
• Display abstract

• FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.

• Zhao Y, Hammond RW, Lee IM, Roe BA, Lin S, Davis RE
• Cell division gene cluster in Spiroplasma kunkelii: functional characterization of ftsZ and the first report of ftsA in mollicutes.
• DNA Cell Biol. 2004; 23: 127-34
• Display abstract

• Spiroplasma kunkelii is a helical, wall-less bacterium that causes corn stunt disease. In adaptation to its phloem-inhabiting parasitic lifestyle, the bacterium has undergone a reductive evolutionary process and, as a result, possesses a compact genome with a gene set approaching the minimal complement necessary for multiplication and pathogenesis. We cloned a much-reduced cell division gene cluster from S. kunkelii and functionally characterized the key division gene, ftsZ(sk). The 1236-bp open reading frame of ftsZ(sk) is capable of encoding a protein with a calculated molecular mass of 44.1 kDa. Protein sequence alignment revealed that FtsZ(sk) is remarkably similar to FtsZ proteins from other eubacteria, and possesses the conserved GTP-binding and hydrolyzing motifs. We demonstrated that overexpression of ftsZ(sk) in Escherichia coli causes transgression of the host cell division, resulting in a filamentous phenotype. We also report, for the first time, the presence of a ftsA gene in the cell division cluster of a mollicute species.

• Jennings LD et al.
• Design and synthesis of indolo[2,3-a]quinolizin-7-one inhibitors of the ZipA-FtsZ interaction.
• Bioorg Med Chem Lett. 2004; 14: 1427-31
• Display abstract

• The binding of FtsZ to ZipA is a potential target for antibacterial therapy. Based on a small molecule inhibitor of the ZipA-FtsZ interaction, a parallel synthesis of small molecules was initiated which targeted a key region of ZipA involved in FtsZ binding. The X-ray crystal structure of one of these molecules complexed with ZipA was solved. The structure revealed an unexpected binding mode, facilitated by desolvation of a loosely bound surface water.

• Sun Q, Margolin W
• Effects of perturbing nucleoid structure on nucleoid occlusion-mediated toporegulation of FtsZ ring assembly.
• J Bacteriol. 2004; 186: 3951-9
• Display abstract

• In Escherichia coli, assembly of the FtsZ ring (Z ring) at the cell division site is negatively regulated by the nucleoid in a phenomenon called nucleoid occlusion (NO). Previous studies have indicated that chromosome packing plays a role in NO, as mukB mutants grown in rich medium often exhibit FtsZ rings on top of diffuse, unsegregated nucleoids. To address the potential role of overall nucleoid structure on NO, we investigated the effects of disrupting chromosome structure on Z-ring positioning. We found that NO was mostly normal in cells with inactivated DNA gyrase or in mukB-null mutants lacking topA, although some suppression of NO was evident in the latter case. Previous reports suggesting that transcription, translation, and membrane insertion of proteins ("transertion") influence nucleoid structure prompted us to investigate whether disruption of these activities had effects on NO. Blocking transcription caused nucleoids to become diffuse, and FtsZ relocalized to multiple bands on top of these nucleoids, biased towards midcell. This suggested that these diffuse nucleoids were defective in NO. Blocking translation with chloramphenicol caused characteristic nucleoid compaction, but FtsZ rarely assembled on top of these centrally positioned nucleoids. This suggested that NO remained active upon translation inhibition. Blocking protein secretion by thermoinduction of a secA(Ts) strain caused a chromosome segregation defect similar to that in parC mutants, and NO was active. Although indirect effects are certainly possible with these experiments, the above data suggest that optimum NO activity may require specific organization and structure of the nucleoid.

• Johnson JE, Lackner LL, Hale CA, de Boer PA
• ZipA is required for targeting of DMinC/DicB, but not DMinC/MinD, complexes to septal ring assemblies in Escherichia coli.
• J Bacteriol. 2004; 186: 2418-29
• Display abstract

• The MinC division inhibitor is required for accurate placement of the septal ring at the middle of the Escherichia coli cell. The N-terminal domain of MinC ((Z)MinC) interferes with FtsZ assembly, while the C-terminal domain ((D)MinC) mediates both dimerization and complex formation with either MinD or DicB. Binding to either of these activators greatly enhances the division-inhibitory activity of MinC in the cell. The MinD ATPase plays a crucial role in the rapid pole-to-pole oscillation of MinC that is proposed to force FtsZ ring formation to midcell. DicB is encoded by one of the cryptic prophages on the E. coli chromosome (Qin) and is normally not synthesized. Binding of MinD or DicB to (D)MinC produces complexes that have high affinities for one or more septal ring-associated targets. Here we show that the FtsZ-binding protein ZipA is required for both recruitment of the (D)MinC/DicB complex to FtsZ rings and the DicB-inducible division block normally seen in MinC(+) cells. In contrast, none of the known FtsZ-associated factors, including ZipA, FtsA, and ZapA, appear to be specifically required for targeting of the (D)MinC/MinD complex to rings, implying that the two MinC/activator complexes must recognize distinct features of FtsZ assemblies. MinD-dependent targeting of MinC may occur in two steps of increasing topological specificity: (i) recruitment of MinC from the cytoplasm to the membrane, and (ii) specific targeting of the MinC/MinD complex to nascent septal ring assemblies on the membrane. Using membrane-tethered derivatives of MinC, we obtained evidence that both of these steps contribute to the efficiency of MinC/MinD-mediated division inhibition.

• Ferguson PL, Shaw GS
• Human S100B protein interacts with the Escherichia coli division protein FtsZ in a calcium-sensitive manner.
• J Biol Chem. 2004; 279: 18806-13
• Display abstract

• S100B is a small, dimeric EF-hand calcium-binding protein abundant in vertebrates. Upon calcium binding, S100B undergoes a conformational change allowing it to interact with a variety of target proteins, including the cytoskeletal proteins tubulin and glial fibrillary acidic protein. In both cases, S100B promotes the in vitro disassembly of these proteins in a calcium-sensitive manner. Despite this, there is little in vivo evidence for the interaction of proteins such as tubulin with S100B. To probe these interactions, we studied the expression of human S100B in Escherichia coli and its interaction with the prokaryotic ancestor of tubulin, FtsZ, the major protein involved in bacterial division. Expression of S100B protein in E. coli results in little change in FtsZ protein levels, causes a filamenting bacterial phenotype characteristic of FtsZ inhibition, and leads to missed rounds of cell division. Further, S100B localizes to positions similar to those of FtsZ in bacterial filaments: the small foci at the poles, the mid-cell positions, and between the nucleoids at regular intervals. Calcium-dependent physical interaction between S100B and FtsZ was demonstrated in vitro by affinity chromatography, and this interaction was severely inhibited by the competitor peptide TRTK-12. Together these results indicate that S100B interacts with the tubulin homologue FtsZ in vivo, modulating its activity in bacterial cell division. This approach will present an important step for the study of S100 protein interactions in vivo.

• Jennings LD et al.
• Combinatorial synthesis of substituted 3-(2-indolyl)piperidines and 2-phenyl indoles as inhibitors of ZipA-FtsZ interaction.
• Bioorg Med Chem. 2004; 12: 5115-31
• Display abstract

• The ZipA-FtsZ protein-protein interaction is a potential target for antibacterial therapy. The design and parallel synthesis of a combinatorial library of small molecules, which target the FtsZ binding area on ZipA are described. Compounds were demonstrated to bind to the FtsZ binding domain of ZipA by HSQC NMR and to inhibit cell division in a cell elongation assay.

• Ogino H et al.
• FtsZ-dependent localization of GroEL protein at possible division sites.
• Genes Cells. 2004; 9: 765-71
• Display abstract

• When Escherichia coli is treated with penicillin, the envelopes bulge at the centre of the cells and the cells then lyse. The bulges expand into vesicle-like structures termed penicillin-induced vesicles. We have developed a method to isolate these structures and have shown that they contain mainly membrane proteins plus a high concentration of a 60 kDa protein. The N-terminal amino acid sequence of the protein is identical to that of GroEL protein. Western blotting analysis using anti-GroEL antibody showed that GroEL is indeed concentrated in the vesicles. Indirect immuno-fluorescence microscopy showed that GroEL protein is localized at the centre of the cells at the site of formation of FtsZ-rings. Localization of GroEL is dependent on FtsZ but not other Fts proteins. GroEL mutants formed elongated cells having no or asymmetrically localized FtsZ-rings at the restrictive temperature. These findings suggest a possible role of the GroEL protein in cell division.

• Varma A, Young KD
• FtsZ collaborates with penicillin binding proteins to generate bacterial cell shape in Escherichia coli.
• J Bacteriol. 2004; 186: 6768-74
• Display abstract

• The mechanisms by which bacteria adopt and maintain individual shapes remain enigmatic. Outstanding questions include why cells are a certain size, length, and width; why they are uniform or irregular; and why some branch while others do not. Previously, we showed that Escherichia coli mutants lacking multiple penicillin binding proteins (PBPs) display extensive morphological diversity. Because defective sites in these cells exhibit the structural and functional characteristics of improperly localized poles, we investigated the connection between cell division and shape. Here we show that under semipermissive conditions the temperature-sensitive FtsZ84 protein produces branched and aberrant cells at a high frequency in mutants lacking PBP 5, and this phenotype is exacerbated by the loss of additional peptidoglycan endopeptidases. Surprisingly, certain ftsZ84 strains lyse at the nonpermissive temperature instead of filamenting, and inhibition of wild-type FtsZ forces some mutants into tightly wound spirillum-like morphologies. The results demonstrate that significant aspects of bacterial shape are dictated by a previously unrecognized relationship between the septation machinery and ostensibly minor peptidoglycan-modifying enzymes and that under certain circumstances improper FtsZ function can destroy the structural integrity of the cell.

• Weiss DS
• Bacterial cell division and the septal ring.
• Mol Microbiol. 2004; 54: 588-97
• Display abstract

• Cell division in bacteria is mediated by the septal ring, a collection of about a dozen (known) proteins that localize to the division site, where they direct assembly of the division septum. The foundation of the septal ring is a polymer of the tubulin-like protein FtsZ. Recently, experiments using fluorescence recovery after photobleaching have revealed that the Z ring is extremely dynamic. FtsZ subunits exchange in and out of the ring on a time scale of seconds even while the overall morphology of the ring appears static. These findings, together with in vitro studies of purified FtsZ, suggest that the rate-limiting step in turnover of FtsZ polymers is GTP hydrolysis. Another component of the septal ring, FtsK, is involved in coordinating chromosome segregation with cell division. Recent studies have revealed that FtsK is a DNA translocase that facilitates decatenation of sister chromosomes by TopIV and resolution of chromosome dimers by the XerCD recombinase. Finally, two murein hydrolases, AmiC and EnvC, have been shown to localize to the septal ring of Escherichia coli, where they play an important role in separation of daughter cells.

• Rico AI, Garcia-Ovalle M, Mingorance J, Vicente M
• Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring.
• Mol Microbiol. 2004; 53: 1359-71
• Display abstract

• The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.

• Anderson DE, Gueiros-Filho FJ, Erickson HP
• Assembly dynamics of FtsZ rings in Bacillus subtilis and Escherichia coli and effects of FtsZ-regulating proteins.
• J Bacteriol. 2004; 186: 5775-81
• Display 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 (approximately 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 approximately 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, approximately 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.

• Haeusser DP, Schwartz RL, Smith AM, Oates ME, Levin PA
• EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ.
• Mol Microbiol. 2004; 52: 801-14
• Display abstract

• In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.

• Piette A, Fraipont C, Den Blaauwen T, Aarsman ME, Pastoret S, Nguyen-Disteche M
• Structural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli.
• J Bacteriol. 2004; 186: 6110-7
• Display abstract

• In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

• Janakiraman A, Goldberg MB
• Recent advances on the development of bacterial poles.
• Trends Microbiol. 2004; 12: 518-25
• Display abstract

• In rod-shaped bacteria, a surprisingly large number of proteins are localized to the cell poles. Polar positioning of proteins is crucial to many fundamental cellular processes. Formation of the pole occurs at the time of a prior cell division event and involves coordination of the cell division machinery with septal placement of newly-synthesized peptidoglycan. Development of polar peptidoglycan and outer membrane depends on the formation of the cytokinetic FtsZ ring at midcell. By contrast, positioning of at least two polar proteins depends on signals independent of both the assembly of the FtsZ ring and the synthesis of septal and polar peptidoglycan. We propose a model for distinct but interrelated developmental pathways for polar cell envelope synthesis and positional information recognized by polar proteins.

• Margalit DN et al.
• Targeting cell division: small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality.
• Proc Natl Acad Sci U S A. 2004; 101: 11821-6
• Display abstract

• FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.

• Mazouni K, Domain F, Cassier-Chauvat C, Chauvat F
• Molecular analysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipN and MinCDE.
• Mol Microbiol. 2004; 52: 1145-58
• Display abstract

• Using a bacterial two-hybrid system and a combination of in vivo and in vitro assays that take advantage of the green fluorescent reporter protein (GFP), we have investigated the localization and the protein-protein interaction of several key components of the cytokinetic machinery of cyanobacteria (i.e. the progenitor of chloroplast). We demonstrate that (i) the ftsZ and zipN genes are essential for the viability of the model cyanobacterium Synechocystis sp. PCC 6803, whereas the minCDE cluster is dispensable for cell growth; (ii) the GTP-binding domain of FtsZ is crucial to FtsZ assembly into the septal ring at mid-cell; (iii) the Z-ring of deeply constricted daughter cells is oriented perpendicularly to the mother Z-ring, showing that Synechocystis divides in alternating perpendicular planes; (iv) the MinCDE system affects the morphology of the cell, as well as the position and the shape of FtsZ structures; and (v) MinD is targeted to cell membranes in a process involving its C-terminal amphipathic helix, but not its ATP-binding region. Finally, we have also characterized a novel Z-interacting protein, ZipN, the N-terminal DnaJ domain of which is critical to the decoration of the Z-ring, and we report that this process is independent of MinCDE.

• Thanedar S, Margolin W
• FtsZ exhibits rapid movement and oscillation waves in helix-like patterns in Escherichia coli.
• Curr Biol. 2004; 14: 1167-73
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• Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis, and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance. Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern. The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings. Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins. The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD. The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.

• Buddelmeijer N, Beckwith J
• A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region.
• Mol Microbiol. 2004; 52: 1315-27
• Display abstract

• Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region.

