Cell Division in Escherichia coli: Analysis of Double Mutants for Filamentation and Abnormal Septation of Deoxyribonucleic Acid-Less Cells

The link between chromosome termination, initiation of cell division, and choice of division sites was studied in Escherichia coli by preparing double mutants. Hybrid mutants containing div52-ts, a cell division initiation mutation, and min, mutations which affect the choice of division sites resulting in the septation of minicells, were characterized. The mutants produced minicells and normal cells coordinately under all conditions studied, although the fraction of minicells is half that of the parental minicell strain. The mutant gradually stopped dividing at both the median and minicell septation sites when transferred from 30 to 41 C in rich medium. A synchronous cell division of filaments was induced 15 min after addition of chloramphenicol to the medium, even at 41 C. Divisions were observed at both normal and minicell sites. These results indicate that div52-ts and min functions share a common step in a cell division pathway. A double mutant containing div52-ts and div27-ts, a dnaB mutant which divides in the absence of DNA synthesis, was characterized. The mutant continues to divide after a shift to the high temperature, although at a reduced rate. The behavior of this hybrid mutant suggests a hypothesis that the chromosome termination signal and div52-ts division initiation signal act on a single membrane site which is altered in div27-ts strains.     

Regulation of Cell Division in a Temperature-Sensitive Division Mutant of Escherichia coli

Escherichia coli fil ts forms multinucleate filaments when suspensions of about 10(7) organisms per ml are shifted from 37 to 43 C in rich medium. Occasional septation continues, chiefly at the poles, and immediately becomes more frequent when the filaments are returned to 37 C. The addition of chloramphenicol (200 mug/ml) at either temperature initially stimulates the formation of polar septa. When very dilute suspensions of the strain (<10(6) organisms per ml) are shifted to the restrictive temperature, the inhibition of septation is more complete and only seldom reversible. Conversely, cell division is little affected when suspensions of >10(8) organisms per ml, or microcolonies of several hundred organisms on agar, are incubated at 43 C; evidence is presented that this is a consequence of a slight reduction in the mutant's growth rate. In certain media, septation is blocked irreversibly by even brief exposure to 43 C, after which cell elongation without division proceeds at 37 C for some hours. Several findings, when considered together, suggest that the cytoplasmic membrane is normal at the restrictive temperature, and that the block in septation is caused by a defect in the cell wall: it is largely overcome by NaCl, but not by sucrose; in some circumstances the filaments become swollen and develop localized bulges in the wall, yet the membrane remains intact and retains its selective permeability; lastly, the strain is insensitive to deoxycholate at both temperatures. The mutation has been mapped between arg B and thr, at a locus which appears to be distinct from others known primarily to influence cell division.     

CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over-replication

We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli . At 30°C, the dnaAcos mutant causes over-replication of the chromosome, and colony formation is inhibited. We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited. This inhibition was independent of SfiA, an inhibitor of the septum-forming protein, FtsZ. To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold-sensitive phenotype of the dnaAcos mutant. One of these proved to be a previously unknown gene, which we named cedA . This gene encoded a 12 kDa protein and resided at 38.9 min on the E. coli genome map. A multicopy supply of the cedA gene to the dnaAcos cells did not repress over-replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number. Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30°C. We propose that over-replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control. In the dnaA ⁺ background, cedA also seems to affect cell division.     

Regulation and SOS induction of division inhibition in Escherichia coli K12

Summary When Escherichia coli is subjected to treatments that damage DNA or perturb DNA replication considerable cell filamentation occurs. It has been postulated that this phenomenon is associated with the presence of a division inhibitor induced coordinately with the SOS functions. The role of this induction would be to delay septation during DNA repair to prevent the formation of DNAless cells. In this communication, we present evidence for such a division inhibitor based on the properties of a division mutant which is hyperactive in the septation delay. Cells of this mutant filament extensively after a nutritional shift-up, have drastically reduced colony-forming abilities on a rich medium but not on a minimal medium following treatment with ultraviolet radiation and, are deficient in the lysogenization of phage lambda; phenotypes which are characteristic of but expressed to a much lower extent in another type of division mutant called lon. Cells harboring the division mutation plus either one of the lexA mutant alleles, spr-51 or tsl-1, are filamentous suggesting that they are permanently derepressed for division inhibition. These results are in agreement with models that assign the regulation of cell division to a division inhibitor which is regulated by the lexA repressor protein.     

