The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide.

The rodA(Sui) mutation allows cell division to take place at 42 degrees C in ftsI23 mutant cells, which produce a thermolabile penicillin-binding protein 3 (PBP3, the septation-specific peptidoglycan transpeptidase). We show here that the mutation in rodA is a single-base change from a glutamine to a chain termination (amber) codon, and that an amber suppressor (supE) present in the strain restores the ability to produce a reduced level of normal RodA protein. The reduced level of RodA is accompanied by an increase in the levels of two other proteins (PBP2 and PBP5) encoded by genes in the rodA operon. We show that an increased level of PBP5 is by itself sufficient to restore cell division to ftsI23 cells at 42 degrees C. Two other treatments were found to restore division capacity to the mutant: an increase in PBP6 (which is a D-alanine carboxypeptidase like PBP5) or suitable concentrations of D-cycloserine. All of the above treatments have the effect of reducing the number of pentapeptide side chains in peptidoglycan and increasing the number of tripeptides. We conclude that the effect of the rodA(Sui) mutation is to indirectly increase the availability of tripeptide side chains, which are used preferentially by PBP3 as acceptors in transpeptidation. A change in the proportions of different kinds of peptide side chain in the peptidoglycan can therefore determine whether cells will divide.     

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.     

Control of cell shape and elongation by the rodA gene in Bacillus subtilis

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli , the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA , encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible P_spac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.     

Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenetic proteins RodA and penicillin-binding protein 2.

The pattern of peptidoglycan (murein) segregation in cells of Escherichia coli with impaired activity of the morphogenetic proteins penicillin-binding protein 2 and RodA has been investigated by the D-cysteine-biotin immunolabeling technique (M. A. de Pedro, J. C. Quintela, J.-V. H?ltje, and H. Schwarz, J. Bacteriol. 179:2823-2834, 1997). Inactivation of these proteins either by amdinocillin treatment or by mutations in the corresponding genes, pbpA and rodA, respectively, leads to the generation of round, osmotically stable cells. In normal rod-shaped cells, new murein precursors are incorporated all over the lateral wall in a diffuse manner, being mixed up homogeneously with preexisting material, except during septation, when strictly localized murein synthesis occurs. In contrast, in rounded cells, incorporation of new precursors is apparently a zonal process, localized at positions at which division had previously taken place. Consequently, there is no mixing of new and old murein. Old murein is preserved for long periods of time in large, well-defined areas. We propose that the observed patterns are the result of a failure to switch off septal murein synthesis at the end of septation events. Furthermore, the segregation results confirm that round cells of rodA mutants do divide in alternate, perpendicular planes as previously proposed (K. J. Begg and W. D. Donachie, J. Bacteriol. 180:2564-2567, 1998).     

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.     

New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.     

FtsZ-spirals and -arcs determine the shape of the invaginating septa in some mutants of Escherichia coli

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

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

FtsW is a dispensable cell division protein required for Z-ring stabilization during sporulation septation in Streptomyces coelicolor

The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.     

The Genes Raw and Ribbon Are Required for Proper Shape of Tubular Epithelial Tissues in Drosophila

The products of two genes, raw and ribbon (rib), are required for the proper morphogenesis of a variety of tissues. Malpighian tubules mutant for raw or rib are wider and shorter than normal tubules, which are only two cells in circumference when they are fully formed. The mutations alter the shape of the tubules beginning early in their formation and block cell rearrangement late in development, which normally lengthens and narrows the tubes. Mutations of both genes affect a number of other tissues as well. Both genes are required for dorsal closure and retraction of the CNS during embryonic development. In addition, rib mutations block head involution, and broaden and shorten other tubular epithelia (salivary glands, tracheae, and hindgut) in much same manner as they alter the shape of the Malpighian tubules. In tissues in which the shape of cells can be observed readily, rib mutations alter cell shape, which probably causes the change in shape of the organs that are affected. In double mutants raw enhances the phenotypes of all the tissues that are affected by rib but unaffected by raw alone, indicating that raw is also active in these tissues.     

Positive selection of mutants with cell envelope defects of a Salmonells typhimurium strain hypersensitive to the products of genes hisF and hisH.

Strain SB564 and its derivative DA78 are hypersensitive to the inhibitory action of the proteins coded for by genes hisF and hisH on cell division. Transduction of hisO1243, a regulatory mutation that elicits a very high level of expression of the histidine operon, into these strains resulted in the production of long filamentous cells carrying large "balloons" and in growth failure. Forty-one hisO1242 derivatives that escaped inhibition were isolated. These strains showed a large variety of alterations, many of which were related to the cell envelope. The more-frequent alterations included: changes in cell shape, increased sensitivity to one or more of several drugs (deoxycholate, cycloserine, penicillin, novobiocin, acridine orange), increased autolytic activity in alkaline buffer, anomalous fermentation of maltose on eosin--methylene blue plates, and temperature-conditional cell division. The alterations are produced, in some of the strains, by pleiotropic mutations in gene envB (Antón, Mol, Gen. Genet. 160:277--286, 1978) or envD (Antón and Orce, Mol. Gen. Genet. 144:97--105, 1976). Strains affected in divC, divD, and rodA loci have also been identified. Genetic analysis has shown that several strains carry more than one envelope mutation. It is assumed that envelope mutations are positively selected because they somehow alleviate the particularly severe inhibition of cell division caused, in strains SB564 and DA78, by the excessive synthesis of hisF and hisH gene products.     