• Koppelman CM et al.
• R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division.
• Mol Microbiol. 2004; 51: 645-57
• Display abstract

• We investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP). FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ. FtsZ-R174D could also localize in an FtsZ depletion strain. However, in contrast to wild-type FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

• Morlot C, Noirclerc-Savoye M, Zapun A, Dideberg O, Vernet T
• The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae.
• Mol Microbiol. 2004; 51: 1641-8
• Display abstract

• Bacterial division requires the co-ordination of membrane invagination, driven by the constriction of the FtsZ-ring, and concomitant cell wall synthesis, performed by the high-molecular-weight penicillin-binding proteins (HMW PBPs). Using immunofluorescence techniques, we show in Streptococcus pneumoniae that this co-ordination requires PBP3, a D,D-carboxypeptidase that degrades the substrate of the HMW PBPs. In a mutant deprived of PBP3, the apparent rings of HMW PBPs and that of FtsZ are no longer co-localized. In wild-type cells, PBP3 is absent at the future division site and present over the rest of the cell surface, implying that the localization of the HMW PBPs at mid-cell depends on the availability of their substrate. FtsW, a putative translocase of the substrate of the PBPs, forms an apparent ring that is co-localized with the septal HMW PBPs throughout the cell cycle of wild-type cells. In particular, the constriction of the FtsW-ring occurs after that of the FtsZ-ring, with the same delay as the constriction of the septal PBP-rings. However, in the absence of PBP3, FtsW remains co-localized with FtsZ in contrast to the HMW PBPs. Our work reveals an unexpected complexity in the relationships between the division proteins. The consequences of the absence of PBP3 indicate that the peptidoglycan composition is central to the co-ordination of the division process.

• Schmidt KL et al.
• A predicted ABC transporter, FtsEX, is needed for cell division in Escherichia coli.
• J Bacteriol. 2004; 186: 785-93
• Display abstract

• FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.

• Cordell SC, Robinson EJ, Lowe J
• Crystal structure of the SOS cell division inhibitor SulA and in complex with FtsZ.
• Proc Natl Acad Sci U S A. 2003; 100: 7889-94
• Display abstract

• SulA halts cell division in Escherichia coli by binding to the major component of the division machinery FtsZ. We have solved the crystal structure of SulA alone and in complex with FtsZ from Pseudomonas aeruginosa. SulA is expressed when the SOS response is induced. This is a mechanism to inhibit cell division and repair DNA in the event of DNA damage. FtsZ is a tubulin-like protein that forms polymers, with the active-site GTPase split across two monomers. One monomer provides the GTP-binding site and the other, through its T7 loop nucleotide hydrolysis. Our structures show that SulA is a dimer, and that SulA inhibits cell division neither by binding the nucleotide-binding site nor by inducing conformational changes in FtsZ. Instead, SulA binds the T7 loop surface of FtsZ, opposite the nucleotide-binding site, blocking polymer formation. These findings explain why GTP hydrolysis and polymer turnover are required for SulA inhibition.

• Sutherland AG et al.
• Structure-based design of carboxybiphenylindole inhibitors of the ZipA-FtsZ interaction.
• Org Biomol Chem. 2003; 1: 4138-40
• Display abstract

• Structural features of two weak inhibitors of the ZipA-FtsZ protein-protein interaction which were found to bind to overlapping but different areas of the key binding site were combined in one new series of carboxybiphenyl-indoles with improved inhibitory activity.

• Di Lallo G, Fagioli M, Barionovi D, Ghelardini P, Paolozzi L
• Use of a two-hybrid assay to study the assembly of a complex multicomponent protein machinery: bacterial septosome differentiation.
• Microbiology. 2003; 149: 3353-9
• Display abstract

• The ability of each of the nine Escherichia coli division proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, FtsN) to interact with itself and with each of the remaining eight proteins was studied in 43 possible combinations of protein pairs by the two-hybrid system previously developed by the authors' group. Once the presumed interactions between the division proteins were determined, a model showing their temporal sequence of assembly was developed. This model agrees with that developed by other authors, based on the co-localization sequence in the septum of the division proteins fused with GFP. In addition, this paper shows that the authors' assay, which has already proved to be very versatile in the study of prokaryotic and eukaryotic protein interaction, is also a powerful instrument for an in vivo study of the interaction and assembly of proteins, as in the case of septum division formation.

• Du Y, Arvidson CG
• Identification of ZipA, a signal recognition particle-dependent protein from Neisseria gonorrhoeae.
• J Bacteriol. 2003; 185: 2122-30
• Display abstract

• A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria gonorrhoeae plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related N. meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in N. gonorrhoeae since it was not possible to generate null mutations in the gene. Although appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene zipA of E. coli. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an E. coli conditional zipA mutant and therefore encodes a functional ZipA homolog in N. gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.

• Weart RB, Levin PA
• Growth rate-dependent regulation of medial FtsZ ring formation.
• J Bacteriol. 2003; 185: 2826-34
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• FtsZ is an essential cell division protein conserved throughout the bacteria and archaea. In response to an unknown cell cycle signal, FtsZ polymerizes into a ring that establishes the future division site. We conducted a series of experiments examining the link between growth rate, medial FtsZ ring formation, and the intracellular concentration of FtsZ in the gram-positive bacterium Bacillus subtilis. We found that, although the frequency of cells with FtsZ rings varies as much as threefold in a growth rate-dependent manner, the average intracellular concentration of FtsZ remains constant irrespective of doubling time. Additionally, expressing ftsZ solely from a constitutive promoter, thereby eliminating normal transcriptional control, did not alter the growth rate regulation of medial FtsZ ring formation. Finally, our data indicate that overexpressing FtsZ does not dramatically increase the frequency of cells with medial FtsZ rings, suggesting that the mechanisms governing ring formation are refractile to increases in FtsZ concentration. These results support a model in which the timing of FtsZ assembly is governed primarily through cell cycle-dependent changes in FtsZ polymerization kinetics and not simply via oscillations in the intracellular concentration of FtsZ. Importantly, this model can be extended to the gram-negative bacterium Escherichia coli. Our data show that, like those in B. subtilis, average FtsZ levels in E. coli are constant irrespective of doubling time.

• Errington J, Daniel RA, Scheffers DJ
• Cytokinesis in bacteria.
• Microbiol Mol Biol Rev. 2003; 67: 52-65
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• Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.

• Rueda S, Vicente M, Mingorance J
• Concentration and assembly of the division ring proteins FtsZ, FtsA, and ZipA during the Escherichia coli cell cycle.
• J Bacteriol. 2003; 185: 3344-51
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• The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.

• Geissler B, Elraheb D, Margolin W
• A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli.
• Proc Natl Acad Sci U S A. 2003; 100: 4197-202
• Display abstract

• ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of Escherichia coli. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with ftsA* in single copy in place of WT ftsA or with ftsA* alone on a multicopy plasmid divide mostly normally, whether they are zipA+ or zipA-. Experiments with ftsQAZ and ftsQA*Z on multicopy plasmids indicate that ftsQAZzipA+ and ftsQA*ZzipA- cells divide fairly normally, whereas ftsQAZzipA- cells divide poorly and ftsQA*ZzipA+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that ftsA* stabilizes Z rings, single-copy ftsA* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by ftsA*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.

• Kenny CH et al.
• Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction.
• Anal Biochem. 2003; 323: 224-33
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• A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide.

• Romberg L, Levin PA
• Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability.
• Annu Rev Microbiol. 2003; 57: 125-54
• Display abstract

• FtsZ is a prokaryotic tubulin homolog that assembles into a ring at the future site of cell division. The resulting "Z ring" forms the framework for the division apparatus, and its assembly is regulated throughout the bacterial cell cycle. A highly dynamic structure, the Z ring exhibits continual subunit turnover and the ability to rapidly assemble, disassemble, and, under certain circumstances, relocalize. These in vivo properties are ultimately due to FtsZ's capacity for guanosine triphosphate (GTP)-dependent, reversible polymerization. FtsZ polymer stability appears to be fine-tuned such that subtle changes in its assembly kinetics result in large changes in the Z ring structure. Thus, regulatory proteins that modulate FtsZ's assembly dynamics can cause the ring to rapidly remodel in response to developmental and environmental cues.

• Hamoen LW, Errington J
• Polar targeting of DivIVA in Bacillus subtilis is not directly dependent on FtsZ or PBP 2B.
• J Bacteriol. 2003; 185: 693-7
• Display abstract

• DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles. Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA. By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.

• Harry EJ, Lewis PJ
• Early targeting of Min proteins to the cell poles in germinated spores of Bacillus subtilis: evidence for division apparatus-independent recruitment of Min proteins to the division site.
• Mol Microbiol. 2003; 47: 37-48
• Display abstract

• The earliest event in bacterial cell division is the assembly of a tubulin-like protein, FtsZ, at mid-cell to form a ring. In rod-shaped bacteria, the Min system plays an important role in division site placement by inhibiting FtsZ ring formation specifically at the polar regions of the cell. The Min system comprises MinD and MinC, which form an inhibitor complex and, in Bacillus subtilis, DivIVA, which ensures that division is inhibited only in the polar regions. All three proteins localize to the division site at mid-cell and to cell poles. Their recruitment to the division site is dependent on localization of both 'early' and 'late' division proteins. We have examined the temporal and spatial localization of DivIVA relative to that of FtsZ during the first and second cell division after germination and outgrowth of B. subtilis spores. We show that, although the FtsZ ring assembles at mid-cell about halfway through the cell cycle, DivIVA assembles at this site immediately before cell division and persists there during Z-ring constriction and completion of division. We also show that both DivIVA and MinD localize to the cell poles immediately upon spore germination, well before a Z ring forms at mid-cell. Furthermore, these proteins were found to be present in mature, dormant spores. These results suggest that targeting of Min proteins to division sites does not depend directly on the assembly of the division apparatus, as suggested previously, and that potential polar division sites are blocked at the earliest possible stage in the cell cycle in germinated spores as a mechanism to ensure that equal-sized daughter cells are produced upon cell division.

• Stricker J, Erickson HP
• In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function.
• J Bacteriol. 2003; 185: 4796-805
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• We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.

• Stricker J, Maddox P, Salmon ED, Erickson HP
• Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching.
• Proc Natl Acad Sci U S A. 2002; 99: 3171-5
• Display abstract

• FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation. FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and in vitro assembly. We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the E. coli Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s. ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic. The Z-ring of the mutant ftsZ84, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ in vitro, showed a 9-fold slower turnover in vivo. This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time.

• Johnson JE, Lackner LL, de Boer PA
• Targeting of (D)MinC/MinD and (D)MinC/DicB complexes to septal rings in Escherichia coli suggests a multistep mechanism for MinC-mediated destruction of nascent FtsZ rings.
• J Bacteriol. 2002; 184: 2951-62
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• The MinC protein is an important determinant of septal ring positioning in Escherichia coli. The N-terminal domain ((Z)MinC) suppresses septal ring formation by interfering with FtsZ polymerization, whereas the C-terminal domain ((D)MinC) is required for dimerization as well as for interaction with the MinD protein. MinD oscillates between the membrane of both cell halves in a MinE-dependent fashion. MinC oscillates along with MinD such that the time-integrated concentration of (Z)MinC at the membrane is minimal, and hence the stability of FtsZ polymers is maximal, at the cell center. MinC is cytoplasmic and fails to block FtsZ assembly in the absence of MinD, indicating that recruitment of MinC by MinD to the membrane enhances (Z)MinC function. Here, we present evidence that the binding of (D)MinC to MinD endows the MinC/MinD complex with a more specific affinity for a septal ring-associated target in vivo. Thus, MinD does not merely attract MinC to the membrane but also aids MinC in specifically binding to, or in close proximity to, the substrate of its (Z)MinC domain. MinC-mediated division inhibition can also be activated in a MinD-independent fashion by the DicB protein of cryptic prophage Kim. DicB shows little homology to MinD, and how it stimulates MinC function has been unclear. Similar to the results obtained with MinD, we find that DicB interacts directly with (D)MinC, that the (D)MinC/DicB complex has a high affinity for some septal ring target(s), and that MinC/DicB interferes with the assembly and/or integrity of FtsZ rings in vivo. The results suggest a multistep mechanism for the activation of MinC-mediated division inhibition by either MinD or DicB and further expand the number of properties that can be ascribed to the Min proteins.

• Mercer KL, Weiss DS
• The Escherichia coli cell division protein FtsW is required to recruit its cognate transpeptidase, FtsI (PBP3), to the division site.
• J Bacteriol. 2002; 184: 904-12
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• The bacterial cell division protein FtsW has been suggested to perform two functions: stabilize the FtsZ cytokinetic ring, and facilitate septal peptidoglycan synthesis by the transpeptidase FtsI (penicillin-binding protein 3). We show here that depleting Escherichia coli cells of FtsW had little effect on the abundance of FtsZ rings but abrogated recruitment of FtsI to potential division sites. Analysis of FtsW localization confirmed and extended these results; septal localization of FtsW required FtsZ, FtsA, FtsQ, and FtsL but not FtsI. Thus, FtsW is a late recruit to the division site and is essential for subsequent recruitment of its cognate transpeptidase FtsI but not for stabilization of FtsZ rings. We suggest that a primary function of FtsW homologues--which are found in almost all bacteria and appear to work in conjunction with dedicated transpeptidases involved in division, elongation, or sporulation--is to recruit their cognate transpeptidases to the correct subcellular location.

• Ding Z, Zhao Z, Jakubowski SJ, Krishnamohan A, Margolin W, Christie PJ
• A novel cytology-based, two-hybrid screen for bacteria applied to protein-protein interaction studies of a type IV secretion system.
• J Bacteriol. 2002; 184: 5572-82
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• DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E. coli and Agrobacterium tumefaciens. DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E. coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP. These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E. coli and A. tumefaciens. Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites. The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments. DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A. tumefaciens T-DNA transfer system, in E. coli and homotypic complexes of VirB10 in A. tumefaciens. VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives. Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.

• Chen JC, Minev M, Beckwith J
• Analysis of ftsQ mutant alleles in Escherichia coli: complementation, septal localization, and recruitment of downstream cell division proteins.
• J Bacteriol. 2002; 184: 695-705
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• FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

• Hale CA, de Boer PA
• ZipA is required for recruitment of FtsK, FtsQ, FtsL, and FtsN to the septal ring in Escherichia coli.
• J Bacteriol. 2002; 184: 2552-6
• Display abstract

• The septal ring in Escherichia coli consists of at least nine essential gene products whose order of assembly resembles a mostly linear dependency pathway: FtsA and ZipA directly bind FtsZ polymers at the prospective division site, followed by the sequential addition of FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN. Recruitment of FtsK and all downstream components requires the prior localization of FtsA. Here we show that recruitment of FtsK, FtsQ, FtsL, and FtsN equally requires ZipA. The results imply that association of both FtsA and ZipA with FtsZ polymers is needed for further maturation of the nascent organelle.

• Pichoff S, Lutkenhaus J
• Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli.
• EMBO J. 2002; 21: 685-93
• Display abstract

• ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ. Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA. We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins. In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized. Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings. These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.

• Gueiros-Filho FJ, Losick R
• A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ.
• Genes Dev. 2002; 16: 2544-56
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• Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

• Scheffers DJ, de Wit JG, den Blaauwen T, Driessen AJ
• GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers.
• Biochemistry. 2002; 41: 521-9
• Display abstract

• The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.