Dual Role for the O-Acetyltransferase OatA in Peptidoglycan Modification and Control of Cell Septation in Lactobacillus plantarum

Until now, peptidoglycan O-acetyl transferases (Oat) were only described for their peptidoglycan O-acetylating activity and for their implication in the control of peptidoglycan hydrolases. In this study, we show that a Lactobacillus plantarum mutant lacking OatA is unable to uncouple cell elongation and septation. Wild-type cells showed an elongation arrest during septation while oatA mutant cells continued to elongate at a constant rate without any observable pause during the cell division process. Remarkably, this defect does not result from a default in peptidoglycan O-acetylation, since it can be rescued by wild-type OatA as well as by a catalytic mutant or a truncated variant containing only the transmembrane domain of the protein. Consistent with a potential involvement in division, OatA preferentially localizes at mid-cell before membrane invagination and remains at this position until the end of septation. Overexpression of oatA or its inactive variants induces septation-specific aberrations, including asymmetrical and dual septum formation. Overproduction of the division inhibitors, MinC or MinD, leads to cell filamentation in the wild type while curved and branched cells are observed in the oatA mutant, suggesting that the Min system acts differently on the division process in the absence of OatA. Altogether, the results suggest that OatA plays a key role in the spatio-temporal control of septation, irrespective of its catalytic activity.     

Localization of Cell Division Protein FtsK to the Escherichia coli Septum and Identification of a Potential N-Terminal Targeting Domain

Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. coli cells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced and ftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize in ftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.     

Streptomyces coelicolor genes ftsL and divIC play a role in cell division but are dispensable for colony formation

We have characterized homologues of the bacterial cell division genes ftsL and divIC in the gram-positive mycelial bacterium Streptomyces coelicolor A3(2). We show by deletion-insertion mutations that ftsL and divIC are dispensable for growth and viability in S. coelicolor. When mutant strains were grown on a conventional rich medium (R2YE, containing high sucrose), inactivation of either ftsL or divIC resulted in the formation of aerial hyphae with partially constricted division sites but no clear separation of prespore compartments. Surprisingly, this phenotype was largely suppressed when strains were grown on minimal medium or sucrose-free R2YE, where division sites in many aerial hyphae had finished constricting and chains of spores were evident. Thus, osmolarity appears to affect the severity of the division defect. Furthermore, double mutant strains deleted for both ftsL and divIC are viable and have medium-dependent phenotypes similar to that of the single mutant strains, suggesting that functions performed by FtsL and DivIC are not absolutely required for septation during growth and sporulation. Alternatively, another division protein may partially compensate for the loss of both FtsL and DivIC on minimal medium or sucrose-free R2YE. Finally, based on transmission electron microscopy observations, we propose that FtsL and DivIC are involved in coordinating symmetrical annular ingrowth of the invaginating septum.     

Separation of processes associated with differentiation of two-celled Fucus embryos

Various inhibitors were used to separate the overlapping processes of polar axis fixation, intracellular localizations forming a polar cell, and cell division, all of which are essential for cellular differentiation in two-celled embryos of Fucus distichus L. Powell. Cycloheximide and sucrose delayed the appearance of a polar cell (rhizoid formation) without inhibiting the fixation of a polar axis. Cytochalasin B, at 10 μg/ml, reversibly inhibited rhizoid formation without altering cell division. At higher concentrations (50–100 μg/ml) given in short pulses, cytochalasin affected the orientation and delayed the fixation of a light-induced polar axis with no qualitative effect on cell division. Disruption of the mitotic apparatus and prevention of cell division by colchicine had no influence on rhizoid formation or on the photopolarization of the developmental axis.     

Effects of cytochalasin B on division and calcium carbonate extrusion in a calcareous alga.

