Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci.

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.     

Early Initiation of Deoxyribonucleic Acid Replication and Shortening of Generation Time Associated with Inhibition of Lateral Wall Formation by Mecillinam

The effects of mecillinam on the growth of rods of the pH-conditional morphology mutant MirM7 was studied. It has been found that mecillinam causes, coincident with transition to coccal shape, a balanced rise in the rate of viable count increase and the rate of macromolecular synthesis which lasts either until the cells enter a stationary growth phase or indefinitely, in the case of continuously diluted cultures. When the antibiotic is removed from cells which have already become coccoid, cells continue to grow at a faster rate until they resume the rod shape. No change in the per-cell rate of protein synthesis has been seen in untreated or mecillinam-treated cells before or after the change in growth rate. Studies with synchronously growing cells have shown that the antibiotic causes a shortening in the I period (initiation of deoxyribonucleic acid replication). Evaluation of the residual divisions in nalidixic acid-treated, exponential-phase cells has shown that mecillinam also shortens the D period (cell division). It is proposed that, in strain MirM7, inhibition of lateral wall elongation by the antibiotic allows the initiation of a new septum, though inhibition is still in progress. The initiation of a new septum is, in turn, responsible for both the early inibition of deoxyribonucleic acid replication and accelerated division. In the parental strain, MirA12, as well as in other sensitive gram-negative rods which divide, become cocci, and stop dividing after addition of the antibiotic, inhibition of lateral wall formation activates a feedback mechanism which prevents insertion of new septa (Satta et al., J. Bacteriol. 142:43-51, 1980). Consequently, no early initiation of deoxyribonucleic acid replication is observed, and the last division allowed by the antibiotic occurs in due time. This negative control is missing in MirM7.     

Control of cell septation by lateral wall extension in a pH-conditional morphology mutant of Klebsiella pneumoniae.

The pH-conditional morphology mutant of Klebsiella pneumoniae strain MirM7 grows as cocci at pH 7 and as rods at pH 5.8. The mutant has a high-level mecillinam resistance (50% lethal dose greater than 200 micrograms/ml) in both forms. When broth cultures of the rod-shaped mutant were grown with 0.7 microgram of mecillinam per ml, cells assumed a round shape and continued to divided at a higher rate than the untreated control. A MirM7 rod-shaped revertant (MirA12), when treated with the same antibiotic concentration, changed to coccal shape and stopped dividing. The penicillin-binding proteins (PBPs) of strains MirA12 and MirM7 were analyzed. K. pneumoniae had six major PBPs quite similar to those of Escherichia coli. No differences were seen in the PBPs of MirM7 cocci and rods and MirA12 cells. In particular, PBP2 was found to be present and similar in MirM7 rods and cocci and MirA12 cells. We suggest that that in gram-negative rods, a control mechanism exists which prevents further septation in the absence of lateral cell wall elongation. The unique behavior of MirM7 is due to the fact that the control mechanism is not active in this strain. This model allows us to explain the preservation of shape in bacterial rods under various conditions of growth and the mechanism of bacterial killing by mecillinam.     

Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of Klebsiella pneumoniae

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.     

Cell shape determination in Escherichia coli

The rigid cell wall peptidoglycan (murein) is a single giant macromolecule whose shape determines the shape of the bacterial cell. Insight into morphogenetic mechanism(s) responsible for determining the shape of the murein sacculus itself has begun to emerge only in recent years. The discovery that MfreB and Mbl are cytoskeletal actin homologues that form helical structures extending from pole to pole in rod-shaped cells has opened an exciting new field of microbial cell biology. MreB (in Gram-negative rods) and Mbl (in Gram-positive species) are essential for murein synthesis along the lateral wall and hence, the rod shape of the cell. Known members of the morphogenetic system include MreB (or Mbl), MreC, MreD and PBP2, but Rod A and murein biosynthetic enzymes involved in peptidoglycan precursor synthesis and assembly are likely to be recruited to the same multimolecular apparatus. However, the actual role of MreB in assembly of the morphogenetic complex is still not clear and little is known about regulatory mechanisms controlling the switch from lateral murein elongation to septa1 murein synthesis at the time of cell division.     

Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.     

Direct interaction of FtsZ and MreB is required for septum synthesis and cell division in Escherichia coli

How bacteria coordinate cell growth with division is not well understood. Bacterial cell elongation is controlled by actin–MreB while cell division is governed by tubulin–FtsZ. A ring‐like structure containing FtsZ (the Z ring) at mid‐cell attracts other cell division proteins to form the divisome, an essential protein assembly required for septum synthesis and cell separation. The Z ring exists at mid‐cell during a major part of the cell cycle without contracting. Here, we show that MreB and FtsZ of Escherichia coli interact directly and that this interaction is required for Z ring contraction. We further show that the MreB–FtsZ interaction is required for transfer of cell‐wall biosynthetic enzymes from the lateral to the mature divisome, allowing cells to synthesise the septum. Our observations show that bacterial cell division is coupled to cell elongation via a direct and essential interaction between FtsZ and MreB.     