• Quardokus EM, Brun YV
• DNA replication initiation is required for mid-cell positioning of FtsZ rings in Caulobacter crescentus.
• Mol Microbiol. 2002; 45: 605-16
• Display abstract

• Polymerization of the GTPase FtsZ to form a structure called the Z-ring is the earliest known step in bacterial cell division. Mid-cell Z-ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z-ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z-rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z-ring assembly at different stages in the cell cycle. Z-ring assembly occurred even when early stages of DNA replication were blocked; however, the Z-rings were localized at a subpolar region of the cell. Z-rings only assembled at the proper mid-cell location if DNA replication had initiated. Z-ring assembly coincided with areas containing little or no DNA, and Z-rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid-cell Z-ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z-ring localization in Caulobacter.

• Buddelmeijer N, Judson N, Boyd D, Mekalanos JJ, Beckwith J
• YgbQ, a cell division protein in Escherichia coli and Vibrio cholerae, localizes in codependent fashion with FtsL to the division site.
• Proc Natl Acad Sci U S A. 2002; 99: 6316-21
• Display abstract

• YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E. coli genome designed to find potential interacting partners for cell division protein FtsL. In V. cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type E. coli cells. Localization of E. coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E. coli. Our results show colocalization of proteins to the bacterial midcell in E. coli and raise the possibility that these proteins interact in a coiled-coil structure.

• Ohashi T, Hale CA, de Boer PA, Erickson HP
• Structural evidence that the P/Q domain of ZipA is an unstructured, flexible tether between the membrane and the C-terminal FtsZ-binding domain.
• J Bacteriol. 2002; 184: 4313-5
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• The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

• Haney SA, Glasfeld E, Hale C, Keeney D, He Z, de Boer P
• Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA.
• J Biol Chem. 2001; 276: 11980-7
• Display abstract

• The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

• Sun Q, Margolin W
• Influence of the nucleoid on placement of FtsZ and MinE rings in Escherichia coli.
• J Bacteriol. 2001; 183: 1413-22
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• We previously presented evidence that replicating but unsegregated nucleoids, along with the Min system, act as topological inhibitors to restrict assembly of the FtsZ ring (Z ring) to discrete sites in the cell. To test if nonreplicating nucleoids have similar exclusion effects, we examined Z rings in dnaA (temperature sensitive) mutants. Z rings were excluded from centrally localized nucleoids and were often observed at nucleoid edges. Cells with nonreplicating nucleoids formed filaments, some of which contained large nucleoid-free areas in which Z rings were positioned at regular intervals. Because MinE may protect FtsZ from the action of the MinC inhibitor in these nucleoid-free zones, we examined the localization of a MinE-green fluorescent protein (GFP) fusion with respect to Z rings and nucleoids. Like Z rings, MinE-GFP appeared to localize independently of nucleoid position, forming rings at regular intervals in nucleoid-free regions. Unlike FtsZ, however, MinE-GFP often localized on top of nucleoids, replicating or not, suggesting that MinE is relatively insensitive to the nucleoid inhibition effect. These data suggest that both replicating and nonreplicating nucleoids are capable of topologically excluding Z rings but not MinE.

• Lowe J, van den Ent F
• Conserved sequence motif at the C-terminus of the bacterial cell-division protein FtsA.
• Biochimie. 2001; 83: 117-20
• Display abstract

• FtsA is an essential part of the septal ring structure in bacterial cell division. Two peptide-protein interactions are known in this process: FtsA and ZipA bind the C-terminus of FtsZ, the bacterial tubulin homologue, which is the first septal component to appear at the septum. Our recent crystal structure of FtsA revealed a possible peptide binding site on FtsA and a long disordered C-terminal region. Here we show that all FtsA proteins contain a conserved 10-13 residue motif at the C-terminal end that may facilitate targeting of downstream septal components.

• Uehara T, Matsuzawa H, Nishimura A
• HscA is involved in the dynamics of FtsZ-ring formation in Escherichia coli K12.
• Genes Cells. 2001; 6: 803-14
• Display abstract

• BACKGROUND: FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non-dividing Escherichia coli. However, it assembles in cytokinetic rings at the early stages of septation. Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined. RESULTS: At 42 degrees C, JE10715 mutant bacteria formed multinucleated filaments with a highly reduced number of FtsZ-rings at potential division sites. The JE10715 phenotype resulted from a mis-sense mutation in the hscA gene which encodes a heat shock Hsp70 family protein, with a single alanine-to-valine substitution at position 192 within the ATPase domain. Both JE10715 and the hscA knockout strain of JE10715 were completely complemented by a plasmid-born, wild-type hscA gene, but not by a mutant-type hscA715 gene. An hscA conditional knockout of the wild-type strain under non-permissive conditions exhibited longer rod cells with an abnormal localization of FtsZ. The over-expression of dnaK partially complemented the JE10715 mutation. In vitro, the ATPase activity of the mutant protein HscA715 was reduced to 63% of wild-type HscA activity. HscA co-sedimented with FtsZ-polymers in the presence of GTP. CONCLUSION: HscA is involved in FtsZ-ring formation, through a chaperon-like interaction with FtsZ. Defects in hscA, however, can partially be compensated for by redundant genes, including the wild-type dnaK.

• Margolin W
• Spatial regulation of cytokinesis in bacteria.
• Curr Opin Microbiol. 2001; 4: 647-52
• Display abstract

• Cytokinesis in bacteria such as Escherichia coli is orchestrated by FtsZ, a tubulin-like protein that forms a circumferential Z ring at the division site. The Z ring then recruits a number of other essential cell division proteins, ultimately assembling the cytokinetic machine that splits the cell. It has been known for some time that the MinCDE proteins and the bacterial nucleoid provide positional information to negatively regulate cytokinesis. Recently, direct visualization of Z rings and Min proteins in whole cells have contributed important new insights into the molecular mechanisms behind this fundamental cellular process. This review summarizes and integrates these insights.

• Chen JC, Beckwith J
• FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division.
• Mol Microbiol. 2001; 42: 395-413
• Display abstract

• During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.

• de la Fuente A, Palacios P, Vicente M
• Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.
• Biochimie. 2001; 83: 109-15
• Display abstract

• Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flardh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

• Pas E, Einav M, Woldringh CL, Zaritsky A
• Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells.
• Biochimie. 2001; 83: 121-4
• Display abstract

• Division planes in Escherichia coli, usually restricted to one dimension of the rod-shaped cell, were induced at all possible planes by transforming the cells to spheroids with mecillinam (inactivating PbpA). Such cells displayed many nucleoids and arcs of FtsZ, genetically tagged to green fluorescent protein, that developed to rings at constriction sites all around their surface. These observations are consistent with the view (Woldringh et al., J. Bacteriol. 176 (1994) 6030-6038) that nucleoids, forced during replication to segregate in the length axis of the cell by the rigid bacillary envelope, induce assembly of FtsZ to division rings in between them.

• Quardokus EM, Din N, Brun YV
• Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus.
• Mol Microbiol. 2001; 39: 949-59
• Display abstract

• Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.

• Mukherjee A, Saez C, Lutkenhaus J
• Assembly of an FtsZ mutant deficient in GTPase activity has implications for FtsZ assembly and the role of the Z ring in cell division.
• J Bacteriol. 2001; 183: 7190-7
• Display 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.

• Meinhardt H, de Boer PA
• Pattern formation in Escherichia coli: a model for the pole-to-pole oscillations of Min proteins and the localization of the division site.
• Proc Natl Acad Sci U S A. 2001; 98: 14202-7
• Display abstract

• Proper cell division requires an accurate definition of the division plane. In bacteria, this plane is determined by a polymeric ring of the FtsZ protein. The site of Z ring assembly in turn is controlled by the Min system, which suppresses FtsZ polymerization at noncentral membrane sites. The Min proteins in Escherichia coli undergo a highly dynamic localization cycle, during which they oscillate between the membrane of both cell halves. By using computer simulations we show that Min protein dynamics can be described accurately by using the following assumptions: (i) the MinD ATPase self-assembles on the membrane and recruits both MinC, an inhibitor of Z ring formation, and MinE, a protein required for MinC/MinD oscillation, (ii) a local accumulation of MinE is generated by a pattern formation reaction that is based on local self-enhancement and a long range antagonistic effect, and (iii) it displaces MinD from the membrane causing its own local destabilization and shift toward higher MinD concentrations. This local destabilization results in a wave of high MinE concentration traveling from the cell center to a pole, where it disappears. MinD reassembles on the membrane of the other cell half and attracts a new accumulation of MinE, causing a wave-like disassembly of MinD again. The result is a pole-to-pole oscillation of MinC/D. On time average, MinC concentration is highest at the poles, forcing FtsZ assembly to the center. The mechanism is self-organizing and does not require any other hypothetical topological determinant.

• Yu XC, Sun Q, Margolin W
• FtsZ rings in mukB mutants with or without the Min system.
• Biochimie. 2001; 83: 125-9
• Display abstract

• The site of cell division in Escherichia coli is defined by formation of the Z ring between the two segregated daughter nucleoids. Positioning of the Z ring, composed of the highly conserved and tubulin-like FtsZ protein, appears to be negatively regulated by both the nucleoid and the oscillating MinCD inhibitor proteins. MukB protein is probably involved in nucleoid condensation, and in the absence of MukB, the negative effect of the nucleoid on Z rings appears to be partially suppressed. In this study, we examined the localization of Z rings in cells lacking both the Min system and MukB. In the Deltamin DeltamukB double null mutant, essentially all nucleoid-free zones, either at the cell poles or at non-polar sites between nucleoids, contained Z rings. However, a significant proportion of Z rings also formed on top of nucleoids. Interestingly, Z ring clusters often formed at gaps between nucleoids, and some of the rings within the clusters were clearly positioned on top of nucleoids. These results provide further evidence that the negative topological effect of nucleoids in cells lacking MukB is partially but not totally suppressed, and that the absence of the Min system allows more promiscuous Z ring formation.

• Pichoff S, Lutkenhaus J
• Escherichia coli division inhibitor MinCD blocks septation by preventing Z-ring formation.
• J Bacteriol. 2001; 183: 6630-5
• Display 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 of ftsZ, 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.

• Erickson HP
• The FtsZ protofilament and attachment of ZipA--structural constraints on the FtsZ power stroke.
• Curr Opin Cell Biol. 2001; 13: 55-60
• Display abstract

• Bacterial cell division protein FtsZ forms protofilaments in vitro that can shift from a straight to a curved conformation. The inside of the curved protofilaments, which corresponds to the carboxyl terminus, should face the center of the cell as curvature increases during constriction of the Z-ring. ZipA, a membrane-tethered division protein, binds to a highly conserved short peptide on the carboxyl terminus of FtsZ. A model is proposed here for how membrane-bound ZipA can reach around the FtsZ protofilament to bind the carboxy-terminal peptide, which faces away from the membrane.

• Feucht A, Lucet I, Yudkin MD, Errington J
• Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis.
• Mol Microbiol. 2001; 40: 115-25
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• The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

• Vinella D, Cashel M, D'Ari R
• Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli.
• Genetics. 2000; 156: 1483-92
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• Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.

• Ghigo JM, Beckwith J
• Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization.
• J Bacteriol. 2000; 182: 116-29
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• In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its Haemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.

• Mosyak L et al.
• The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography.
• EMBO J. 2000; 19: 3179-91
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• In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded beta-sheet packed against three alpha-helices and contains the split beta-alpha-beta motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended beta-strand followed by alpha-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.

• Salimnia H, Radia A, Bernatchez S, Beveridge TJ, Dillon JR
• Characterization of the ftsZ cell division gene of Neisseria gonorrhoeae: expression in Escherichia coli and N. gonorrhoeae.
• Arch Microbiol. 2000; 173: 10-20
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• We cloned the cell division gene ftsZ of the gram-negative coccus Neisseria gonorrhoeae (Ng) strain CH811, characterized it genetically and phenotypically, and studied its localization in N. gonorrhoeae and Escherichia coli (Ec). The 1,179-bp ORF of ftsZ(Ng) encodes a protein with a predicted molecular mass of 41.5 kDa. Protein sequence alignments indicate that FtsZ(Ng) is similar to other FtsZ proteins and contains the conserved GTP binding motif. FtsZ homologues were identified in several N. gonorrhoeae strains and in Neisseria lactamica, Neisseria sicca, Neisseria polysaccharae and Neisseria cinerea either by Western blot or by PCR-Southern blot analysis. Attempts to inactivate the ftsZ(Ng) on the chromosome failed, indicating that it is essential for gonococcal growth. FtsZ(Ng) was synthesized in an in vitro transcription/translation system and was shown to be 43 kDa, the same size as in Western blots. Expression of the ftsZ(Ng) gene from nongonococcal promoters resulted in a filamentous phenotype in E. coli. Under controlled expression, the FtsZ(Ng)-GFP fusion protein localized at the mid-cell division site in E. coli. E. coli expressing high levels of the FtsZ(Ng)-GFP fusion protein formed filaments and exhibited different fluorescent structures including helices, spiral tubules extending from pole to pole, and regularly spaced dots or bands that did not localize at the middle of the cell. Expression of the FtsZ(Ng)-GFP fusion protein in N. gonorrhoeae resulted in abnormal cell division as shown by electron microscopy. FtsZ(Ng)-GFP fusions were also expressed in a gonococcal background using a unique shuttle vector.

• Ishii Y et al.
• Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli.
• Biosci Biotechnol Biochem. 2000; 64: 799-807
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• The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.

• Justice SS, Garcia-Lara J, Rothfield LI
• Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery.
• Mol Microbiol. 2000; 37: 410-23
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• SulA and MinCD are specific inhibitors of cell division in Escherichia coli. In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented the formation of high-molecular-weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo. However, MinCD did prevent the formation of FtsA rings. Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.

• Yan K, Pearce KH, Payne DJ
• A conserved residue at the extreme C-terminus of FtsZ is critical for the FtsA-FtsZ interaction in Staphylococcus aureus.
• Biochem Biophys Res Commun. 2000; 270: 387-92
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• FtsZ, a tubulin-like protein with GTPase activity, and FtsA, an actin-like protein with ATPase activity, are two proteins known to play critical roles in the later stages of the bacterial cell cycle. It is well documented that FtsA-FtsZ co-localization at the septum of dividing bacteria is essential for successful cell division in E. coli. We have validated and characterized this interaction by co-expressing FtsA and FtsZ, from both E. coli and S. aureus, in the yeast two-hybrid system. We demonstrate for the first time a specific association between S. aureus FtsA and FtsZ proteins and self interaction of S. aureus FtsZ. These observations are consistent with the conserved role of FtsA and FtsZ in bacterial cell division. Using deletion mutagenesis, we have shown that a highly conserved motif as small as 10 residues in the extreme C-terminus of S. aureus FtsZ is critical for the interaction with FtsA. Further dissection of this motif by alanine scanning mutagenesis showed that Phe376 likely plays a major role in the FtsA-FtsZ interaction. This work has important implications for the design of antagonists and agonists of the FtsA-FtsZ interaction as such agents could provide a novel approach for tackling multi-resistant Gram positive pathogens.