Cytochalasin B (CB) (100 μg/ml) reversibly blocked cell division and cuased the formation of abnormal cytoplasmic bodies in the alga Cricosphaera carterae. Concentrations of 20 μg/ml and 40 μg/ml CB were without effects. In the presence of CB, calcified bodies (coccoliths) which form in Golgi vesicles and are normally extruded through the plasma membrane were not extruded and accumulated within the cell. CB appeared to alter the membranes of Golgi vesicles containing coccoliths. DMSO (10% $\text{v}\text{v}$), the solvent for CB, was without effect on cell division and coccolith extrusion. A concentration of 20% $\text{v}\text{v}$ DMSO inhibited cell division irreversibly.     

Regulation of Bacterial Cell Division: Genetic and Phenotypic Analysis of Temperature-Sensitive, Multinucleate, Filament-Forming Mutants of Escherichia coli

Three mutants of Escherichia coli K-12 which form filaments during 42 C incubation have been characterized. The mutant strains AX621, AX629, and AX655 continued to grow and to synthesize deoxyribonucleic acid at 42 C for 150 to 180 min, after which time growth ceased. When cultures of the mutants were transferred from 42 to 28 C, septation of the filaments began after a 25- to 30-min period and continued at a greater than normal rate until no filaments remained. Addition of chloramphenicol at the time of transfer from 42 to 28 C prevented cell division in strain AX655 and caused lysis of strains AX621 and AX629. The temperature sensitivity mutation in each strain mapped near leu. For strain AX621, the mutation was specifically located between leu and nadC by P1 transduction. Properties of these strains are compared with those of other cell division mutants.     

Effect of cis-platinum(II)diamminodichloride on cell division of Hyphomicrobium and Caulobacter.

Low concentrations of the radiomimetic agent cis-platinum(II)diamminodichloride (PDD) inhibited cell division in Caulobacter crescentus (0.1 mug/ml) and Hyphomicrobium sp. strain B-522 (1.0 mug/ml) without altering the length of prosthecae. After exposure, cells of C. crescentus appeared as long filaments, whereas only the bud portion of Hyphomicrobium underwent elongation. PDD-treated cells of both species were multinucleated. After the removal of PDD by washing, filaments of C. crescentus fragmented unequally and then normal growth resumed. In Hyphomicrobium (where division involves release of swarmer cells that arise as buds on the distal ends of hyphae), potential septation sites formed in the presence of PDD remained inactive after washing. Reinitiation of cell division in this species was dependent upon the synthesis of new hyphae that could arise from either end of the elongated bud. This finding suggests that the PDD-induced lesion at a given septation site is irreversible and, upon removal of this compound, alternate sites must be synthesized for the subsequent occurrence of cell division.     

Effects of some platinum IV complexes on cell division of Escherichia coli.

A comparison was made of the effects of cis-tetrachlorodiaminoplatinum (IV) (cis-TCDPt), rans-TCDPt), and hexachloroplatinum (HCP) on growth and cell division of Escherichia coli strains D21 and D22. At or below 40 microgram/mL, cis-TCDPt inhibited cell division but not growth, DNA, or protein synthesis, although areas of increased electron density could be demonstrated in treated cells. In contrast, 40 microgram/mL of trans-TCDPt or HCP inhibited growth. Trans-TCDPt-treated cells developed condensed nucleoids; HCP-treated cells showed no obvious cytological changes to correlate with growth inhibition. Combination of cis-TCDPt with nalidixic acid, both at one-half the lowest filament-forming concentrations, resulted in formation of filaments, suggesting an additive effect. Combination of cis-TCDPt followed by ampicillin on E. coli B/r resulted in single bulges near the center of the filaments. Cis-TCDPt could therefore inhibit an initial step in the septation sequence, possibly at the level of the regulation of the hydrolytic enzymes. Whether cis-TCDPt exerts its effect by interreaction with DNA or with a membrane target is still uncertain.     

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.     

