The ftsA gene product participates in formation of the Escherichia coli septum structure.

The patterns of septation in filaments of Escherichia coli, formed as a consequence of the lack of an active ftsA gene product and then returned to permissive conditions, were analyzed in isogenic strains containing three different mutated alleles of ftsA. Septation was blocked for at least one doubling time at the potential septation sites that presumably contained inactive FtsA protein but not at those sites containing either the active gene product or no gene product at all. These results suggested a possible structural role for the ftsA gene product in the construction of the E. coli septum.     

Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery

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.     

Interaction of FtsA and PBP3 proteins in the Escherichia coli septum.

Mutations in the ftsA gene of Escherichia coli conferred a higher resistance to lysis induced by penicillin or by a combination of cefsulodin and furazlocillin. The ftsA2 allele codes for an FtsA protein which is inactive at 42 degrees C but is able to regain its activity once it is transferred back to 30 degrees C; ftsA2 filaments formed at 42 degrees C in the presence of penicillin divided once the penicillin was removed and the temperature was lowered to 30 degrees C. Potential septation sites in the filaments of wild-type cells treated in the same way remained inactive. The binding of a radioactively labeled derivative of ampicillin to penicillin-binding protein 3 (PBP3) was significantly decreased in strain D-3, containing the mutant allele ftsA3, when the binding assay was performed at the restrictive temperature. A molecular species able to cross-react with an anti-PBP3 serum was nevertheless found to be present in the envelope of D-3 cells. These observations suggested that the FtsA protein, a protein with a structural and regulatory role in septation, and PBP3, a protein enzymatically active in the synthesis of murein for septation, interact with each other.     

Concentration and assembly of the division ring proteins FtsZ,FtsA, and ZipA during the Escherichia coli cell cycle

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.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

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.     

Coupling between DNA replication and cell division mediated by the FtsA protein in Escherichia coli: a pathway independent of the SOS response, the "TER" pathway

Inhibition of DNA synthesis prevented the recovery of cell division in filaments of D-3R [ftsA3(Ts) recA56] returned to the permissive temperature. The FtsA protein may be a signal involved in the "TER" pathway, a series of events that coordinate cell division with DNA replication, that is independent of the SOS pathway.     

Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression.

Aminoacyl-tRNA synthetase mutants of Escherichia coli are resistant to amdinocillin (mecillinam), a beta-lactam antibiotic which specifically binds penicillin-binding protein 2 (PBP2) and prevents cell wall elongation with concomitant cell death. The leuS(Ts) strain, in which leucyl-tRNA synthetase is temperature sensitive, was resistant to amdinocillin at 37 degrees C because of an increased guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool resulting from partial induction of the stringent response, but it was sensitive to amdinocillin at 25 degrees C. We constructed a leuS(Ts) delta (rodA-pbpA)::Kmr strain, in which the PBP2 structural gene is deleted. This strain grew as spherical cells at 37 degrees C but was not viable at 25 degrees C. After a shift from 37 to 25 degrees C, the ppGpp pool decreased and cell division was inhibited; the cells slowly carried out a single division, increased considerably in volume, and gradually lost viability. The cell division inhibition was reversible when the ppGpp pool increased at high temperature, but reversion required de novo protein synthesis, possibly of septation proteins. The multicopy plasmid pZAQ, overproducing the septation proteins FtsZ, FtsA, and FtsQ, conferred amdinocillin resistance on a wild-type strain and suppressed the cell division inhibition in the leuS(Ts) delta (rodA-pbpA)::Kmr strain at 25 degrees C. The plasmid pAQ, in which the ftsZ gene is inactivated, did not confer amdinocillin resistance. These results lead us to hypothesize that the nucleotide ppGpp activates ftsZ expression and thus couples cell division to protein synthesis.     

DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli.

In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.     