• Hu Z, Lutkenhaus J
• Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ.
• J Bacteriol. 2000; 182: 3965-71
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• In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the min 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 min function, 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.

• Katis VL, Wake RG, Harry EJ
• Septal localization of the membrane-bound division proteins of Bacillus subtilis DivIB and DivIC is codependent only at high temperatures and requires FtsZ.
• J Bacteriol. 2000; 182: 3607-11
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• Using immunofluorescence microscopy, we have examined the dependency of localization among three Bacillus subtilis division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.

• Gullbrand B, Nordstrom K
• FtsZ ring formation without subsequent cell division after replication runout in Escherichia coli.
• Mol Microbiol. 2000; 36: 1349-59
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• In this report, we have investigated cell division after inhibition of initiation of chromosome replication in Escherichia coli. In a culture grown to the stationary phase, cells containing more than one chromosome were able to divide some time after restart of growth, under conditions not allowing initiation of chromosome replication. This shows that there is no requirement for cell division to take place within a certain time after initiation of chromosome replication. Continued growth without initiation of replication resulted in filamented cells that generally did not have any constrictions. Interestingly, FtsZ rings were formed in a majority of these cells as they reached a certain cell length. These rings appeared and were maintained for some time at the cell quarter positions on both sides of the centrally localized nucleoid. These results confirm previous findings that cell division sites are formed independently of chromosome replication and indicate that FtsZ ring assembly is dependent on cell size rather than on the capacity of the cell to divide. Disruption of the mukB gene caused a significant increase in the region occupied by DNA after the replication runout, consistent with a role of MukB in chromosome condensation. The aberrant nucleoid structure was accompanied by a shift in FtsZ ring positioning, indicating an effect of the nucleoid on the positioning of the FtsZ ring. A narrow cell length interval was found, under and over which primarily central and non-central FtsZ rings, respectively, were observed. This finding correlates well with the previously observed oscillatory movement of MinC and MinD in short and long cells.

• Moy FJ, Glasfeld E, Mosyak L, Powers R
• Solution structure of ZipA, a crucial component of Escherichia coli cell division.
• Biochemistry. 2000; 39: 9146-56
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• ZipA, an essential component of cell division in Escherichia coli, interacts with the FtsZ protein at the midcell in one of the initial steps of septum formation. The high-resolution solution structure of the 144-residue C-terminal domain of E. coli ZipA (ZipA(185)(-)(328)) has been determined by multidimensional heteronuclear NMR. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2758 experimental NMR restraints. The atomic root means square distribution about the mean coordinate positions for residues 6-142 for the 30 structures is 0.37 +/- 0.04 A for the backbone atoms, 0. 78 +/- 0.05 A for all atoms, and 0.45 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of ZipA(185)(-)(328) is composed of three alpha-helices and a beta-sheet consisting of six antiparallel beta-strands where the alpha-helices and the beta-sheet form surfaces directly opposite each other. A C-terminal peptide from FtsZ has been shown to bind ZipA(185)(-)(328) in a hydrophobic channel formed by the beta-sheet providing insight into the ZipA-FtsZ interaction. An unexpected similarity between the ZipA(185)(-)(328) fold and the split beta-alpha-beta fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.

• Hale CA, Rhee AC, de Boer PA
• ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains.
• J Bacteriol. 2000; 182: 5153-66
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• FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.

• Poplawski A, Gullbrand B, Bernander R
• The ftsZ gene of Haloferax mediterranei: sequence, conserved gene order, and visualization of the FtsZ ring.
• Gene. 2000; 242: 357-67
• Display abstract

• We sequenced the ftsZ gene region of the halophilic archaeon Haloferax mediterranei and mapped the transcription start sites for the ftsZ gene. The gene encoded a 363-amino-acid long FtsZ protein with a predicted molecular mass of 38 kDa and an isoelectric point of 4.2. A high level of similarity to the FtsZ protein of Haloferax volcanii was apparent, with 97 and 90% identity at the amino acid and nucleotide levels, respectively. Structural conservation at the protein level was shown by visualization of the FtsZ ring structure in H. mediterranei cells using an antiserum raised against FtsZ of H. volcanii. FtsZ rings were observed in cells in different stages of division, including cells with pleomorphic shapes and cells that appeared to be undergoing asymmetric division. Cells were also observed that displayed constriction-like invaginations in the absence of an FtsZ ring, indicating that morphological data are not sufficient to determine whether pleomorphic Haloferax cells are undergoing cell division. Both the upstream and downstream gene order in the ftsZ region was found to be conserved within the genus Haloferax. Furthermore, the downstream gene order, which includes the secE and nusG genes, is conserved in almost all euryarchaea sequenced to date. The secE and nusG genes are likely to be transcriptionally and translationally coupled in Haloferax, and this co-expression may have been a selective force that has contributed to keeping the gene cluster intact.

• Yu XC, Margolin W
• Deletion of the min operon results in increased thermosensitivity of an ftsZ84 mutant and abnormal FtsZ ring assembly, placement, and disassembly.
• J Bacteriol. 2000; 182: 6203-13
• Display abstract

• To investigate the interaction between FtsZ and the Min system during cell division of Escherichia coli, we examined the effects of combining a well-known thermosensitive mutation of ftsZ, ftsZ84, with DeltaminCDE, a deletion of the entire min locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing minCDE might at least partially suppress the thermosensitivity of ftsZ84, which can form colonies below 42 degrees C but not at or above 42 degrees C. Contrary to expectations, the double mutant was significantly more thermosensitive than the ftsZ84 single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22 degrees C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the ftsZ84 single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.

• Daniel RA, Harry EJ, Errington J
• Role of penicillin-binding protein PBP 2B in assembly and functioning of the division machinery of Bacillus subtilis.
• Mol Microbiol. 2000; 35: 299-311
• Display abstract

• We have characterized the role of the penicillin-binding protein PBP 2B in cell division of Bacillus subtilis. We have shown that depletion of the protein results in an arrest in division, but that this arrest is slow, probably because the protein is relatively stable. PBP 2B-depleted filaments contained, at about their mid-points, structures resembling partially formed septa, into which most, if not all, of the division proteins had assembled. Although clearly deficient in wall material, membrane invagination seemed to continue, indicating that membrane and wall ingrowth can be uncoupled. At other potential division sites along the filaments, no visible ingrowths were observed, although FtsZ rings assembled at regular intervals. Thus, PBP 2B is apparently required for both the initiation of division and continued septal ingrowth. Immunofluorescence microscopy showed that the protein is recruited to the division site. The pattern of localization suggested that this recruitment occurs continually during septal ingrowth. During sporulation, PBP 2B was present transiently in the asymmetrical septum of sporulating cells, and its availability may play a role in the regulation of sporulation septation.

• Chen JC, Weiss DS, Ghigo JM, Beckwith J
• Septal localization of FtsQ, an essential cell division protein in Escherichia coli.
• J Bacteriol. 1999; 181: 521-30
• Display abstract

• Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.

• Den Blaauwen T, Buddelmeijer N, Aarsman ME, Hameete CM, Nanninga N
• Timing of FtsZ assembly in Escherichia coli.
• J Bacteriol. 1999; 181: 5167-75
• Display abstract

• The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

• Yu XC, Margolin W
• FtsZ ring clusters in min and partition mutants: role of both the Min system and the nucleoid in regulating FtsZ ring localization.
• Mol Microbiol. 1999; 32: 315-26
• Display abstract

• To understand further the role of the nucleoid and the min system in selection of the cell division site, we examined FtsZ localization in Escherichia coli cells lacking MinCDE and in parC mutants defective in chromosome segregation. More than one FtsZ ring was sometimes found in the gaps between nucleoids in min mutant filaments. These multiple FtsZ rings were more apparent in longer cells; double or triple rings were often found in the nucleoid-free gaps in ftsI min and ftsA min double mutant filaments. Introducing a parC mutation into the ftsA min double mutant allowed the nucleoid-free gaps to become significantly longer. These gaps often contained dramatic clusters of FtsZ rings. In contrast, filaments of the ftsA parC double mutant, which contained active MinCDE, assembled only one or two rings in most of the large nucleoid-free gaps. These results suggest that all positions along the cell length are competent for FtsZ ring assembly, not just sites at mid-cell or at the poles. Consistent with previous results, unsegregated nucleoids also correlated with a lack of FtsZ localization. A model is proposed in which both the inhibitory effect of the nucleoid and the regulation by MinCDE ensure that cells divide precisely at the midpoint.

• Ma X, Margolin W
• Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ.
• J Bacteriol. 1999; 181: 7531-44
• Display abstract

• In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coli FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.

• Hu Z, Mukherjee A, Pichoff S, Lutkenhaus J
• The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization.
• Proc Natl Acad Sci U S A. 1999; 96: 14819-24
• Display abstract

• Positioning of the Z ring at the midcell site in Escherichia coli is assured by the min system, which masks polar sites through topological regulation of MinC, an inhibitor of division. To study how MinC inhibits division, we have generated a MalE-MinC fusion that retains full biological activity. We find that MalE-MinC interacts with FtsZ and prevents polymerization without inhibiting FtsZ's GTPase activity. MalE-MinC19 has reduced ability to inhibit division, reduced affinity for FtsZ, and reduced ability to inhibit FtsZ polymerization. These results, along with MinC localization, suggest that MinC rapidly oscillates between the poles of the cell to destabilize FtsZ filaments that have formed before they mature into polar Z rings.

• Weiss DS, Chen JC, Ghigo JM, Boyd D, Beckwith J
• Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL.
• J Bacteriol. 1999; 181: 508-20
• Display abstract

• Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of Escherichia coli, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of ftsI at the normal chromosomal locus and a genetic fusion of ftsI to the green fluorescent protein (gfp) at the lambda attachment site. gfp-ftsI was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of beta-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.

• Raskin DM, de Boer PA
• MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli.
• J Bacteriol. 1999; 181: 6419-24
• Display abstract

• By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in Escherichia coli. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell's middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.

• Pedersen LB, Angert ER, Setlow P
• Septal localization of penicillin-binding protein 1 in Bacillus subtilis.
• J Bacteriol. 1999; 181: 3201-11
• Display abstract

• Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

• Levin PA, Kurtser IG, Grossman AD
• Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis.
• Proc Natl Acad Sci U S A. 1999; 96: 9642-7
• Display abstract

• During the bacterial cell cycle, the tubulin-like cell-division protein FtsZ polymerizes into a ring structure that establishes the location of the nascent division site. We have identified a regulator of FtsZ ring formation in Bacillus subtilis. This protein, EzrA, modulates the frequency and position of FtsZ ring formation. The loss of ezrA resulted in cells with multiple FtsZ rings located at polar as well as medial sites. Moreover, the critical concentration of FtsZ required for ring formation was lower in ezrA null mutants than in wild-type cells. EzrA was associated with the cell membrane and also colocalized with FtsZ to the nascent septal site. We propose that EzrA interacts either with FtsZ or with one of its binding partners to promote depolymerization.

• RayChaudhuri D
• ZipA is a MAP-Tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division.
• EMBO J. 1999; 18: 2372-83
• Display abstract

• The first visible event in prokaryotic cell division is the assembly of the soluble, tubulin-like FtsZ GTPase into a membrane-associated cytokinetic ring that defines the division plane in bacterial and archaeal cells. In the temperature-sensitive ftsZ84 mutant of Escherichia coli, this ring assembly is impaired at the restrictive temperature causing lethal cell filamentation. Here I present genetic and morphological evidence that a 2-fold higher dosage of the division gene zipA suppresses thermosensitivity of the ftsZ84 mutant by stabilizing the labile FtsZ84 ring structure in vivo. I demonstrate that purified ZipA promotes and stabilizes protofilament assembly of both FtsZ and FtsZ84 in vitro and cosediments with the protofilaments. Furthermore, ZipA organizes FtsZ protofilaments into arrays of long bundles or sheets that probably represent the physiological organization of the FtsZ ring in bacterial cells. The N-terminal cytoplasmic domain of membrane-anchored ZipA contains sequence elements that resemble the microtubule-binding signature motifs in eukaryotic Tau, MAP2 and MAP4 proteins. It is postulated that the MAP-Tau-homologous motifs in ZipA mediate its binding to FtsZ, and that FtsZ-ZipA interaction represents an ancient prototype of the protein-protein interaction that enables MAPs to suppress microtubule catastrophe and/or to promote rescue.

• Binenbaum Z, Klyman E, Fishov I
• Division-associated changes in membrane viscosity of Escherichia coli.
• Biochimie. 1999; 81: 921-9
• Display abstract

• Septum formation is initiated by the FtsZ ring assembly in the middle of rod-shape bacteria. The mechanism which determines the division site in the membrane and makes it recognizable by FtsZ is still unknown. We have recently demonstrated that the putative division membrane domains can be visualized by a fluorescent membrane probe (Fishov and Woldring, Mol. Microbiol., 1999) and that these domains can be dissipated by interrupting the process of coupled transcription and translation of proteins (Binenbaum et al., Mol. Microbiol., 1999). Here, we examined the membrane dynamics of Escherichia coli during division and after a reversible division arrest. Anisotropy of DPH fluorescence, used as an indicator of membrane dynamics (viscosity), correlated with the rate of division in synchronous cells. It decreased during filamentation caused by drugs or by temperature, but not in the ftsZ mutant and when DNA replication was blocked by nalidixic acid. Based on previous data, we incline to interpret these results as reflecting formation and dissipation of putative membrane domains marking the division sites; domains are formed by partitioning nucleoids and dissipate while used for constriction or after the nucleoids have been segregated too far in a filament.

• Ghigo JM, Weiss DS, Chen JC, Yarrow JC, Beckwith J
• Localization of FtsL to the Escherichia coli septal ring.
• Mol Microbiol. 1999; 31: 725-37
• Display abstract

• In Escherichia coli, nine gene products are known to be essential for assembly of the division septum. One of these, FtsL, is a bitopic membrane protein whose precise function is not understood. Here we use fluorescence microscopy to study the subcellular localization of FtsL, both in a wild-type strain and in a merodiploid strain that expresses a GFP-FtsL fusion protein. We show that FtsL localizes to the cell septum where it forms a ring analogous to the cytoplasmic FtsZ ring. FtsL localization is dependent upon the function of FtsZ, FtsA and FtsQ, but not FtsI. In a reverse approach, we use fusions of green fluorescent protein (GFP) to FtsZ, FtsA and ZipA to show that these proteins localize to the division site in an FtsL-independent fashion. We propose that FtsL is a relatively late recruit to the ring structure that mediates septation.