The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate.

Mecillinam, a beta-lactam antibiotic which binds specifically to penicillin-binding protein 2 (PBP2), blocks lateral cell-wall elongation, induces spherical morphology and ultimately kills bacteria. We describe here a new mecillinam-resistant mutant of Escherichia coli, the lov mutant. It possesses active PBP2, as evidenced by its rod shape in the absence of mecillinam (but not in its presence), its ability to filament when septation is inhibited, and its penicillin-binding ability. The lov mutant grows slowly but seems to regulate its macromolecular parameters properly: cell volume, RNA content (ribosome concentration), and DNA content are appropriate for the growth rate, and the growth yield is identical to that of wild type. The lov mutation is located at 41 min on the E.coli genetic map and is recessive. Certain rpsL (StrR) mutations suppress the lov mutant's mecillinam resistance. The allele specificity of the suppression suggests that the lov gene product may interact directly with the ribosomes. The lov gene product thus seems to define a link between PBP2 (the mecillinam target) and the ribosomes; we propose that this link is involved in transmitting information on the growth rate (ribosome concentration) to the peptidoglycan synthesizing apparatus.     

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division.     

Cell cycle parameters of Proteus mirabilis: interdependence of the biosynthetic cell cycle and the interdivision cycle.

We investigated the time periods of DNA replication, lateral cell wall extension, and septum formation within the cell cycle of Proteus mirabilis. Cells were cultivated under three different conditions, yielding interdivision times of approximately 55, 57, and 160 min, respectively. Synchrony was achieved by sucrose density gradient centrifugation. The time periods were estimated by division inhibition studies with cephalexin, mecillinam, and nalidixic acid. In addition, DNA replication was measured by thymidine incorporation, and murein biosynthesis was measured by incorporation of N-acetylglucosamine into sodium dodecyl sulfate-insoluble murein sacculi. At interdivision times of 55 to 57 min murein biosynthesis for reproduction of a unit cell lasted longer than the interdivision time itself, whereas DNA replication finished within 40 min. Surprisingly, inhibition of DNA replication by nalidixic acid did not inhibit the subsequent cell division but rather the one after that. Because P. mirabilis fails to express several reactions of the recA-dependent SOS functions known from Escherichia coli, the drug allowed us to determine which DNA replication period actually governed which cell division. Taken together, the results indicate that at an interdivision time of 55 to 57 min, the biosynthetic cell cycle of P. mirabilis lasts approximately 120 min. To achieve the observed interdivision time, it is necessary that two subsequent biosynthetic cell cycles be tightly interlocked. The implications of these findings for the regulation of the cell cycle are discussed.     

Taking shape: control of bacterial cell wall biosynthesis

The characteristic shape of a bacterial cell is a function of the three dimensional architectures of the cell envelope and is determined by the balance between lateral wall extension and synthesis of peptidoglycan at the division septum. The three dimensional patterns of cell wall synthesis in the bacterium Bacillus subtilis is influenced by actin-like proteins that form helical coils in the cell and by the MreCD membrane proteins that link the cytoskeletal elements with the penicillin-binding proteins that carry out peptidoglycan synthesis. Recent genetic studies have provided important clues as to how these proteins are arranged in the cell and how they function to regulate cell shape.     

DivIVA is required for polar growth in the MreB-lacking rod-shaped actinomycete Corynebacterium glutamicum

The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA(Cg)) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA(Cg)-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA(Cg) localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.     

Shape determination in Bacillus subtilis

The discovery of cytoskeletal elements in prokaryotes has dramatically changed the way we think about bacterial cell morphogenesis. The rod shape of Bacillus subtilis is maintained by the two major polymers (peptidoglycan and teichoic acids) of its thick cell wall and determined by the way these are inserted during growth. The current view is that the dynamic tubulin-like (FtsZ) and actin-like (MreB) cytoskeletons orchestrate, both in time and space, the assembly of macromolecular machineries that effect cell wall synthesis and hydrolysis during cell division and cell elongation, respectively.     