Mechanism for the Regulation of Cell Division in Agmenellum

We describe a nonlethal temperature-conditional mutant of Agmenellum quadruplicatum which allows dissociation of the processes of growth and cell division. With this system, evidence has been obtained for the regulation of one step in the process of cell division by a small effector molecule. The effector molecule is apparently released into the surrounding medium and can be obtained from lyophilized spent medium by extraction with 80% ethanol. The addition of this extract to serpentine filaments of the SN29 mutant strain stimulates cell division in these filaments and leads to the production of cells approximating normal dimensions within one generation time. The degree of stimulation of cell division is directly related to the amount of extract added. A general hypothesis is presented for the positive regulation of the initiation of cell wall and cell membrane invagination in this organism.     

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.     

Properties of a thermosensitive asporogenous filamentous mutant of Bacillus megaterium

Mutant TH14 of Bacillus megaterium ATCC 19213 is thermosensitive and defective in cell-division septation and spore formation at the restrictive temperature (39 C). As a consequence, the mutant forms multinucleate aseptate filaments and is asporogenic. The mutation does not result in any qualitative compositional changes in extractable membrane proteins. At the restrictive temperature, the mutant membrane has a reduced content of a small molecular weight protein(s). A membrane protein(s) with a molecular weight of nearly 80,000 appears to be partially derepressed in the mutant grown at the restrictive temperature. In addition, numerous unidentified spherical inclusions of fairly uniform size (diameter approximately 100 nm) are present in the cytoplasm at the restrictive temperature. They are especially concentrated at only one pole of each filament. Filamentous growth of the mutant is less sensitive to penicillin than growth in the rod form. Growth in either form is equally sensitive to d-cycloserine at the concentrations used for selection of the mutant. Temperature shift-up experiments suggest that one to two rounds of deoxyribonucleic acid (DNA) replication occur before the phenotypic expression of the mutation occurs. The septations after these replication events can be either two-division septations or a single-division septation plus a subsequent sporulation septation. This conclusion, coupled with previously reported work, supports the hypothesis that the early stages of sporulation represent a modified cell division.     

Sodium dodecyl sulfate-sensitive septation in a mitomycin C-sensitive, mtc, mutant of Escherichia coli.

A mitomycin C-sensitive, mtc, mutant of Escherichia coli has an altered cell surface and is sensitive to sodium dodecyl sulfate (SDS). The mutant, M27, formed multinucleate nonseptated filaments in the presence of a low concentration of SDS (50 microgram/ml). When the culture grown at that concentration of SDS was diluted with an SDS-free medium, the filaments began to divide at a very rapid rate after a lag of about 20 min. Chloramphenicol inhibited this recovery division when added within 10 min after SDS dilution but did not inhibit the division when added 20 min after dilution. Penicillin G at a low concentration, which is enough to cause filamentation, had virtually no effect on the recovery division of SDS-induced filaments. The division of penicillin G-induced filaments was inhibited by SDS.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Reversal by sodium chloride of envelope protein changes related to DNA replication and cell division of Escherichia coli

Further evidence is presented here for previously reported connections between the syntheses of two envelope proteins X and Y, cell division and DNA replication, respectively. On addition of 1% NaCl to an Escherichia coli temperature-sensitive mutant at 41 °C (non-permissive temperature) the phenotype, inability to synthesize DNA but continued ability to divide at 41 °C in the absence of NaCl, becomes wild-type. The syntheses of proteins X and Y also are converted to the wild-type pattern by NaCl. Furthermore, inhibition of DNA synthesis by thymidine starvation at 41 °C in the presence of NaCl changes the cell envelope proteins as with the wild-type, in contrast to altered syntheses in the absence of NaCl. The effect of nalidixic acid on the mutant and a recA strain are also studied for changes in envelope proteins. All of these changes are consistent with the originally proposed relationships.     

Microinjection of ricin entrapped in unilamellar liposomes into a ricin-resistant mutant of baby hamster kidney cells

Fluorescein-labelled Ricin was entrapped in unilamellar liposomes; 14 μg of protein was entrapped by 1 mg of lipids. Liposomes added to cells in culture in low serum medium can deliver entrapped Ricin to a Ricin-resistant mutant of baby hamster kidney(BHK)cells. Ricin entrapped in unilamellar liposomes inhibits protein biosynthesis at a concentration of 1.75 μg/ml in Ricin-resistant cells. Ricin dissolved in medium at 50 μg/ml does not affect protein synthesis in these cells.