• Dassain M, Leroy A, Colosetti L, Carole S, Bouche JP
• A new essential gene of the 'minimal genome' affecting cell division.
• Biochimie. 1999; 81: 889-95
• Display abstract

• The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.

• Carballes F, Bertrand C, Bouche JP, Cam K
• Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB.
• Mol Microbiol. 1999; 34: 442-50
• Display abstract

• Genes rcsC and rcsB form a two-component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB-dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co-regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.

• Sossong TM Jr, Brigham-Burke MR, Hensley P, Pearce KH Jr
• Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ.
• Biochemistry. 1999; 38: 14843-50
• Display abstract

• The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin. We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 microM. At 5 microM FtsZ, we have determined a K(m) of 82 microM GTP and a V(max) of 490 nmol of P(i) min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg(2+) and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 microM total protein concentration. Chemical cross-linking of FtsZ also shows that monomer, dimer, trimer, and tetramer species are present at higher (>2 microM) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization. We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg(2+) and Ca(2+) had significant effects on secondary structure. Taken together, our results support the hypothesis that Mg(2+)-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ. On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.

• Witte A, Brand E, Mayrhofer P, Narendja F, Lubitz W
• Mutations in cell division proteins FtsZ and FtsA inhibit phiX174 protein-E-mediated lysis of Escherichia coli.
• Arch Microbiol. 1998; 170: 259-68
• Display abstract

• Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites. These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites. Therefore, E. coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis. The ftsZ84 and the ftsA12 cell division mutant strains of E. coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division. The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division - rather than specific functions of cell division - plays an essential role in protein-E-mediated lysis. SulA-overproducing cells had a lysis-positive phenotype, the ring structure - but not the GTPase function - of FtsZ was impaired.

• Daniel RA, Harry EJ, Katis VL, Wake RG, Errington J
• Characterization of the essential cell division gene ftsL(yIID) of Bacillus subtilis and its role in the assembly of the division apparatus.
• Mol Microbiol. 1998; 29: 593-604
• Display abstract

• We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL, of Escherichia coli. Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, sigmaF. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.

• Sun Q, Yu XC, Margolin W
• Assembly of the FtsZ ring at the central division site in the absence of the chromosome.
• Mol Microbiol. 1998; 29: 491-503
• Display abstract

• The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division. To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli, we investigated its localization in parC and mukB mutants that are defective for chromosome segregation. Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate. In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature. Filamentous mukB cells were usually longer and lacked many potential rings. At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids. However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal. The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints. This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.

• Mukherjee A, Cao C, Lutkenhaus J
• Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli.
• Proc Natl Acad Sci U S A. 1998; 95: 2885-90
• Display abstract

• The bacterial cell division protein FtsZ assembles into the cytokinetic Z ring that directs cytokinesis in prokaryotes. In Escherichia coli the formation of the Z ring is prevented by induction of the cell division inhibitor SulA (SfiA), a component of the SOS response. Here we show that a MalE-SulA fusion that retains this inhibitory function in vivo inhibits the GTPase activity and polymerization of FtsZ in vitro. MalE-SulA10, which does not block Z ring formation in vivo, is unable to inhibit the GTPase activity and polymerization in vitro. Furthermore, FtsZ114, which is refractory to SulA in vivo, is not inhibited by MalE-SulA. These results indicate that SulA blocks Z ring formation by blocking FtsZ polymerization.

• Begg K, Nikolaichik Y, Crossland N, Donachie WD
• Roles of FtsA and FtsZ in activation of division sites.
• J Bacteriol. 1998; 180: 881-4
• Display abstract

• Increasing FtsZ induces the formation of minicells at cell poles but does not increase the frequency or timing of central divisions. A coordinate increase in both FtsZ and FtsA, however, increases the frequency of both polar and central divisions.

• Flardh K, Palacios P, Vicente M
• Cell division genes ftsQAZ in Escherichia coli require distant cis-acting signals upstream of ddlB for full expression.
• Mol Microbiol. 1998; 30: 305-15
• Display abstract

• A transcriptional reporter fusion has been introduced into the chromosomal ftsZ locus in such a way that all transcription that normally reaches ftsZ can be monitored. The new Phi(ftsZ-lacZ ) fusion yields four times more beta-galactosidase activity than a ddlB-ftsQAZ-lacZ fusion on a lambda prophage vector. A strongly polar ddlB ::Omega insertion prevents contributions from signals upstream of the ftsQAZ promoters and decreases transcription of the chromosomal Phi(ftsZ-lacZ ) fusion by 66%, demonstrating that around two-thirds of total ftsZ transcription require cis-acting elements upstream of ddlB. We suggest that those elements are distant promoters, and thus that the cell division and cell wall synthesis genes in the dcw gene cluster are to a large extent co-transcribed. The ddlB ::Omega insertion is lethal unless additional copies of ftsQA are provided or a compensatory decrease in FtsZ synthesis is made. This shows that ddlB is a dispensable gene, and reinforces the critical role of the FtsA/FtsZ ratio in septation. Using the new reporter fusion, it is demonstrated that ftsZ expression is not autoregulated.

• Sun Q, Margolin W
• FtsZ dynamics during the division cycle of live Escherichia coli cells.
• J Bacteriol. 1998; 180: 2050-6
• Display abstract

• The dynamics and assembly of bacterial cell division protein FtsZ were monitored in individual, growing and dividing Escherichia coli cells in real time by microculture of a merodiploid strain expressing green fluorescent protein (GFP)-tagged FtsZ. Cells expressing FtsZ-GFP at levels less than or equivalent to that of wild-type FtsZ were able to grow and divide over multiple generations, with their FtsZ rings visualized by fluorescence. During the late stages of cytokinesis, which constituted the last one-fourth of the cell cycle, the lumen of the FtsZ ring disappeared as the whole structure condensed. At this time, loops of FtsZ-GFP polymers emanated outward from the condensing ring structure and other unstable fluorescent structures elsewhere in the cell were also observed. Assembly of FtsZ rings at new division sites occurred within 1 min, from what appeared to be single points. Interestingly, this nucleation often took place in the predivisional cell at the same time the central FtsZ ring was in its final contraction phase. This demonstrates directly that, at least when FtsZ-GFP is being expressed, new division sites have the capacity to become fully functional for FtsZ targeting and assembly before cell division of the mother cell is completed. The results suggest that the timing of FtsZ assembly may be normally controlled in part by cellular FtsZ concentration. The use of wide-field optical sectioning microscopy to obtain sharp fluorescence images of FtsZ structures is also discussed.

• Pogliano J et al.
• Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA* mutants.
• J Bacteriol. 1998; 180: 3486-90
• Display abstract

• In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.

• Lutkenhaus J
• Organelle division: from coli to chloroplasts.
• Curr Biol. 1998; 8: 61921-61921
• Display abstract

• FtsZ, an ancestral homolog of eukaryotic tubulin, assembles into the cytokinetic Z ring that directs cell division in bacteria. Recent results indicate that FtsZ is also used for division by chloroplasts, though not by mitochondria.

• Lu C, Stricker J, Erickson HP
• FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly.
• Cell Motil Cytoskeleton. 1998; 40: 71-86
• Display abstract

• We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ (and some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml. This is just above the plateau for full GTPase activity in vitro.

• Bouche JP, Pichoff S
• On the birth and fate of bacterial division sites.
• Mol Microbiol. 1998; 29: 19-26
• Display abstract

• Thanks to genetics, to the study of protein-protein interactions and to direct viewing of subcellular structures by the use of immunofluorescence and green fluorescent protein (GFP) fusions, the organization of the constriction apparatus of walled bacteria is gradually coming to light. The tubulin-like protein FtsZ assembles as a ring around the site of constriction and operates as an organizer and activator of septum-shaping proteins. Much less is known about the factors specifying the location of FtsZ rings. Circumstantial evidence favours the presence at future ring positions of fixed elements, the potential division sites (PDS), before FtsZ assembles. FtsZ polymerization is initiated from a point on a PDS, the nucleation site, still to be identified, and proceeds bidirectionally around the cell. We hypothesize that new PDS are specified in a manner that depends on the functioning of an active chromosome partition apparatus. This view is supported by the fact that formation of mid-cell PDS requires initiation of DNA replication, and by recent studies supporting the existence of a specialized partition apparatus in a variety of microorganisms. Although PDS may be specified directly by the partition apparatus, indirect localization linked to compartmentalized gene expression during chromosome segregation is also possible. Once created, PDS are used in a regulated manner, and several mechanisms normally operate to direct constriction to selected PDS at the correct time. One, dedicated to the permanent suppression of polar PDS, rests on the minicell suppression system and involves a protein that is able to discriminate between polar and non-polar sites. Another is involved in asymmetric site selection at the early stages of sporulation in Bacillus subtilis. Finally, a mechanism observed only in certain multi-nucleated cells appears to favour division at non-polar PDS related to the most ancient replication/DNA segregation events.

• Mukherjee A, Lutkenhaus J
• Purification, assembly, and localization of FtsZ.
• Methods Enzymol. 1998; 298: 296-305

• Trusca D, Scott S, Thompson C, Bramhill D
• Bacterial SOS checkpoint protein SulA inhibits polymerization of purified FtsZ cell division protein.
• J Bacteriol. 1998; 180: 3946-53
• Display abstract

• Cell division of Escherichia coli is inhibited when the SulA protein is induced in response to DNA damage as part of the SOS checkpoint control system. The SulA protein interacts with the tubulin-like FtsZ division protein. We investigated the effects of purified SulA upon FtsZ. SulA protein inhibits the polymerization and the GTPase activity of FtsZ, while point mutant SulA proteins show little effect on either of these FtsZ activities. SulA did not inhibit the polymerization of purified FtsZ2 mutant protein, which was originally isolated as insensitive to SulA. These studies define polymerization assays for FtsZ which respond to an authentic cellular regulator. The observations presented here support the notion that polymerization of FtsZ is central to its cellular role and that direct, reversible inhibition of FtsZ polymerization by SulA may account for division inhibition.

• Mileykovskaya E, Sun Q, Margolin W, Dowhan W
• Localization and function of early cell division proteins in filamentous Escherichia coli cells lacking phosphatidylethanolamine.
• J Bacteriol. 1998; 180: 4252-7
• Display abstract

• Escherichia coli cells that contain the pss-93 null mutation are completely deficient in the major membrane phospholipid phosphatidylethanolamine (PE). Such cells are defective in cell division. To gain insight into how a phospholipid defect could block cytokinesis, we used fluorescence techniques on whole cells to investigate which step of the cell division cycle was affected. Several proteins essential for early steps in cytokinesis, such as FtsZ, ZipA, and FtsA, were able to localize as bands to potential division sites in pss-93 filaments, indicating that the generation and localization of potential division sites was not grossly affected by the absence of PE. However, there was no evidence of constriction at most of these potential division sites. FtsZ and green fluorescent protein (GFP) fusions to FtsZ and ZipA often formed spiral structures in these mutant filaments. This is the first report of spirals formed by wild-type FtsZ expressed at normal levels and by ZipA-GFP. The results suggest that the lack of PE may affect the correct interaction of FtsZ with membrane nucleation sites and alter FtsZ ring structure so as to prevent or delay its constriction.

• Buddelmeijer N, Aarsman ME, Kolk AH, Vicente M, Nanninga N
• Localization of cell division protein FtsQ by immunofluorescence microscopy in dividing and nondividing cells of Escherichia coli.
• J Bacteriol. 1998; 180: 6107-16
• Display abstract

• The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the gamma form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

• Baumann L, Baumann P
• Characterization of ftsZ, the cell division gene of Buchnera aphidicola (endosymbiont of aphids) and detection of the product.
• Curr Microbiol. 1998; 36: 85-9
• Display abstract

• Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, contains the gene ftsZ, which codes for a protein involved in the initiation of septum formation during cell division. With immunological techniques, this protein has been detected in cell-free extracts of the endosymbiont. Nucleotide sequence determination of a 6.4-kilobase B. aphidicola DNA fragment has indicated that, as in E. coli, ftsZ is adjacent to genes coding for other cell division proteins as well as genes involved in murein synthesis (murC-ddlB-ftsA-ftsZ). Although B. aphidicola ftsZ is expressed in E. coli, it cannot complement E. coli ftsZ mutants. High levels of B. aphidicola FtsZ results in the formation of long filamentous E. coli cells, suggesting that this protein interferes with cell division. The presence of FtsZ indicates that in this, as well as in many other previously described properties, B. aphidicola resembles free-living bacteria.

• Din N, Quardokus EM, Sackett MJ, Brun YV
• Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA.
• Mol Microbiol. 1998; 27: 1051-63
• Display abstract

• The cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N-terminal region of approximately 320 amino acids; a variable spacer region; and a conserved C-terminal region of eight amino acids. We show that FtsZ mutants missing different C-terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C-terminal conserved region, the linker, and 40 amino acids from the end of the N-terminal conserved region (FtsZdeltaC281) prevents the localization or the polymerization of FtsZ. Because two-hybrid analysis indicates that FtsZdeltaC281 does not interact with FtsZ, we hypothesize that FtsZdeltaC281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C-terminus of FtsZ (FtsZdeltaC485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C-terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.

• Yu XC, Tran AH, Sun Q, Margolin W
• Localization of cell division protein FtsK to the Escherichia coli septum and identification of a potential N-terminal targeting domain.
• J Bacteriol. 1998; 180: 1296-304
• Display abstract

• Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. coli cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and ftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in ftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.

• Wang L, Khattar MK, Donachie WD, Lutkenhaus J
• FtsI and FtsW are localized to the septum in Escherichia coli.
• J Bacteriol. 1998; 180: 2810-6
• Display abstract

• The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

• Mukherjee A, Lutkenhaus J
• Dynamic assembly of FtsZ regulated by GTP hydrolysis.
• EMBO J. 1998; 17: 462-9
• Display abstract

• FtsZ forms a cytokinetic ring, designated the Z ring, that directs cytokinesis in prokaryotes. It has limited sequence similarity to eukaryotic tubulins and, like tubulin, it has GTPase activity and the ability to assemble into various structures including protofilaments, bundles and minirings. By using both electron microscopy and sedimentation, we demonstrate that FtsZ from Escherichia coli undergoes a strictly GTP-dependent polymerization and the polymers disappear as the GTP is consumed. Thus, FtsZ polymerization, like that of tubulin, is dynamic and regulated by GTP hydrolysis. These results provide the basis for the dynamics of the Z ring and favor a model in which the Z ring is formed by a nucleation event.

• Lockhart A, Kendrick-Jones J
• Interaction of the N-terminal domain of MukB with the bacterial tubulin homologue FtsZ.
• FEBS Lett. 1998; 430: 278-82
• Display abstract

• The MukB protein is involved in the process of chromosome partition in Escherichia coli and has a domain structure reminiscent of the eukaryotic motor proteins kinesin and myosin. This has led to the suggestion that MukB may function as a motor protein in vivo. In order to test this idea we have recombinantly expressed the N-terminal domain of MukB (residues 1-342) as a poly-His tagged fusion protein for biochemical characterisation. The purified protein (Muk342) is monomeric and has low basal Mg-ATPase (1.23 min(-1)) and Mg-GTPase (0.17 min(-1)) activities. Muk342 binds with high affinity to the prokaryotic tubulin homologue FtsZ and we have evidence that FtsZ can stimulate nucleotide turnover by Muk342. These properties are consistent with MukB functioning as a motor protein using FtsZ as a track or anchor for generating force within E. coli.

