The ftsA gene product: a possible connection between DNA replication and septation in Escherichia coli

The study of Escherichia coli strain D-2, which harbours the ftsA2(ts) allele, has shown that temperature-induced filaments of this strain can divide, at 30 degrees C, in the absence of DNA replication and translation. Strain D-2 is thermosensitive during a period coincident with that in which the termination protein should be synthesized and exert its action. The ftsA gene product, which participates in the structure of the septum, needs for its synthesis a short period of DNA replication. The FtsA protein could be involved in a mechanism that coordinates chromosome replication and cell division by a pathway different from and independent of the SOS-induced response.     

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.     

The spoIIN279(ts) mutation affects the FtsA protein of Bacillus subtilis.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.     

High-level expression of the FtsA protein inhibits cell septation in Escherichia coli K-12.

DNA fragments encoding the ftsA gene were subcloned into plasmids downstream of a lac promoter or a tac promoter. These plasmid constructs, when transformed into wild-type and mutant strains, inhibited normal cell septation, causing the formation of long nonseptate filaments. This phenotype is due to overproduction of the FtsA protein.     

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.     

Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family.

Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state. The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP. A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP. Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one. This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles. Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA. Alternatively they may have a regulatory role on the action of FtsA in the septator.     

Phage-display and correlated mutations identify an essential region of subdomain 1C involved in homodimerization of Escherichia coli FtsA

FtsA plays an essential role in Escherichia coli cell division and is nearly ubiquitous in eubacteria. Several evidences postulated the ability of FtsA to interact with other septation proteins and with itself. To investigate these binding properties, we screened a phage-display library with FtsA. The isolated peptides defined a degenerate consensus sequence, which in turn displayed a striking similarity with residues 126-133 of FtsA itself. This result suggested that residues 126-133 were involved in homodimerization of FtsA. The hypothesis was supported by the analysis of correlated mutations, which identified a mutual relationship between a group of amino acids encompassing the ATP-binding site and a set of residues immediately downstream to amino acids 126-133. This information was used to assemble a model of a FtsA homodimer, whose accuracy was confirmed by probing multiple alternative docking solutions. Moreover, a prediction of residues responsible for protein-protein interaction validated the proposed model and confirmed once more the importance of residues 126-133 for homodimerization. To functionally characterize this region, we introduced a deletion in ftsA, where residues 126-133 were skipped. This mutant failed to complement conditional lethal alleles of ftsA, demonstrating that amino acids 126-133 play an essential role in E. coli.     

Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three beta-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.     

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.     

Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments.

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.     

Differential effect of mutational impairment of penicillin-binding proteins 1A and 1B on Escherichia coli strains harboring thermosensitive mutations in the cell division genes ftsA, ftsQ, ftsZ, and pbpB.

To study the functional differences between penicillin-binding proteins (PBPs) 1A and 1B, as well as their recently postulated involvement in the septation process (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989), a series of isogenic strains with mutations in the genes coding for PBP 1A (ponA) or PBP 1B (ponB) or in the cell division-specific genes ftsA, ftsQ, pbpB, and ftsZ was constructed and used as the start point to produce double mutants combining the ponA or ponB characters with mutations in cell division genes. PBP 1A seemed to be unable to preserve cell integrity by itself, requiring the additional activities of PBP 2, PBP 3, and FtsQ. PBP 1B was apparently endowed with a more versatile biosynthetic potential that permitted a substantial enlargement of PBP 1A-deficient cells when PBP 2 or 3 was inhibited or when FtsQ was inactive. beta-Lactams binding to PBP 2 (mecillinam) or 3 (furazlocillin) caused rapid lysis in a ponB background. The lytic effect of furazlocillin to ponB cell division double mutants was suppressed at the restrictive temperature irrespective of the identity of the mutated cell division gene. These results indicate that PBPs 1A and 1B play distinct roles in cell wall synthesis and support the idea of a relevant involvement of PBP 1B in peptidoglycan synthesis at the time of septation.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

Cell shape and division in Escherichia coli: experiments with shape and division mutants.

