Interaction between the min locus and ftsZ.

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.     

A new essential gene of the 'minimal genome' affecting cell division

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.     

Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin.

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.     

The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli.

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.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

ftsZ is an essential cell division gene in Escherichia coli.

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.     

Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ.

The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.     

Isolation and characterization of ftsZ alleles that affect septal morphology.

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.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

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.     

Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA).

In Escherichia coli, the ftsZ gene is thought to be an essential cell division gene. Several dominant mutations that make lon mutant cells refractory to the cell division inhibitor SulA, sulB9, sulB25, and sfiB114, have been mapped to the ftsZ gene. DNA sequence analysis of these mutations and the sfiB103 mutation confirmed that all of these mutations mapped within the ftsZ gene and revealed that the two sulB mutations were identical and by selection for resistance to higher levels of SulA, contained a second mutation within the ftsZ gene. We therefore propose that these mutations be redesignated ftsZ(Rsa) for resistance to SulA. A procedure involving mutagenesis of ftsZ cloned on low-copy-number vectors was used to isolate three additional ftsZ(Rsa) mutations. DNA sequence analysis of these mutations revealed that they were distinct from the previously isolated mutations. One of these mutations, ftsZ3(Rsa), led to an altered FtsZ protein that could no longer support cell growth but still conferred the Rsa phenotype in the presence of ftsZ+. In addition to being resistant to SulA, all ftsZ(Rsa) mutations also conferred resistance to a LacZ-FtsZ hybrid protein (ZZ). One possibility is that FtsZ functions as a multimer and that FtsZ(Rsa) mutant proteins have an increased ability for multimerization, making them resistant to SulA and ZZ.     

Developmentally regulated association of plastid division protein FtsZ1 with thylakoid membranes in Arabidopsis thaliana

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.     

New temperature-sensitive alleles of ftsZ in Escherichia coli

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

Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene.

The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.     

Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules

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.     

A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae

Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae (CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of alpha-Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results of Western analysis suggest that CmFtsZ1 is localized in mitochondria.     

An evolutionary puzzle: chloroplast and mitochondrial division rings

Consistent with their bacterial origin, chloroplasts and primitive mitochondria retain a FtsZ ring for division. However, chloroplasts and mitochondria have lost most of the proteins required for bacterial division other than FtsZ and certain homologues of the Min proteins, but they do contain plastid and mitochondrion dividing rings, which were recently shown to be distinct from the FtsZ ring. Moreover, recent studies have revealed that rings of the eukaryote-specific dynamin-related family of GTPases regulate the division of chloroplasts and mitochondria, and these proteins emerged early in eukaryotic evolution. These findings suggest that the division of chloroplasts and primitive mitochondria involve very similar systems, consisting of an amalgamation of rings from bacteria and eukaryotes.     

Isolation and characterization of dcw cluster from Streptomyces collinus producing kirromycin

A 4.5-kb BamHI fragment of chromosomal DNA of Streptomyces collinus containing gene ftsZ was cloned and sequenced. Upstream of ftsZ are localized genes ftsQ, murG, and ftsW, and downstream is yfiH. Gene ftsA is not adjacent to ftsZ or other genes of the cloned fragment. Protein FtsZ was isolated and characterized with respect to its binding to GTP and GTPase activity. The binding of GTP to FtsZ was Ca(2+) or Mg(2+) dependent with an optimum at 10 mM. The rate of GTP hydrolysis by FtsZ was stimulated by KCl. The presence of Ca(2+) (3-5 mM) resulted in a significant increase of GTPase activity. Higher concentrations of Ca(2+) than 5 mM had an inhibitory effect on GTPase activity. These results indicate that divalent ions (Ca(2+) or Mg(2+)) can be involved in regulation of GTP binding and hydrolysis of FtsZ. The maximum level of FtsZ was detected in aerial mycelium when spiral loops and sporulation septa were formed. FtsZ is degraded after finishing sporulation septa.     

Characterization of the ftsZ gene from Mycoplasma pulmonis, an organism lacking a cell wall.

The ftsZ gene is required for cell division in Escherichia coli and Bacillus subtilis. In these organisms, FtsZ is located in a ring at the leading edge of the septum. This ring is thought to be responsible for invagination of the septum, either causing invagination of the cytoplasmic membrane or activating septum-specific peptidoglycan biosynthesis. In this paper, we report that the cell division gene ftsZ is present in two mycoplasma species, Mycoplasma pulmonis and Acholeplasma laidlawii, which are eubacterial organisms lacking a cell wall. Sequencing of the ftsZ homolog from M. pulmonis revealed that it was highly homologous to other known FtsZ proteins. The M. pulmonis ftsZ gene was overexpressed, and the purified M. pulmonis FtsZ bound GTP. Using antisera raised against this purified protein, we could demonstrate that it was expressed in M. pulmonis. Expression of the M. pulmonis ftsZ gene in E. coli inhibited cell division, leading to filamentation, which could be suppressed by increasing expression of the E. coli ftsZ gene. The implications of these results for the role of ftsZ in cell division are discussed.