• Guzman LM, Weiss DS, Beckwith J
• Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.
• J Bacteriol. 1997; 179: 5094-103
• Display abstract

• FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.

• Pichoff S, Vollrath B, Bouche JP
• MinCD-independent inhibition of cell division by a protein that fuses MalE to the topological specificity factor MinE.
• J Bacteriol. 1997; 179: 4616-9
• Display abstract

• We report that MinE, the topological specificity factor of cell division in Escherichia coli, inhibits septation when fused to the C terminus of the maltose-binding protein MalE. This contrasts with overexpression of MinE alone, which affects growth but has no effect on division. Inhibition by MalE-MinE was minCD independent and depended on MinE segments involved in dimerization and prevention of MinCD division inhibition. The SOS and the heat shock responses were not involved, suggesting that the inhibition comes from a direct interaction of MalE-MinE with the septation apparatus. MalE-MinE lethality was suppressed by overexpression of ftsZ, as well as by overexpression of ftsN, a suppressor of temperature-sensitive mutations in genes ftsQ, ftsA, and ftsI. We also report that high-level synthesis of MalE disturbs nucleoid partitioning.

• Raskin DM, de Boer PA
• The MinE ring: an FtsZ-independent cell structure required for selection of the correct division site in E. coli.
• Cell. 1997; 91: 685-94
• Display abstract

• E. coli cell division is mediated by the FtsZ ring and associated factors. Selection of the correct division site requires the combined action of an inhibitor of FtsZ ring formation (MinCD) and of a topological specificity factor that somehow prevents MinCD action at the middle of the cell (MinE). Here we show that a biologically active MinE-Gfp fusion accumulates in an annular structure near the middle of young cells. Formation of the MinE ring required MinD but was independent of MinC and continued in nondividing cells in which FtsZ function was inhibited. The results indicate that the MinE ring represents a novel cell structure, which allows FtsZ ring formation at midcell by suppressing MinCD activity at this site.

• de Pedro MA, Quintela JC, Holtje JV, Schwarz H
• Murein segregation in Escherichia coli.
• J Bacteriol. 1997; 179: 2823-34
• Display abstract

• Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.

• Khattar MM, Addinall SG, Stedul KH, Boyle DS, Lutkenhaus J, Donachie WD
• Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function.
• J Bacteriol. 1997; 179: 784-93
• Display abstract

• Two new mutations in the cell division gene ftsW have been isolated and characterized. The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation. The ftsW1640(Ts) mutation, however, causes a block to division at a later stage. The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent. Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site. Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure. We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations.

• Schwedock J, McCormick JR, Angert ER, Nodwell JR, Losick R
• Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor.
• Mol Microbiol. 1997; 25: 847-58
• Display abstract

• In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 microm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 microm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.

• Pogliano J, Pogliano K, Weiss DS, Losick R, Beckwith J
• Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites.
• Proc Natl Acad Sci U S A. 1997; 94: 559-64
• Display abstract

• A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the beta-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.

• Addinall SG, Cao C, Lutkenhaus J
• FtsN, a late recruit to the septum in Escherichia coli.
• Mol Microbiol. 1997; 25: 303-9
• Display abstract

• The localization of FtsN in Escherichia coli was inves tigated by immunofluorescence microscopy. FtsN is an essential cell division protein with a simple bitopic topology, a short N-terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain. FtsN was found to localize to the septum in a ring pattern similar to that observed for FtsZ and FtsA, although the frequency of cells with rings was less. A MalG-FtsN fusion was also localized to the septum, indicating that the information for FtsN localization is supplied by its periplasmic domain. FtsN localization was dependent upon the prior localization of FtsZ and FtsA and required the function of FtsI and FtsQ. Consistent with FtsN functioning after FtsZ, Z rings were observed in a mutant depleted of FtsN.

• Wang X, Huang J, Mukherjee A, Cao C, Lutkenhaus J
• Analysis of the interaction of FtsZ with itself, GTP, and FtsA.
• J Bacteriol. 1997; 179: 5551-9
• Display abstract

• The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.

• Ma X, Sun Q, Wang R, Singh G, Jonietz EL, Margolin W
• Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring.
• J Bacteriol. 1997; 179: 6788-97
• Display abstract

• FtsZ and FtsA are essential for cell division in Escherichia coli and colocalize to the septal ring. One approach to determine what regions of FtsA and FtsZ are important for their interaction is to identify in vivo interactions between FtsA and FtsZ from different species. As a first step, the ftsA genes of Rhizobium meliloti and Agrobacterium tumefaciens were isolated and characterized. In addition, an FtsZ homolog that shared the unusual C-terminal extension of R. meliloti FtsZ1 was found in A. tumefaciens. In order to visualize their localization in cells, we tagged these proteins with green fluorescent protein (GFP). When R. meliloti FtsZ1-GFP or A. tumefaciens FtsZ-GFP was expressed at low levels in E. coli, they specifically localized only to the E. coli FtsZ ring, possibly by coassembly. When A. tumefaciens FtsA-GFP or R. meliloti FtsA-GFP was expressed in E. coli, they failed to localize detectably to the E. coli FtsZ ring. However, when R. meliloti FtsZ1 was coexpressed with them, fluorescence localized to a band at the midcell division site, strongly suggesting that FtsA from either A. tumefaciens or R. meliloti can bind directly to its cognate FtsZ. As expected, GFP-tagged FtsZ1 and FtsA from either R. meliloti or A. tumefaciens localized to the division site in A. tumefaciens cells. Therefore, the 61 amino acid changes between A. tumefaciens FtsA and R. meliloti FtsA do not prevent their direct interaction with FtsZ1 from either species, suggesting that those residues are not essential for protein-protein contacts. Moreover, the failure of the two non-E. coli FtsA derivatives to interact strongly with E. coli FtsZ in this in vivo system unless their cognate FtsZ was also present suggests that FtsA-FtsZ interactions have coevolved and that the residues which differ between the E. coli proteins and those of the two other species may be important for specific interactions.

• Lutkenhaus J, Addinall SG
• Bacterial cell division and the Z ring.
• Annu Rev Biochem. 1997; 66: 93-116
• Display abstract

• Bacterial cell division occurs through the formation of an FtsZ ring (Z ring) at the site of division. The ring is composed of the tubulin-like FtsZ protein that has GTPase activity and the ability to polymerize in vitro. The Z ring is thought to function in vivo as a cytoskeletal element that is analogous to the contractile ring in many eukaryotic cells. Evidence suggests that the Z ring is utilized by all prokaryotic organisms for division and may also be used by some eukaryotic organelles. This review summarizes our present knowledge about the formation, function, and evolution of the Z ring in prokaryotic cell division.

• Boyle DS, Khattar MM, Addinall SG, Lutkenhaus J, Donachie WD
• ftsW is an essential cell-division gene in Escherichia coli.
• Mol Microbiol. 1997; 24: 1263-73
• Display abstract

• In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD. We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW, in the presence of the plasmid-borne wild-type ftsW gene under the control of P(BAD). In the absence of arabinose, the ftsW-null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW-null filaments, the FtsZ ring is absent in 50-60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P(BAD) is sufficient for complementation of the ftsW-null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli, and that it plays a role in the stabilization of the FtsZ ring during cell division.

• D'Ari R
• The Escherichia coli cell cycle, cell division and ppGpp: regulation and mechanisms.
• Folia Microbiol (Praha). 1997; 42: 161-4
• Display abstract

• The literature demonstrating tight regulation of the Escherichia coli cell cycle is reviewed. Recent evidence is presented indicating that the normal rod cell shape can be abandoned, allowing growth as a coccus, either by increasing the amount of the division proteins FtsZ, FtsA and FtsQ, or by increasing the pool of the nucleotide ppGpp. It is argued that ppGpp may be a cell cycle signal in E. coli.

• Addinall SG, Cao C, Lutkenhaus J
• Temperature shift experiments with an ftsZ84(Ts) strain reveal rapid dynamics of FtsZ localization and indicate that the Z ring is required throughout septation and cannot reoccupy division sites once constriction has initiated.
• J Bacteriol. 1997; 179: 4277-84
• Display abstract

• FtsZ is an essential division protein in bacteria that functions by forming a ring at midcell that mediates septation. To further study the function of the Z ring the effect of a temperature-sensitive mutation, ftsZ84(Ts), on ring dynamics and septal progression was examined. Shifting a strain carrying an ftsZ84(Ts) mutation to the nonpermissive temperature led to loss of Z rings within 1 min. Septal ingrowth was immediately inhibited, and sharply demarcated septa, present at the time of the shift, were gradually replaced by blunted septa. These results indicate that the Z ring is required throughout septation. Shifting filaments to permissive temperature led to a rapid localization of FtsZ84 at regular intervals. Included in these localization events were complete and partial rings as well as spots, although some of these eventually aborted. These results reveal the rapid dynamics of FtsZ localization and indicate that nucleation sites are formed in the absence of FtsZ function. Interestingly, Z rings could not reform at division sites that were constricted although they could reform at sites that had not begun constriction.

• Yu XC, Margolin W
• Ca2+-mediated GTP-dependent dynamic assembly of bacterial cell division protein FtsZ into asters and polymer networks in vitro.
• EMBO J. 1997; 16: 5455-63
• Display abstract

• FtsZ, a tubulin-like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis. It is not known what triggers FtsZ ring assembly. In this work, we use a FtsZ-green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy. We show that FtsZ polymers can assemble dynamically in solution in a GTP-dependent manner. Initially, FtsZ nucleation centers grow into aster-like structures that dramatically resemble microtubule organizing centers. As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo. Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly. FtsZ can undergo repeated GTP-dependent assembly and disassembly in solution by sequential addition and removal of Ca2+. In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration. Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell.

• Addinall SG, Lutkenhaus J
• FtsZ-spirals and -arcs determine the shape of the invaginating septa in some mutants of Escherichia coli.
• Mol Microbiol. 1996; 22: 231-7
• Display abstract

• The essential cell division protein FtsZ forms a dynamic ring structure at the future division site. This Z-ring contracts during cell division while maintaining a position at the leading edge of the invaginating septum. Using immunofluorescence microscopy we have characterized two situations in which non-ring FtsZ structures are formed. In ftsZ26 (temperature sensitive, Ts) mutant cells, FtsZ-spirals are formed and lead to formation of spirally invaginating septa, which in turn cause morphological abnormalities. In rodAoul mutant cells, which grow as spheres instead of rods, FtsZ-arcs are formed where asymmetric septal invaginations are initiated. The FtsZ-arcs later mature into complete FtsZ-rings. Our data show that Z-spirals and Z-arcs can contract and that in doing so, they determine the shape of the invaginating septa. These results also strongly suggest that in normal cell division, FtsZ is positioned to a single nucleation site on the inner membrane, from which it polymerizes bidirectionally around the cell circumference to form the Z-ring.

• Addinall SG, Lutkenhaus J
• FtsA is localized to the septum in an FtsZ-dependent manner.
• J Bacteriol. 1996; 178: 7167-72
• Display abstract

• The localization of the cell division protein FtsA in E. coli was examined. FtsA was found to localize to the septum in a ring pattern as previously shown for FtsZ. The localization of FtsA was completely dependent on the localization of FtsZ. Under a variety of conditions that prevented formation of the Z ring, FtsA was unable to localize. In mutants where FtsZ forms structures in addition to Z rings, the pattern of FtsA duplicated these structures. These results suggest that the Z ring recruits FtsA to the septum.

• Addinall SG, Bi E, Lutkenhaus J
• FtsZ ring formation in fts mutants.
• J Bacteriol. 1996; 178: 3877-84
• Display abstract

• The formation of FtsZ rings (Z rings) in various fts mutants was examined by immunoelectron microscopy and immunofluorescence. In two temperature-sensitive ftsZ mutants which form filaments with smooth morphology, the Z ring was unable to form. In ftsA, ftsI, and ftsQ mutants, which form filaments with an indented morphology, Z rings formed but their contraction was blocked. These results indicate that fully functional ftsA, ftsQ, and ftsI genes are not required for Z-ring formation and are unlikely to have a role in localization of the Z ring. The results also suggest that one function of the Z ring is to localize the activity of other fts gene products.

• Ma X, Ehrhardt DW, Margolin W
• Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein.
• Proc Natl Acad Sci U S A. 1996; 93: 12998-3003
• Display abstract

• In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GEP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ-GFP or with FtsA-GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ-GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ-GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA-GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

• Voskuil JL, Nanninga N
• How does FtsZ find its location?
• Microb Drug Resist. 1996; 2: 55-61
• Display abstract

• The conformational flexibility of FtsZ and the properties of its epitopes have been studied. Cellular fractions of Escherichia coli have been treated with Triton X-114. FtsZ distributed in the polar as well as in the non-polar phase. This has been interpreted to mean that FtsZ can change its conformation. For the nonpolar conformation it has been assumed that the putative hydrophobic pocket of FtsZ (cf. Voskuil et al., J. Bacteriol. 176:1886-1893) is being turned inside out upon interaction with the cytoplasmic membrane. In a tentative model we suggest that FtsA mediates this interaction. Immunoprecipitations of FtsZ with various monoclonal antibodies in the presence or absence of 1 M NaCl gave a clue concerning the hydrophobicity and hydrophilicity of FtsZ's epitopes. Immunogold-labeling also showed differences with respect to the accessibility of FtsZ.

• Wang X, Lutkenhaus J
• FtsZ ring: the eubacterial division apparatus conserved in archaebacteria.
• Mol Microbiol. 1996; 21: 313-9
• Display abstract

• FtsZ is a tubulin-like protein that is essential for cell division in eubacteria. It functions by forming a ring at the division site that directs septation. The archaebacteria constitute a kingdom of life separate from eubacteria and eukaryotes. Like eubacteria, archaebacteria are prokaryotes, although they are phylogenetically closer to eukaryotes. Here it is shown that archaebacteria also possess FtsZ and that it is biochemically similar to eubacterial FtsZs. Significantly, FtsZ from the archaebacterium Haloferax volcanii is a GTPase that is localized to a ring that coincides with the division constriction. These results indicate that the FtsZ ring was part of the division apparatus of a common prokaryotic ancestor that was retained by both eubacteria and archaebacteria.