Role of Escherichia coli FtsN protein in the assembly and stability of the cell division ring

Deprivation of FtsN, the last protein in the hierarchy of divisome assembly, causes the disassembly of other elements from the division ring, even extending to already assembled proto‐ring proteins. Therefore the stability and function of the divisome to produce rings active in septation is not guaranteed until FtsN is recruited. Disassembly follows an inverse sequential pathway relative to assembly. In the absence of FtsN, the frequencies of FtsN and FtsQ rings are affected similarly. Among the proto‐ring components, ZipA are more sensitive than FtsZ or FtsA rings. In contrast, removal of FtsZ leads to an almost simultaneous disappearance of the other elements from rings. Although restoration of FtsN allows for a quick reincorporation of ZipA into proto‐rings, the de novo joint assembly of the three components when FtsZ levels are restored to FtsZ‐deprived filaments is even faster. This suggests that the recruitment of ZipA into FtsZ‐FtsA incomplete proto‐rings may require first a period for the reversal of these partial assemblies.     

Cell division in Bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-Ring assembly

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.     

Reversibility of SOS-associated division inhibition in Escherichia coli.

In Escherichia coli the SOS response, induced by DNA-damaging treatments, includes two systems of cell division inhibition, SfiA and SfiC, which are thought to prevent cell division by interacting with the division protein FtsZ. It is shown here that SfiA-mediated division inhibition is readily reversible, even in the absence of de novo protein synthesis, suggesting that functional FtsZ molecules can be recovered from SfiA-FtsZ complexes. The action of SfiC, on the other hand, is essentially irreversible; induction by expression of the recA (Tif) mutation for 60 min results in division inhibition that continues for at least 180 min after the end of the induction period. An excess of the presumed target molecule FtsZ, furnished by a multicopy plasmid, suppresses the action of SfiA but not SfiC. Simultaneous induction of SfiA and SfiC results in irreversible division inhibition, showing that SfiC is epistatic to SfiA. The irreversibility of SfiC action is most readily accounted for by assuming that the SfiC product, unlike SfiA, is stable. The reversibility of SfiA action is slower in a lon mutant, in which the SfiA protein is partially stabilized. From the kinetics of division resumption in the absence of protein synthesis, we estimated the in vivo half-life of the SfiA protein to be 10 min in a lon+ strain and 170 min in a lon mutant.     

lon incompatibility associated with mutations causing SOS induction: null uvrD alleles induce an SOS response in Escherichia coli

The uvrD gene in Escherichia coli encodes a 720-amino-acid 3'-5' DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible with lon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lon incompatibility was suppressed by sulA, lexA3(Ind(-)), or recA (Def) mutations. Other mutations, such as priA, dam, polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lon incompatible. SOS induction was not observed in uvrC and mutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor was uvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB and recF). These data suggest that SOS induction in uvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in uvrD strains.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring.

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.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

Mapping and characterization of mutants of the Escherichia coli cell division gene, ftsA

Eight independent temperature-sensitive mutants of the cell division protein FtsA have been studied. They fall into two classes in terms of their behaviour at 42 degrees C and recovery at 30 degrees C. The first class shows salt-dependent temperature-sensitivity and reversible inactivation of FtsA protein at 42 degrees C. The second shows irreversible inactivation which is not prevented by salt. Recovery of the ability to divide at 30 degrees C is rapid in mutants of the first group, but is delayed for approximately a generation time in the second group. This suggests that irreversible inactivation of FtsA causes extensive damage to the division machinery. The amino acid substitutions show clustering to a limited domain of the protein, and one particular substitution is found in three of the mutants.     

Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.     

A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli

The E. coli minicell locus (minB) was shown to code for three gene products (MinC, MinD, and MinE) whose coordinate action is required for proper placement of the division spetum. Studies of the phenotypic effects of expression of the three genes, alone and in all possible combinations, indicated the following: cell poles contain potential division sites that will support additional septation events unless specifically inactivated; the minC and minD gene products act in concert to form a nonspecific inhibitor of septation that is capable of blocking cell division at all potential division sites; and the minE gene codes for a topological specificity factor that, in wild-type cells, prevents the division inhibitor from acting at internal division sites while permitting it to block septation at polar sites.