Control of Deoxyribonucleic Acid Synthesis in a Bacillus subtilis Mutant Temperature Sensitive for Initiation of Chromosome Replication

We used a Bacillus subtilis mutant described previously, which is temperature sensitive for initiation of replication. The inhibition of deoxyribonucleic acid synthesis occurring at 45 C was shown to be reversed when the temperature is lowered even in the absence of protein synthesis. If the bacteria are returned to 30 C, after a prior period at 45 C, they are able to initiate the first round of replication in the presence of chloramphenicol, but the initiation of the second round still requires protein synthesis. This paper shows that the proteins necessary to initiate the second round of replication can be present in bacteria long before this round is initiated. In addition, the appearance of these proteins seems to be influenced by the length of the previous 45 C period. Although similar reinitiation kinetics are observed at 30 C after prior 45 C periods of 30 or 65 min, the ability to initiate the second round without further protein synthesis appears much earlier after a longer exposure at 45 C. To explain these results, a hypothesis is presented which assumes that two different proteins are both necessary for initiation. Only one of these proteins could be accumulated at 45 C during the inhibition of deoxyribonucleic acid synthesis. A peculiarity of initiation material in mutant Ts 37 is that it may be active at 45 C if it has been exposed previously at 30 C.     

Mutant of Escherichia coli with Thermosensitive Protein in the Process of Cellular Division

A new thermosensitive mutant of Escherichia coli deficient in cell division was isolated by means of membrane filtration after nitrosoguanidine mutagenesis. The mutant cells grow normally at 30 C but stop dividing immediately after shift to 42 C, resulting in multinucleated filaments lacking septa. The number of colony-forming units does not decrease for at least 6 hr at 42 C. The maximum length of the filaments is 10 to 16 times that of normal cells. Addition of a high concentration of NaCl fails to stimulate cell division at 42 C. The filaments formed at 42 C divide abruptly 30 min after shift to 30 C, and synchronous increase of cell number is shown for 3 hr. The macromolecular synthesis of protein and nucleic acids at 42 C is normal on the whole. The cell division shown after the shift from 42 to 30 C is observed in the absence of thymine, but not in the presence of chloramphenicol or in a medium deficient in amino acids. However, the filament can divide to some extent in the presence of chloramphenicol if some protein synthesis is allowed to proceed at 30 C before the addition of the antibiotic. The elongated cells divide at 42 C provided that they are exposed to 30 C before being shifted to high temperature.     

Cell growth and division in Escherichia coli: a common genetic control involved in cell division and minicell formation

Escherichia coli Div 124(ts) is a conditional-lethal cell division mutant formed from a cross between a mutant that produces polar anucleated minicells and a temperature-sensitive cell division mutant affected in a stage of cross-wall synthesis. Under permissive growth temperature (30 C), Div 124(ts) grows and produces normal progeny cells and anucleated minicells from its polar ends. When transferred to nonpermissive growth temperature (42 C), growth and macromolecular synthesis continue, but cell division and minicell formation are inhibited. Growth at 42 C results in formation of filamentous cells showing some constrictions along the length of the filaments. Return of the filaments from 42 to 30 C results in cell division and minicell formation in association with the constrictions and other areas along the length of the filaments. This gives rise to a "necklace-type" array of cells and minicells. Recovery of cell division is observed after a lag and is followed by a burst in cell division and finally by a return to the normal growth characteristic of 30 C cultures. Recovery of cell division takes place in the presence of chloramphenicol or nalidixic acid when these are added at the time of shift from 42 to 30 C, and indicates that a division potential for filament fragmentation is accumulated while the cells are at 42 C. This division potential is used for the production of both minicells and cells of normal length. The conditional-lethal temperature sensitive mutation controls a step(s) in cross-wall synthesis common to cell division and minicell formation.     

Initiation of deoxyribonucleic acid replication in a temperature-sensitive mutant of B. subtilis: evidence for a transcriptional step

A mutant of Bacillus subtilis unable to initiate a new round of replication at 45 C has been described. Here we show that inhibition of DNA synthesis in this mutant is reversible and that DNA synthesis is resumed at low temperature, even in the presence of chloramphenicol. Initiation of a new replication cycle thus can occur in the absence of protein synthesis. A thermolabile component required for initiation therefore appears to be synthesized at 45 C in an inactive form and can be activated at 30 C in the presence of an inhibitor of protein synthesis. Although resistant to chloramphenicol, the reinitiation of replication occurring after lowering the temperature is sensitive to rifampin and streptolydigin.     