• Palacios P, Vicente M, Sanchez M
• Dependency of Escherichia coli cell-division size, and independency of nucleoid segregation on the mode and level of ftsZ expression.
• Mol Microbiol. 1996; 20: 1093-8
• Display abstract

• Expression of ftsZ in strain VIP205 is dissociated from its natural promoters, and is under the control of an inducible tac promoter. This abolishes the oscillation in ftsZ transcription observed in the wild type, allowing different levels of ftsZ expression. We demonstrate that this construction does not affect the expression of other genes, and has no effects on replication or nucleoid segregation. A shift in IPTG from 30 microM, that supports division at wild-type sizes, to lower (6 microM) or higher (100 microM) concentrations, indicates that VIP205 cells can divide within a broad range of FtsZ concentrations. Analysis of the morphological parameters during the transition from one IPTG concentration to another suggests that the correct timing of ftsZ expression, and the correct FtsZ concentration, are required for division to occur at normal cell sizes. After a transient division delay during the transition to lower IPTG concentrations, cells in which ftsZ is expressed continuously (yielding 80% of the wild-type FtsZ levels) divide with the same division time as the wild type, but at the expense of becoming 1.5 times larger. A precise control of ftsZ expression is required for normal division, but the existence of additional regulators to maintain the correct timing during the cell cycle cannot be ruled out.

• Quardokus E, Din N, Brun YV
• Cell cycle regulation and cell type-specific localization of the FtsZ division initiation protein in Caulobacter.
• Proc Natl Acad Sci U S A. 1996; 93: 6314-9
• Display abstract

• Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation of FtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.

• Conter A, Bouche JP, Dassain M
• Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.
• J Bacteriol. 1996; 178: 5100-4
• Display abstract

• A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.

• Margolin W, Wang R, Kumar M
• Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin.
• J Bacteriol. 1996; 178: 1320-7
• Display abstract

• We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.

• Cam K, Rome G, Krisch HM, Bouche JP
• RNase E processing of essential cell division genes mRNA in Escherichia coli.
• Nucleic Acids Res. 1996; 24: 3065-70
• Display abstract

• The ratio of the FtsZ to FtsA proteins determines the correct initiation of cell division in Escherichia coli. The genes for these proteins are contiguous on the chromosome. Although both genes are transcribed from common promoters, the presence of ftsZ-specific promoters, along with differences in the efficiency of translation of their respective mRNAs, contribute to the increased relative expression of ftsZ. We report here that the polycistronic ftsA-ftsZ transcripts are cleaved by RNase E and that this cleavage affects the decay of ftsA and ftsZ mRNA. As a consequence of the cleavage, RNase E also contributes to the differential expression of the two genes.

• Huang J, Cao C, Lutkenhaus J
• Interaction between FtsZ and inhibitors of cell division.
• J Bacteriol. 1996; 178: 5080-5
• Display abstract

• The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.

• Vinella D, D'Ari R
• Overview of controls in the Escherichia coli cell cycle.
• Bioessays. 1995; 17: 527-36
• Display abstract

• The harmonious growth and cell-to-cell uniformity of steady-state bacterial populations indicate the existence of a well-regulated cell cycle, responding to a set of internal signals. In Escherichia coli, the key events of this cycle are the initiation of DNA replication, nucleoid segregation and the initiation of cell division. The replication initiator is the DnaA protein. In nucleoid segregation, the MukB protein, required for proper partitioning, may be a member of the myosin-kinesin superfamily of mechanoenzymes. In cell division, the FtsZ protein has a tubulin motif, is a GTPase and polymerizes in a ring around midcell during septation; the FtsA protein has an actin-like structure. The nature of the internal signals triggering these events is not known but candidates include cell mass, the superhelical density of the chromosome and the concentration of two regulatory nucleotides, cyclic AMP and ppGpp. The involvement of cytoskeletal-like proteins in key cycle events encourages the notion of a fundamental biological unity in cell cycle regulation in all organisms.

• Higashitani A, Higashitani N, Horiuchi K
• A cell division inhibitor SulA of Escherichia coli directly interacts with FtsZ through GTP hydrolysis.
• Biochem Biophys Res Commun. 1995; 209: 198-204
• Display abstract

• E. coli SulA is an SOS-inducible protein that inhibits cell division. FtsZ is a protein that plays a central role in bacterial cell division. Using purified SulA protein that was fused to the maltose binding protein, we demonstrate in vitro that SulA interacts with FtsZ to form a stable complex. The reaction requires GTP and Mg ion. GDP and GTP gamma S cannot substitute for GTP, which suggests that hydrolysis of GTP is required for the reaction. The complex is formed in a molar ratio of approximately one to one of the two proteins. It is likely that the complex formation represents the in vivo mechanism by which SulA inhibits cell division.

• Cook WR, Rothfield LI
• Early stages in development of the Escherichia coli cell-division site.
• Mol Microbiol. 1994; 14: 485-95
• Display abstract

• Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZnull and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.

• Cook WR, Rothfield LI
• Development of the cell-division site in FtsA- filaments.
• Mol Microbiol. 1994; 14: 497-503
• Display abstract

• Early changes at cell-division sites were studied in non-septate filaments induced by growth of ftsATs mutant cells under non-permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division-site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.

• Mukherjee A, Lutkenhaus J
• Guanine nucleotide-dependent assembly of FtsZ into filaments.
• J Bacteriol. 1994; 176: 2754-8
• Display abstract

• FtsZ is an essential cell division protein that is localized to the leading edge of the bacterial septum in a cytokinetic ring. It contains the tubulin signature motif and is a GTP binding protein with a GTPase activity. Further comparison of FtsZ with eukaryotic tubulins revealed some additional sequence similarities, perhaps indicating a similar GTP binding site. Examination of FtsZ incubated in vitro by electron microscopy revealed a guanine nucleotide-dependent assembly into protein filaments, supporting the hypothesis that the FtsZ ring is formed through self-assembly. FtsZ3, which is unable to bind GTP, does not polymerize, whereas FtsZ2, which binds GTP but is deficient in GTP hydrolysis, is capable of polymerization.

• Bramhill D, Thompson CM
• GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules.
• Proc Natl Acad Sci U S A. 1994; 91: 5813-7
• Display abstract

• The FtsZ protein is a GTPase that is essential for cell division in Escherichia coli. During cytokinesis, FtsZ localizes to a ring at the leading edge of septum synthesis. We report the GTP-dependent polymerization of purified FtsZ measured by sedimentation and light scattering. Electron microscopy of polymerized FtsZ revealed structures including tubules 14-20 nm in diameter with longitudinal arrays of protofilaments. FtsZ depolymerized upon removal of GTP and repolymerized after subsequent GTP addition. Mutant FtsZ84 protein polymerized inefficiently, suggesting that polymerization is important for the cellular role of FtsZ in division. The possibility that tubules of FtsZ protein form a cytoskeleton involved in septum synthesis is consistent with our data.

• Khattar MM, Begg KJ, Donachie WD
• Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.
• J Bacteriol. 1994; 176: 7140-7
• Display abstract

• The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.

• Dai K, Mukherjee A, Xu Y, Lutkenhaus J
• Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.
• J Bacteriol. 1994; 176: 130-6
• Display abstract

• Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.

• RayChaudhuri D, Park JT
• A point mutation converts Escherichia coli FtsZ septation GTPase to an ATPase.
• J Biol Chem. 1994; 269: 22941-4
• Display abstract

• The cell division protein FtsZ, essential to initiate septum formation in Escherichia coli, is a GTPase. The thermosensitive ftsZ84 mutation, which impairs the ability of FtsZ to bind and hydrolyze GTP in vitro, maps to a short glycine-rich FtsZ segment. This region is conserved in eubacterial FtsZ homologs and is strikingly similar to the proposed GTP binding motif in the eukaryotic cytoskeletal protein tubulin. Here we show that in contrast to FtsZ, FtsZ84 protein has a Mg(2+)-dependent ATPase activity in vitro. This activity, unlike the wild-type GTPase, is specifically inhibited by sodium azide, a known antagonist of F-type ATPases and the bacterial SecA protein translocation ATPase (Oliver, D., Cabelli, R. J., Dolan, K. M., and Jarosik, G. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8227-8231). Conversely, aluminum fluoride abolishes FtsZ GTPase activity but only partially affects FtsZ84 ATPase. Affinity-purified anti-FtsZ antibody blocks FtsZ84 ATPase activity, indicating that this enzymatic function is intrinsic to the mutant protein. This is, to our knowledge, the first example of a missense mutation that converts a GTPase to an ATPase.

• Garrido T, Sanchez M, Palacios P, Aldea M, Vicente M
• Transcription of ftsZ oscillates during the cell cycle of Escherichia coli.
• EMBO J. 1993; 12: 3957-65
• Display abstract

• The FtsZ protein is a key element controlling cell division in Escherichia coli. A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells. The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates. This cell cycle dependency is specifically due to the two proximal ftsZ promoters. A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer. In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division. Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated. This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles.

• Hiraga S
• Chromosome partition in Escherichia coli.
• Curr Opin Genet Dev. 1993; 3: 789-801
• Display abstract

• The past year has seen important genetic and biochemical advances in our understanding of the mechanisms that are involved in chromosome partition into two daughter cells in Escherichia coli. Topoisomerase IV and XerCD recombinase have been shown to be required for the unlinking of replicated chromosomes. MukB, an alpha-helical coiled-coil protein, has been shown to be involved in chromosome partition, and this is the first candidate for a bacterial motor protein. Another protein, FtsZ, has been shown to form a constriction ring in cell division and may also relate to chromosome partition.

• Mukherjee A, Dai K, Lutkenhaus J
• Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein.
• Proc Natl Acad Sci U S A. 1993; 90: 1053-7
• Display abstract

• FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control. The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin. In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity. A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides. A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.

• Bi E, Lutkenhaus J
• Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring.
• J Bacteriol. 1993; 175: 1118-25
• Display abstract

• Immunoelectron microscopy was used to assess the effects of inhibitors of cell division on formation of the FtsZ ring in Escherichia coli. Induction of the cell division inhibitor SulA, a component of the SOS response, or the inhibitor MinCD, a component of the min system, blocked formation of the FtsZ ring and led to filamentation. Reversal of SulA inhibition by blocking protein synthesis in SulA-induced filaments led to a resumption of FtsZ ring formation and division. These results suggested that these inhibitors block cell division by preventing FtsZ localization into the ring structure. In addition, analysis of min mutants demonstrated that FtsZ ring formation was also associated with minicell formation, indicating that all septation events in E. coli involve the FtsZ ring.

• Wang X, Lutkenhaus J
• The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration.
• Mol Microbiol. 1993; 9: 435-42
• Display abstract

• The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.

• Dewar SJ, Donachie WD
• Antisense transcription of the ftsZ-ftsA gene junction inhibits cell division in Escherichia coli.
• J Bacteriol. 1993; 175: 7097-101
• Display abstract

• A 490-bp DNA segment spanning the junction between the ftsA and ftsZ genes inhibits cell division when present in high copy number. We show that this segment contains an antisense promoter and an antisense transcription terminator which define a new gene, stfZ.

• Lutkenhaus J
• Escherichia coli cell division.
• Curr Opin Genet Dev. 1993; 3: 783-8
• Display abstract

• Recent progress in the molecular analysis of bacterial septation and chromosome partitioning suggests that these processes may involve cytoskeletal elements previously thought to be present only in eukaryotic cells. The continued biochemical and genetic analysis of key proteins, such as the tubulin-like FtsZ, should lead to further unravelling of the regulation and mechanism of bacterial cell division.

• Donachie WD
• The cell cycle of Escherichia coli.
• Annu Rev Microbiol. 1993; 47: 199-230
• Display abstract

• For E. coli cells, growth to a critical mass leads to initiation of chromosome replication. Initiation requires ATP-bound DnaA protein, together with replication proteins. Replication is followed by decatenation and monomerization of sister-chromosome molecules. Sister chromosomes are rapidly partitioned into opposite halves of the cell, perhaps by a kinesin-like protein (MukB). Cytokinesis starts with the formation of a ring of a GTP-binding protein (FtsZ), usually around the cell center. A specific enzyme (PBP3) and other proteins (FtsQ, FtsA, FtsL, FtsW) are responsible for the coordinate ingrowth of the peptidoglycan cell wall at this location. EnvA protein is required to split the resultant cross-wall to form new cell poles. The formation of the FtsZ ring is inhibited by activated MinC protein, but the MinE protein reverses this inhibition at potential division sites. A minimum cell length is probably required for partition and a minimum-size DNA-free zone for septum formation.

• Lutkenhaus J
• FtsZ ring in bacterial cytokinesis.
• Mol Microbiol. 1993; 9: 403-9
• Display abstract

• FtsZ is localized to a cytokinetic ring at the cell division site in bacteria. In this review a model is discussed that suggests that FtsZ self assembles into a ring at a nucleation site formed on the cytoplasmic membrane under cell-cycle control. This model suggests that formation of the cytokinetic FtsZ ring initiates and coordinates the circumferential invagination of the cytoplasmic membrane and cell wall, leading to formation of the septum. It is also suggested that this process may be conserved among the peptidoglycan-containing eubacteria. In addition, similarities between FtsZ and tubulin are discussed.

• Tetart F, Bouche JP
• Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules.
• Mol Microbiol. 1992; 6: 615-20
• Display abstract

• We show that the 53-nucleotide RNA molecule encoded by gene dicF blocks cell division in Escherichia coli by inhibiting the translation of ftsZ mRNA. Such a role for dicF had been predicted on the basis of the complementarity of DicF RNA with the ribosome-binding region of the ftsZ mRNA. An analysis of ftsZ expression at its chromosomal locus, and of an ftsZ-lacZ translational fusion controlled by promoters ftsZ1p and ftsZ2p only, indicates that ftsZ is not autoregulated. Partial inhibition of FtsZ synthesis leads to increased cell size. However, the number of FtsZ molecules per cell can be reduced threefold without affecting the division rate significantly. Our results suggest that septation is not triggered by a fixed number of newly synthesized FtsZ molecules per cell.

• de Boer P, Crossley R, Rothfield L
• The essential bacterial cell-division protein FtsZ is a GTPase.
• Nature. 1992; 359: 254-6
• Display abstract

• Cytokinesis defines the last stage in the division cycle, in which cell constriction leads to the formation of daughter cells. The biochemical mechanisms responsible for this process are poorly understood. In bacteria, the ftsZ gene product, FtsZ, is required for cell division, playing a prominent role in cytokinesis. The cellular concentration of FtsZ regulates the frequency of division and genetic studies have indicated that it is the target of several endogenous division inhibitors. At the time of onset of septal invagination, the FtsZ protein is recruited from the cytoplasm to the division site, where it assembles into a ring that remains associated with the leading edge of the invaginating septum until septation is completed. Here we report that FtsZ specifically binds and hydrolyses GTP. The reaction can be dissociated into a GTP-dependent activation stage that is markedly affected by the concentration of FtsZ, and a hydrolysis stage in which GTP is hydrolysed to GDP. The results indicate that GTP binding and hydrolysis are important in enabling FtsZ to support bacterial cytokinesis, either by facilitating the assembly of the FtsZ ring and/or by catalysing an essential step in the cytokinetic process itself.