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.     

Investigation of the role of cell-cell interactions in division plane determination during maize leaf development through mosaic analysis of the tangled mutation

Most plant cells divide in planes that can be predicted from their shapes according to simple geometrical rules, but the division planes of some cells appear to be influenced by extracellular cues. In the maize leaf, some cells divide in orientations not predicted by their shapes, raising the possibility that cell-cell communication plays a role in division plane determination in this tissue. We investigated this possibility through mosaic analysis of the tangled (tan) mutation, which causes a high frequency of cells in all tissue layers to divide in abnormal orientations. Clonal sectors of tan mutant tissue marked by a closely linked albino mutation were examined to determine the phenotypes of cells near sector boundaries. We found that tan mutant cells always showed the mutant phenotype regardless of their proximity to wild-type cells, demonstrating that the wild-type Tan gene acts cell-autonomously in both lateral and transverse leaf dimensions to promote normally oriented divisions. However, if the normal division planes of wild-type cells depend on cell-cell communication involving the products of genes other than Tan, then aberrantly dividing tan mutant cells might send abnormal signals that alter the division planes of neighboring cells. The cell-autonomy of the tan mutation allowed us to investigate this possibility by examining wild-type cells near the boundaries of tan mutant sectors for evidence of aberrantly oriented divisions. We found that wild-type cells near tan mutant cells did not divide differently from other wild-type cells. These observations argue against the idea that the division planes of proliferatively dividing maize leaf epidermal cells are governed by short-range communication with their nearest neighbors.     

Inhibition of growth of ftsQ, ftsA, and ftsZ mutant cells of Escherichia coli by amplification of a chromosomal region encompassing closely aligned cell division and cell growth genes.

Amplification of a 2.6-kilobase chromosomal fragment of the mra region of Escherichia coli encompassing the ftsI(pbpB) gene and an open reading frame upstream with lethal to E. coli strains with mutations of the flanking cell division genes ftsQ, ftsA, and ftsZ. A shortened fragment in which the major portion of ftsI was deleted also had lethal effects on ftsQ and ftsZ mutants.     

A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW.

We constructed a null allele of the ftsI gene encoding penicillin-binding protein 3 of Escherichia coli. It caused blockage of septation and loss of viability when expression of an extrachromosomal copy of ftsI was repressed, providing a final proof that ftsI is an essential cell division gene. In order to complement this null allele, the ftsI gene cloned on a single-copy mini-F plasmid required a region 1.9 kb upstream, which was found to contain a promoter sequence that could direct expression of a promoterless lacZ gene on a mini-F plasmid. This promoter sequence lies at the beginning of the mra cluster in the 2 min region of the E. coli chromosome, a cluster of 16 genes which, except for the first 2, are known to be involved in cell division and cell envelope biosynthesis. Disruption of this promoter, named the mra promoter, on the chromosome by inserting the lac promoter led to cell lysis in the absence of a lac inducer. The defect was complemented by a plasmid carrying a chromosomal fragment ranging from the mra promoter to ftsW, the fifth gene downstream of ftsI, but not by a plasmid lacking ftsW. Although several potential promoter sequences in this region of the mra cluster have been reported, we conclude that the promoter identified in this study is required for the first nine genes of the cluster to be fully expressed.     

Coordination of the initiation of recombination and the reductional division in meiosis in Saccharomyces cerevisiae

Early exchange (EE) genes are required for the initiation of meiotic recombination in Saccharomyces cerevisiae. Cells with mutations in several EE genes undergo an earlier reductional division (MI), which suggests that the initiation of meiotic recombination is involved in determining proper timing of the division. The different effects of null mutations on the timing of reductional division allow EE genes to be assorted into three classes: mutations in RAD50 or REC102 that confer a very early reductional division; mutations in REC104 or REC114 that confer a division earlier than that of wild-type (WT) cells, but later than that of mutants of the first class; and mutations in MEI4 that do not significantly alter the timing of MI. The very early mutations are epistatic to mutations in the other two classes. We propose a model that accounts for the epistatic relationships and the communication between recombination initiation and the first division. Data in this article indicate that double-strand breaks (DSBs) are not the signal for the normal delay of reductional division; these experiments also confirm that MEI4 is required for the formation of meiotic DSBs. Finally, if a DSB is provided by the HO endonuclease, recombination can occur in the absence of MEI4 and REC104.     

Genetic analysis of the cell division protein FtsI (PBP3): amino acid substitutions that impair septal localization of FtsI and recruitment of FtsN

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened approximately 7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids-R23, L39, or Q46-that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by approximately 150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.     

Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli.

Two closely linked genes, mrdA and mrdB, located at ca. 14.2 min on the Escherichia coli chromosomal linkage map, seen to be responsible for the normal rod shape and mecillinam sensitivity of E. coli. The product of mrdA was concluded to be penicillin-binding protein 2, because mrdA mutations caused formation of thermosensitive penicillin-binding protein 2. The product of the mrdB gene is unknown. At 42 degrees, C, mutation in either of these genes caused formation of spherical cells and mecillinam resistance. Both mutations was recessive, and complementation, as detected in +-/-+ meroheterodiploids having the wild-type phenotype, provided strong evidence that the two mutations are in different complementation groups. P1 transduction suggested that the most plausible gene order is leuS-mrdA-mrdB-lip. The rodA mutation reported previously seems to be similar to the mrdB murations, but the identities of the two have not yet been proven.