Double mutants which carry mutations in genes (rodA, pbpA) required for cell elongation (i.e., maintenance of rod shape) in combination with mutations in genes (ftsA, ftsI, ftsQ, or ftsZ) required for septation were constructed. Such mutants were able to grow for about two mass doublings at a normal rate at the restrictive temperature (42 degrees C). The morphology of the cells formed under these conditions was interpreted by assuming the existence of a generalized system for peptidoglycan growth together with two additional systems which modify the shape of the growing peptidoglycan layer. The results also showed that different fts genes probably control different stages in septation. ftsZ (sulB or sfiB) appears to be required for the earliest step in septation, ftsQ and ftsI (pbpB or sep) are required for a later step or steps, and ftsA is required only for the latest stages in septation.     

Structural inhibition and reactivation of Escherichia coli septation by elements of the SOS and TER pathways.

The inhibition of cell division caused by induction of the SOS pathway in Escherichia coli structurally blocks septation, as deduced from two sets of results. Potential septation sites active at the time of SOS induction became inactivated, while those initiated during the following doubling time were active. Penicillin resistance increased in wild-type UV light-irradiated cells, a behavior similar to that observed in mutants in which structural blocks were introduced by inactivation of FtsA. Potential septation sites that have been structurally blocked by either the SOS division inhibitor, furazlocillin inhibition of PBP3, or inactivation of a TER pathway component, FtsA3, could be reactivated one doubling time after removal of the inhibitory agent in the presence of an active lon gene product. Reactivation of potential septation sites blocked by the presence of an inactivated FtsA3 was significantly lower when the lon protease was not active, suggesting that Lon plays a role in the removal of inactivated TER pathway products from the blocked potential septation sites.     

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.     

Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [(14)C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30 degrees C, and the ability to bind [(14)C]benzylpenicillin was rapidly lost on incubation at 42 degrees C. The other PBPs were normal in both mutants. At 30 degrees C both mutants were slightly longer than their parents and on shifting to 42 degrees C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42 degrees C division slowly recommenced, but at 44 degrees C this did not occur. The inhibition of division at 42 degrees C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl(2). PBP3 was not stabilized in vitro at 42 degrees C by these concentrations of sucrose or MgCl(2). Revertants that grew as normal rods at 42 degrees C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42 degrees C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli. It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.     

Z-ring-independent interaction between a subdomain of FtsA and late septation proteins as revealed by a polar recruitment assay

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.     

Inhibition of Escherichia coli Division by Protein X

We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced. Rifampin selectively blocks protein X production. This drug was shown to allow cells whose DNA had been damaged by nalidixic acid to resume septation. Several mutants formed septa-less filaments and also produced protein X at 42 degrees C; rifampin both inhibited their production of protein X and permitted them to form septa and divide. Essentially complementary results were obtained with a dnaA mutant which at 42 degrees C stopped making DNA, did not produce protein X, and continued to divide; added bleomycin degraded DNA, induced protein X, and inhibited septation. These results, as well as previous observations, are all consistent with the proposal that protein X is produced as a consequence of DNA damage and is an inhibitor of septation. We suggest that septation could require binding of a single-stranded region of DNA to a septum site in the membrane. Protein X could block this binding by combining with the DNA. This control could provide an emergency mechanism in addition to the usually proposed coordination in which completion of DNA synthesis creates a positive effector for a terminal step of septation. Or it could be the sole coordinating mechanism, even under unperturbed growth conditions.     

A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface

In Escherichia coli, initial assembly of the Z ring for cell division requires FtsZ plus the essential Z ring‐associated proteins FtsA and ZipA. Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP binding pocket and result in cell division arrest at non‐permissive temperatures. We found that purified wild‐type FtsA bound and hydrolysed ATP, whereas FtsA27 was defective in both activities. FtsA27 was also less able to localize to the Z ring in vivo. To investigate the role of ATP transactions in FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably, suppressors outside of the ATP binding site, including some mapping to the FtsA‐FtsA subunit interface, also enhanced ATP transactions and exhibited gain of function phenotypes in vivo. These results suggest that allosteric effects, including changes in oligomeric state, may influence the ability of FtsA to bind and/or hydrolyse ATP.     

Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site

Summary In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.