Formation of Filaments and Synthesis of Macromolecules at Temperatures Below the Minimum for Growth of Escherichia coli

When Escherichia coli ML30 was transferred during exponential growth at a temperature near the minimum for growth to temperatures just below the minimum for growth, optical density increased for a considerable period of time and considerable synthesis of ribonucleic acid, deoxyribonucleic acid, protein and mucopeptide also occurred. Synthesis of deoxyribonucleic acid was inhibited slightly before the cessation of synthesis of other macromolecules. At 6 C, filaments up to 300 mu in length were formed. Cross walls were not formed, but on transfer to 30 C the filaments rapidly fragmented into short, single cells. The filaments had abundant nuclear material distributed along their length, in contrast to filaments formed by E. coli 15T(-) in the absence of thymine. There was evidence for false division points and incomplete septum formation.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

Cell Division of Escherichia coli BUG-6: Effect of Varying the Temperature Used as the Nonpermissive Growth Condition

When Escherichia coli BUG-6 is shifted from 30 C to 36 or 38 C, division does not stop, but the rate of division of the cell population is initially decreased followed by a period of increased rate of division before the rates characteristic of growth at 36 and 38 C are obtained. After a shift from 30 to 40 C, the rate of cell division gradually decreases over a 10-min period and then stops. The inhibition continues for 25 min, and then the cells divide rapidly before the division rate characteristic of 40 C is obtained. If filaments produced by 45 min of growth at 42 C are temporarily replaced at 30 C and then returned to 42 C, division occurs at 42 C. The amount of division is dependent on the length of the period at 30 C and can be decreased by a 3-min pulse of chloramphenicol immediately before the 42 to 30 C shift.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

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.     

Regulation of Cell Division in Escherichia coli: Characterization of Temperature-Sensitive Division Mutants

A temperature-sensitive division mutant of Escherichia coli was isolated by using differential filtration to select for filaments at 42 C and normal cells at 30 C. Cells shifted from 30 to 42 C stop dividing almost immediately, suggesting the temperature-sensitive element is required for cell division late in the cell cycle. Cells returned to 30 from 42 C divide abruptly, suggesting accumulation of division potential at 42 C. Inhibitors of protein, deoxyribonucleic acid, and ribonucleic acid synthesis do not block division during the recovery period at 30 C. Cycloserine does not stop cell division, vancomycin shows some effect on cell division, whereas penicillin completely stops cell division during this period. The addition of high concentrations of NaCl to filaments at 42 C results in a burst of cell division. The final cell number is equivalent to the control which is grown at 30 C if sufficient salt is added (11 g/liter, final concentration). After the original burst, cell division ceases at the nonpermissive temperature even at increased osmolality. Chloramphenicol, puromycin, vancomycin, and penicillin prevent division during the recovery in the presence of NaCl. Kinetic data indicate division potential decays to a reversible inactive intermediate which rapidly decays to an irreversible inactive form. Conversion of division potential to the inactive form is correlated with a 100- to 1,000-fold derepression of the synthesis of division potential. The mutation appears to involve a stage in cross-wall synthesis which is required during the terminal stages of division.     

Ampicillin induced septum formation in Bacillus cereus

Cells of Bacillus cereus grown in the presence of subinhibitory concentrations of ampicillin at either 30 degrees or 45 degrees C exhibited an increase in the numbers of centres of septum formation per unit cell length. Under identical conditions of cultivation, cells of Escherichia coli grew as aseptate filaments. In general, untreated B. cereus cells grown at 45 degrees C were longer than those grown at 30 degrees C. The strain of E. coli used was unaffected in terms of filamentation by elevated growth temperature. Results are discussed in terms of the presence and availability of penicillin binding proteins and autolysins involved in cell growth, division and separation.     

Growth, Cell Division, and Fragmentation in a Species of Flexibacter

Flexibacter FS-1, a gram-negative gliding bacterium was grown in liquid culture as long (over 100-mum) filaments. The filaments possessed a triple-track wall which resembled that found in other gram-negative bacteria. Although phase-contrast microscopy indicated that the long filaments were nonseptate, electron microscopy revealed three or four septa along the length of each filament. The septa contained lysozyme-sensitive, electron-opaque material, presumed to be peptidoglycan, sandwiched between cell membranes. The outer triple track wall was not part of the septum. Mesosomes were seen in various areas of the cell and frequently were observed attached to septa in different stages of completion. Studies of the organism in slide culture revealed that individual filaments grew in an exponential fashion and divided in the middle despite the long length and multiseptate condition. When the temperature of a liquid culture growing exponentially with a generation time of 90 minutes was shifted from 30 to 35 C, the filaments fragmented into three or four shorter cells within 120 min. The short cells continued to grow exponentially at 35 C at approximately the same rate as at 30 C. When the culture was shifted back to 30 C, the cells immediately stopped dividing and began to elongate. After a period of 2 to 3 hr, cell division resumed. It is suggested that the shift-up in temperature induced the completion of the cross wall (centripetal growth of the triple-track wall) and cell separation at the sites of previously formed septa, whereas the shift-down in temperature caused a transient inhibition of cross-wall formation but not of growth. Fragmentation was inhibited by sodium azide but took place despite the inhibition of protein synthesis by chloramphenicol or the inhibition of deoxyribonucleic acid synthesis by mitomycin C.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Duplication of Escherichia coli during inhibition of net phospholipid synthesis.