Sublethal High Hydrostatic Pressure Treatment Reveals the Importance of Genes Coding Cytoskeletal Protein in Escherichia Coli Morphogenesis

We studied morphologic changes after sublethal high hydrostatic pressure treatment (HPT) of Escherichia coli K-12 strains in which genes related to the cytoskeleton, cell wall, and cell division had been deleted. Some long filamentous and swelling cells were observed in wild-type bacteria, while some spherical, branched, or collapsed cells were observed in deletion mutants. In particular, ΔzapA and ΔrodZ showed distinguished morphologies. ZapA supports FtsZ, a cytoskeletal protein, forming ring with ZapB. RodZ, a cytoskeletal protein, interacts with MreB, also a cytoskeletal protein, and both factors are necessary for maintaining the rod shape of the cell. These results showed that insufficient formation of FtsZ rings induced cell elongation and that insufficient formation of MreB induced a branched and collapsed cell shape. Therefore, the correct formation of the bacteria cytoskeleton by FtsZ rings and MreB is important for keeping normal cell shape during growth after HPT, and the polymerization of cytoskeletal proteins was a critical target of sublethal HPT. These results indicate that sublethal HPT induces bacterial cell morphologic change and provide important information on the role of genes involved in morphogenesis. Therefore, sublethal HPT may be a good tool for studying the morphogenesis of bacterial cells.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Polarity and the diversity of growth mechanisms in bacteria

Bacterial cell growth is a complex process consisting of two distinct phases: cell elongation and septum formation prior to cell division. Although bacteria have evolved several different mechanisms for cell growth, it is clear that tight spatial and temporal regulation of peptidoglycan synthesis is a common theme. In this review, we discuss bacterial cell growth with a particular emphasis on bacteria that utilize tip extension as a mechanism for cell elongation. We describe polar growth among diverse bacteria and consider the advantages and consequences of this mode of cell elongation.     

Electron microscope study of the rod-to-coccus shape change in a temperature-sensitive rod- mutant of Bacillus subtilis.

The changes in cell morphology of Bacillus subtilis rodB during a temperature shift from 20 to 42 degrees C, in the absence of added anions, are described. At 20 degrees C the organisms grow as rods but gradually become spherical in shape when placed at 42 degrees C. The shape change is initiated by an increase in diameter at the cell equator, resulting in a bulged morphology, which is further modified to the morphology of a coccus. This change may involve a modification of the pattern of normal cylindrical extension such that incorporation of newly synthesized wall leads only to increase in diameter, perhaps from a growth zone of limited extent. The pattern of surface growth was followed by reconstructing the sequence of cross wall formation and pole construction in rods grown at 20 degrees C and in organisms incubated at 42 degrees C for 75 and 150 min. In thin section, wall forming the septum and nascent poles can be distinguished from the surface distal to the division site by the presence of raised tears, perhaps analogous to the wall bands of streptococci. By using an analog rotation technique involving the three-dimensional reconstruction of cells by mathematical rotation of axial thin sections about their longitudinal axis, it is shown that the proportion of septal wall increases during the shape change. In the coccal forms, all surface growth may arise from septal growth sites.     

On the origin of branches in Escherichia coli.

Some Escherichia coli strains with impaired cell division form branched cells at high frequencies during certain growth conditions. Here, we show that neither FtsI nor FtsZ activity is required for the development of branches. Buds did not form at specific positions along the cell surface during high-branching conditions. Antibiotics affecting cell wall synthesis had a positive effect on branch formation in the case of ampicillin, cephalexin, and penicillin G, whereas mecillinam and D-cycloserine had no substantial effect. Altering the cell morphology by nutritional shifts showed that changes in morphology preceded branching, indicating that the cell's physiological state rather than specific medium components induced branching. Finally, there was no increased probability for bud formation in the daughters of a cell with a bud or branch, showing that bud formation is a random event. We suggest that branch formation is caused by abnormalities in cell wall elongation rather than by aberrant cell division events.     

Incorporation of diaminopimelic acid into the old poles of Escherichia coli

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.     

The GpsB files: the truth is out there

Peptidoglycan (PG), an essential stress‐bearing component of the bacterial cell wall, is synthesised by penicillin binding proteins (PBPs). PG synthesis at the cell division septum is necessary for constructing new poles of progeny cells, and cells cannot elongate without inserting new PG in the side‐wall. The cell division regulator GpsB appears to co‐ordinate PG synthesis at the septum during division and at the side‐wall during elongation in rod‐shaped and ovococcoid Gram‐positive bacteria. How the control over PG synthesis is exerted is unknown. In this issue of Molecular Microbiology, Rued et al. show that in pneumococci GpsB forms complexes with PBP2a and PBP2b, and that deletion or depletion of GpsB prevents closure of the septal ring that in itself is PBP2x‐dependent. Loss of GpsB can be suppressed by spontaneous mutations, including within the gene encoding the only PP2C Ser/Thr phosphatase in Streptococcus pneumoniae, indicating that GpsB plays a key – but unknown – role in protein phosphorylation in pneumococci. Rued et al. combine phenotypic and genotypic analyses of mutant strains that suggest discrepancies in the literature concerning GpsB might have arisen from accumulation of unidentified suppressors, highlighting the importance and power of strain validation and whole genome sequencing in this context.