• Dewar SJ, Begg KJ, Donachie WD
• Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ.
• J Bacteriol. 1992; 174: 6314-6
• Display abstract

• Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ. In contrast, overexpression of FtsA and FtsZ together does not block division. A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.

• RayChaudhuri D, Park JT
• Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein.
• Nature. 1992; 359: 251-4
• Display abstract

• Escherichia coli divides by forming a septum across the middle of the cell. The biochemical mechanism underlying this process is unknown. Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E. coli cell division, ftsZ plays a central role at the earliest known step of septation. Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements. In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired. Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein. FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins. We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP. The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E. coli.

• Bi E, Lutkenhaus J
• Isolation and characterization of ftsZ alleles that affect septal morphology.
• J Bacteriol. 1992; 174: 5414-23
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• The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery.

• Dai K, Lutkenhaus J
• The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.
• J Bacteriol. 1992; 174: 6145-51
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• Interactions among cell division genes in Escherichia coli were investigated by examining the effect on cell division of increasing the expression of the ftsZ, ftsA, or ftsQ genes. We determined that cell division was quite sensitive to the levels of FtsZ and FtsA but much less so to FtsQ. Inhibition of cell division due to an increase in FtsZ could be suppressed by an increase in FtsA. Inhibition of cell division due to increased FtsA could be suppressed by an increase in FtsZ. In addition, although wild-type strains were relatively insensitive to overexpression of ftsQ, we observed that cell division was sensitized to ftsQ overexpression in ftsI, ftsA, and ftsZ mutants. Among these, the ftsI mutant was the most sensitive. These results suggest that these gene products may interact and that the proper ratio of FtsZ to FtsA is critical for cell division to occur.

• Tetart F, Albigot R, Conter A, Mulder E, Bouche JP
• Involvement of FtsZ in coupling of nucleoid separation with septation.
• Mol Microbiol. 1992; 6: 621-7
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• The cell-cycle parameters of an Escherichia coli strain expressing essential division gene ftsZ at one-fifth of its normal level, because of antisense regulation by DicF RNA, have been analysed. Inhibition of FtsZ expression affects neither the generation time nor the replication initiation mass, the C period, or the constriction period, but it does dramatically retard the initiation of constriction relative to replication termination. Separation of the nucleoids is equally postponed, indicating that division is not coupled to termination of replication, but to partitioning. The severe inhibition of nucleoid separation by DicF RNA, and its suppression by overproduction of FtsZ, suggest a role for FtsZ in the control of separation, and consequently in the coupling of separation and division. We suggest that the normal pattern of nucleoid separation previously found in cells deficient in ftsZ function was a consequence of the loss of a negative effect exerted by FtsZ on separation. In agreement with this view, we find that nucleoid separation is temporarily inhibited after arrest of FtsZ synthesis, but is later resumed as FtsZ is further diluted into the elongating filaments.

• Cook WR, Rothfield LI
• Biogenesis of cell division sites in ftsA and ftsZ filaments.
• Res Microbiol. 1991; 142: 321-4
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• The development of nascent cell division sites was studied in Escherichia coli strains containing ftsAts and ftsZts mutations in which septal development is arrested after shift to the restrictive temperature. Division sites were studied by measuring positions of plasmolysis bays, a visible marker for periseptal annuli. Annuli are circumferential zones of membrane adhesion which represent the earliest known structural differentiation at developing septal sites. Two patterns of annulus development were observed in the mutant strains. Normal numbers of new annuli were generated in filaments of both mutants, but in ftsZ filaments annuli failed to mature and to become properly localized, suggesting that ftsZ gene product is first required for maturation of annuli. In contrast, mature annuli accumulated at division sites in ftsA filaments, suggesting that the ftsA gene product is required at a stage after maturation and localization of nascent annuli.

• Rothfield LI, Cook WR, de Boer PA
• Biogenesis of the Escherichia coli cell division system.
• Cold Spring Harb Symp Quant Biol. 1991; 56: 751-6

• Begg K
• Cell Division. Ring of bright metal.
• Nature. 1991; 354: 109-10

• Casaregola S et al.
• Analysis of a myosin-like protein and the role of calcium in the E. coli cell cycle.
• Res Microbiol. 1991; 142: 201-7
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• For a number of years now, we have argued that current models for the control of initiation of DNA synthesis, chromosomal partitioning and septum formation in Escherichia coli are unsatisfactory. Indeed, we could argue that despite considerable efforts, with the possible exception of dnaA and ftsZ, no genes specifically implicated in these control processes have been identified. In the cases of DnaA and FtsZ, no evidence has appeared to indicate how such molecules might be regulated to act once per cycle. In 1988, we formulated a specific proposal that the timing of cell cycle events in E. coli might be determined by a Ca++ flux, mediated by calcium-binding proteins and protein kinases and culminating, in the case of chromosome segregation and division, in the action of force-generating proteins such as myosin (Norris et al., 1988). In formulating this proposal, we took the view that the fundamental elements of cell cycle regulation are likely to be highly conserved across all species including prokaryotes. In this presentation, we shall describe the approaches we have been taking in order to test this hypothesis and to summarize the data obtained, in particular in relation to new genes identified which may play a role in the E. coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis.

• Bi EF, Lutkenhaus J
• FtsZ ring structure associated with division in Escherichia coli.
• Nature. 1991; 354: 161-4
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• Genes for cell division have been identified in Escherichia coli by the isolation of conditional lethal mutations that block cell division, but do not affect DNA replication or segregation. Of these genes, ftsZ is of great interest as it acts earliest in the division pathway, is essential, its level dictates the frequency of division, and it is thought to be the target of two cell-division inhibitors, SulA, produced in response to DNA damage, and MinCD, which prevents division at old sites. Here we have used immunoelectronmicroscopy to localize the FtsZ protein to the division site. The results suggest that FtsZ self-assembles into a ring structure at the future division site and may function as a cytoskeletal element. The formation of this ring may be the point at which division is regulated.

• Dai K, Lutkenhaus J
• ftsZ is an essential cell division gene in Escherichia coli.
• J Bacteriol. 1991; 173: 3500-6
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• The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.

• Pla J, Sanchez M, Palacios P, Vicente M, Aldea M
• Preferential cytoplasmic location of FtsZ, a protein essential for Escherichia coli septation.
• Mol Microbiol. 1991; 5: 1681-6
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• An ftsZ thermonull mutant has been constructed in which the ftsZ gene has been deleted from the Escherichia coli chromosome while maintaining a wild-type copy of the gene in a thermosensitive plasmid. Under conditions in which the ftsZ+ allele is unable to be replicated at the same pace as the chromosome, the cells become non-viable and grow as filaments, indicating that, contrary to other reports, FtsZ performs a function essential for cell survival. Antibodies raised against FtsZ have been used to detect the cellular location of FtsZ and its contents per cell. Fractionation experiments indicate that most of the total FtsZ present in the cell stays in the cytoplasm.

• Gollop N, March PE
• A GTP-binding protein (Era) has an essential role in growth rate and cell cycle control in Escherichia coli.
• J Bacteriol. 1991; 173: 2265-70
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• Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40 degrees C but completely repressed at 27 degrees C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTP-binding proteins as important regulators of cell growth and division may be ubiquitous in nature.

• Bi E, Lutkenhaus J
• Interaction between the min locus and ftsZ.
• J Bacteriol. 1990; 172: 5610-6
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• In Escherichia coli, distinct but similar minicell phenotypes resulting from mutation at the minB locus and increased expression of ftsZ suggested a possible interaction between these genes. A four- to fivefold increase in FtsZ resulting from increased gene dosage was found to suppress the lethality of minCD expressed from the lac promoter. Since increased MinCD did not affect the level of FtsZ, this suggested that MinCD may antagonize FtsZ to inhibit its cell division activity. This possibility was supported by the finding that alleles of ftsZ isolated as resistant to the cell division inhibitor SulA were also resistant to MinCD. Among the ftsZ(Rsa) alleles, two appeared to be completely resistant to MinCD as demonstrated by the lack of an effect of MinCD on cell length and a minicell phenotype observed in the absence of a significant increase in FtsZ. It was shown that SulA inhibits cell division independently of MinCD.

• Bi E, Lutkenhaus J
• FtsZ regulates frequency of cell division in Escherichia coli.
• J Bacteriol. 1990; 172: 2765-8
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• Cell division is regulated so that it occurs only once per cell cycle. In Escherichia coli, a rod-shaped bacterium, division normally takes place at the center of the long axis of the cell; however, in the minicell mutant, division can also take place at the cell pole. Such divisions take place at the expense of normal divisions, resulting in an overall increase in nucleated cell length. We report here that increasing the level of FtsZ can completely suppress the cell length of the minicell mutant by increasing the frequency at which cell division events take place. This result suggests that the level of FtsZ controls the frequency of cell division in E. coli.

• Holland IB, Casaregola S, Norris V
• Cytoskeletal elements and calcium: do they play a role in the Escherichia coli cell cycle?
• Res Microbiol. 1990; 141: 131-6

• Foley M et al.
• Compartmentalization of the periplasm at cell division sites in Escherichia coli as shown by fluorescence photobleaching experiments.
• Mol Microbiol. 1989; 3: 1329-36
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• Morphological evidence has previously indicated that the periplasmic space of Escherichia coli is compartmentalized at sites corresponding to future sites of cell division. The borders of these morphological compartments are formed by localized zones of adhesion (periseptal annuli). In the present study, the technique of fluorescence recovery after photobleaching was used to determine whether these structures act as barriers to the free movement of proteins within the periplasm. The recovery of fluorescence in the ftsA filaments was found to be uniformly low over at potential sites of cell division and at the cell poles, indicating that these regions are biochemically sequestered from the remainder of the periplasmic space. Our results provide direct evidence for local compartments within the periplasm, primarily located at the sites of past or future cell divisions. The implications of this finding for cell division and other periplasmic processes are discussed.

• Taschner PE, Huls PG, Pas E, Woldringh CL
• Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.
• J Bacteriol. 1988; 170: 1533-40
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• Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments. To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM). Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass. The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period. All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process. The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions. Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly. Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature. The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells. Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3. The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature. After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed. Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions.

• Kepes F, D'Ari R
• Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli.
• J Bacteriol. 1987; 169: 4036-40
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• A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium). Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery. The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay. Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up. Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay. These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted.

• Holland IB, Jones C
• The role of the FtsZ protein (SfiB) in UV-induced division inhibition and in the normal Escherichia coli cell division cycle.
• Ann Inst Pasteur Microbiol. 1985; 136: 165-71
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• This paper describes some of the major characteristics of the SOS-dependent division arrest which occurs in Escherichia coli during repair of DNA damage following UV irradiation. We shall review the evidence that the inducible division inhibitor, SfiA, interacts directly with an essential division protein, FtsZ. On the basis of its pivotal role in division inhibition during the UV stress response and other properties of ftsZ, we propose that this is a key gene involved in the actual regulation of the division cycle in E. coli. We also propose that at least some components of the division machinery interact to form a specific complex which we designate as a "septalsome". We shall discuss the possibility that a critical concentration of FtsZ is required to trigger the formation of an active septalsome prior to division. Alternatively, FtsZ might act to inhibit the formation of an active septalsome until a critical point in the cell cycle is reached.

• Ward JE Jr, Lutkenhaus J
• Overproduction of FtsZ induces minicell formation in E. coli.
• Cell. 1985; 42: 941-9
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• The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli.

• Yi QM, Lutkenhaus J
• The nucleotide sequence of the essential cell-division gene ftsZ of Escherichia coli.
• Gene. 1985; 36: 241-7
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• The nucleotide sequence of a 1.8-kb fragment of Escherichia coli DNA containing the essential cell division gene ftsZ is reported. The FtsZ protein has an Mr of 40294 and has 23% charged residues with a calculated isoelectric point of 4.9. The codon usage of the ftsZ gene reflects that of a highly expressed gene. Also located on this DNA fragment is the 3' end of the ftsA gene and the 5' end of the envA gene. These designations were confirmed by locating Tn5 insertions within the ends of these genes that inactivate each of these genes. A potential promoter for ftsZ overlapped the 3' end of the ftsA gene. A Tn5 insertion was located within the 3' end of the ftsA and within this potential promoter. No transcription terminators were evident between ftsA and ftsZ or between ftsZ and envA.

• Jones C, Holland IB
• Role of the SulB (FtsZ) protein in division inhibition during the SOS response in Escherichia coli: FtsZ stabilizes the inhibitor SulA in maxicells.
• Proc Natl Acad Sci U S A. 1985; 82: 6045-9
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• Induction of the SOS response in Escherichia coli by DNA-damaging treatments results in the synthesis of the SulA polypeptide, and this is sufficient to cause the resulting inhibition of cell division. Mutations at either sulA (sfiA) or sulB (sfiB) suppress this division inhibition. The SulB protein is identical to FtsZ, a protein required for normal division in E. coli. In the presence of FtsZ, the half-life of SulA synthesized in maxicells is approximately 12 min. In contrast, in the absence of FtsZ or in the presence of a mutant form of FtsZ (SulB114) that prevents division inhibition in vivo, SulA is extremely unstable with a half-life of only 3 min. Both FtsZ and SulA are isolated with the inner membrane of E. coli maxicells in the presence of MgCl2. We propose that the SulA inhibitor interacts directly with FtsZ in vivo to block the essential division function of this protein.

• Jones CA, Holland IB
• Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli.
• EMBO J. 1984; 3: 1181-6
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• A dominant sfiB allele has been cloned which renders partial diploids of an sfiB + Escherichia coli host resistant to division inhibition mediated by the SOS response. Transpositional mutagenesis was used to map the position of this sfiB114 allele, carried by a plasmid pLG552 , to an approximately 0.6-kb region overlapping the coding regions for ftsA and ftsZ , two genes essential for normal division. Most Tn 1000 insertions which inactivated sfiB114 also inactivated the ftsA function and caused the disappearance of both a 47-K polypeptide and reduced levels of a 42-K polypeptide in maxi-cells carrying pLG552 . An additional insertion inactivating sfiB114 was mapped to the right of ftsA and resulted in loss of the 42-K but not the 47-K polypeptide in maxi-cells. Moreover, a 2.1-kb BamHI-EcoRI DNA fragment was subcloned which carried ftsA and coded for a 47-K polypeptide but did not carry sfiB114 and did not complement ftsZ . We conclude that sfiB114 is located within ftsZ coding for a 42-K polypeptide. Nevertheless, insertions into ftsZ coding the 47-K polypeptide suppress the sfiB114 allele by substantially reducing the synthesis of the FtsZ ( SfiB114 ) polypeptide. The level of residual FtsZ synthesis was minimal when Tn 1000 was inserted closest to the distal end of ftsA , indicating the presence of a regulatory region essential for maximal expression of ftsZ .

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