In Escherichia coli BB26-36, the inhibition of net phospholipid synthesis during glycerol starvation affected cell duplication in a manner that was similar in some respects to that observed during the inhibition of protein synthesis. Ongoing rounds of chromosome replication continued, and cells in the D period divided. The initiation of new rounds of chromosome replication and division of cells in the C period were inhibited. Unlike the inhibition of protein synthesis, however, the accumulation of initiation potential in dnaA and dnaC mutants at the nonpermissive temperature was not affected by the inhibition of phospholipid synthesis. Furthermore, proteins synthesized during the inhibition of phospholipid synthesis can be utilized later for division. The results are consistent with a dual requirement for protein and phospholipid synthesis for both the inauguration of new rounds of chromosome replication and the initiation of septum formation. Once initiated, both processes progress to completion independent of continuous phospholipid and protein synthesis.     

Temperature control of initiation of protein synthesis in Escherichia coli

When an exponentially growing culture of Escherichia coli is cooled to below 8 °C, initiation of protein synthesis appears to be blocked, while the elongation of initiated proteins continues until they are completed. This is demonstrated here by showing that nascent polypeptide chains increase in size during a 5 °C incubation and that f2 viral coat protein is completed, but not initiated. Upon rewarming, the cells initiate protein synthesis synchronously. This is demonstrated by a transient rise in the incorporation of methionine which is used to initiate protein synthesis.     

Process of cellular division in Escherichia coli: physiological study on thermosensitive mutants defective in cell division

Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described.     

Timing and other features of the action of the ts1 division initiation gene product of Bacillus subtilis.

The ts1 division initiation mutation of Bacillus subtilis 160 was transferred into a thymine-requiring strain of B. subtilis 168. Aspects of the role and timing of the action of the ts1 gene product in relation to septum formation were studied by comparing the behavior of this new strain with that of the isogenic wild type after outgrowth of germinated spores. The ts1 gene product was shown to be required for the asymmetric division which occurs in the absence of chromosome replication, in addition to normal division septation. The time interval between completion of the action of the ts1 gene product and initiation of the first central division septum was estimated to be less than 4 min at 34 degrees C, and it is possible that an active ts1 gene product is required until the commencement of septal growth. Recovery of septa after transfer of outgrown spores (filaments) from the nonpermissive to the permissive temperature was also examined. During recovery, septa formed at sites which were discrete fractional lengths of the filaments, with the first septum located at the most polar of these sites. The data have been interpreted in terms of the formation of potential division sites at the nonpermissive temperature and the preferred utilization, upon recovery, of the most recently formed site. Recovery of septa at the permissive temperature occurred in the absence of DNA synthesis but was blocked completely by inhibitors of RNA and protein synthesis. It is possible that the only protein synthesis required for recovery of septa is that of the ts1 gene product itself.     

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.     

The physics of filopodial protrusion

Filopodium, a spike-like actin protrusion at the leading edge of migrating cells, functions as a sensor of the local environment and has a mechanical role in protrusion. We use modeling to examine mechanics and spatial-temporal dynamics of filopodia. We find that >10 actin filaments have to be bundled to overcome the membrane resistance and that the filopodial length is limited by buckling for 10-30 filaments and by G-actin diffusion for >30 filaments. There is an optimal number of bundled filaments, approximately 30, at which the filopodial length can reach a few microns. The model explains characteristic interfilopodial distance of a few microns as a balance of initiation, lateral drift, and merging of the filopodia. The theory suggests that F-actin barbed ends have to be focused and protected from capping (the capping rate has to decrease one order of magnitude) once every hundred seconds per micron of the leading edge to initiate the observed number of filopodia. The model generates testable predictions about how filopodial length, rate of growth, and interfilopodial distance should depend on the number of bundled filaments, membrane resistance, lamellipodial protrusion rate, and G-actin diffusion coefficient.