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.     

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.     

Induction of cell division in a temperature-sensitive division mutant of Escherichia coli by inhibition of protein synthesis.

Synchronous cells of the thermosensitive division-defective Escherichia coli strain MACI (divA) divided at the restrictive temperature (42 degrees C) if they were allowed to grow at 42 degrees C for a certain period before protein synthesis was inhibited by adding chloramphenicol (CAP) or rifampicin. The completion of chromosome replication was not required for such divA-independent division. Synchronous cells of strain MACI divided in the presence of an inhibitor of DNA synthesis, nalidixic acid, if they were shifted to 42 degrees C and CAP or rifampicin was added after some time; cells of the parent strain MC6 (div A+) treated in the same way did not divide. These data suggest that coupling of cell division to DNA synthesis depends on the divA function. The ability to divide at 42 degrees C, whether or not chromosome termination was allowed, was directly proportional to the mean cell volume of cultures at the time of CAP addition, suggesting that cells have to be a certain size to divide under these conditions. The period of growth required for CAP-induced division had to be at the restrictive temperature; when cells were grown at 30 degrees C, in the presence of nalidixic acid to prevent normal division, they did not divide on subsequent transfer to 42 degrees C followed, after a period, by protein synthesis inhibition. A model is proposed in which the role of divA as a septation initiator gene is to differentiate surface growth sites by converting a primary unregulated structure, with the capacity to make both peripheral wall and septum, to a secondary structure committed to septum formation.     

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.     

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.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Analysis of the Multiseptate Potential of Bacillus subtilis

Bacillus subtilis was grown at 30 C in 15 media which produced generation times of between 200 and 40 min. A correlation was observed between the growth rate and the number of septa per cell. The time between the production of a septum and its involvement in cell division was not related to the growth rate, but, for the 15 populations, had a mean value of 138 min. Multiseptate cells became progressively evident when the organism was grown at rates in excess of a 138-min generation time.     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55 degrees C for 25 min increased by several orders of magnitude when cells grown at 37 degrees C were pre-incubated at 42 degrees, 45 degrees or 48 degrees C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Pre-incubation of cells at 48 degrees C for 30 min increased their resistance to subsequent heating at 50 degrees, 52 degrees, 55 degrees, 57 degrees or 59 degrees C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures

The heat stress response was studied in Lactobacillus helveticus PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20 degrees C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42 degrees C. Proteinase, peptidase, and acidification activities of L. helveticus PR4 were found to be higher in cells harvested when 40 degrees C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42 degrees C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55 degrees C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20 degrees C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42 degrees C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40 degrees C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.     

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.     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55°C for 25 min increased by several orders of magnitude when cells grown at 37°C were pre-incubated at 42°, 45° or 48°C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Preincubation of cells at 48°C for 30 min increased their resistance to subsequent heating at 50°, 52°, 55°, 57° or 59°C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

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

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

Some Effects of Temperature on the Growth of F Pili

The effect of temperature on the production of F pili by an F(+) strain of Escherichia coli B/r was studied by electron microscopy and by a technique involving serum-blocking power. The latter method is based on the ability of F pili to adsorb F pili antibody which inhibits male-specific phage infection. The total amount of pili in a sample was estimated by serum-blocking power; the length of F pili and number per cell was determined by electron microscopy. Cell extracts prepared by sonic oscillation lacked serum-blocking power, suggesting that F pili are not present in the cytoplasm. The number of F pili per cell varied with the growth temperature, but the average length of F pili remained constant. Maximum number of pili per cell occurs between 37 and 42 C; below 37 C the number decreases, reaching zero at about 25 C. When cells are grown at 37 C, blended, and resuspended in fresh media at 25 C, they make F pili. These pili are probably assembled from a pool of subunits that were synthesized during growth at 37 C. The rates of assembly at 25 and 37 C, as judged by the rate of increase in length of F pili, are similar. When cells were grown at 25 C and shifted up to 37 C, there was a 30-min lag in pili production followed by a period of rapid outgrowth. When cells were shifted down from 37 to 20 C, outgrowth (assembly) of pili ceased, and approximately 50% of the attached pili were released in 2 min. No release was observed when cells were shifted to 0 C. This suggests that pili may be released from the cell by a mechanism that requires metabolic activity, but not the outgrowth of F pili.     

Septum Formation in Escherichia coli: Characterization of Septal Structure and the Effects of Antibiotics on Cell Division

Septa can be demonstrated in sections of Escherichia coli strains B and B/r after fixation with acrolein and glutaraldehyde. The septum consists of an ingrowth of the cytoplasmic membrane and the mucopeptide layer; the outer membrane is excluded from the septum until the cells begin to separate. Mesosomes have also been observed. The septum is highly labile and, except in the chain-forming strains, E. coli D22 env A and CRT 97, not easily preserved by standard procedures. The labile nature of the septum may be due to the presence of autolysin(s) located at the presumptive division site. Blocking division by addition of ampicillin (2 to 5 mug/ml) to cells of E. coli B/r produces a bulge at the middle of the cells; bulge formation is stopped by addition of chloramphenicol. Cephalosporins also induce bulge formation but may stop cell elongation as well as division. Bulge formation, due to the presumed action of an autolysin(s), may be an initial step in the septation sequence when the mucopeptide is modified to allow construction of the septum. In a nonseptate filament-forming strain, PAT 84, which ceases to divide at 42 C, bulge formation only occurs in the presence of ampicillin at the time of a shift-down at 30 C or at 42 C in the presence of NaCl (0.25 to 0.34 M). Experiments with chloramphenicol suggest that the filaments are fully compartmentalized but fail to divide owing to the inactivation, rather than loss of synthesis, of an autolysin at 42 C.     

Nuclear Division in the Ameboflagellate Adelphamoeba galeacystis

Nuclear division in synchronized cultures of the ameboflagellate Adelphamoeba galeacystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stains is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronization of amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.     

Heat shock inactivates a supernatant factor(s) specifically required for efficient expression of the amp gene in Escherichia coli

A 160,000 x g supernatant of E. coli extract prepared from cells grown at 30 degrees C stimulated specifically the expression of the amp gene on pBR322 and pBR328 in an in vitro gene expression system from E. coli. This activity of the supernatant was markedly reduced when the cells were exposed to 42 degrees C for 30 min prior to preparing the supernatant. These results are consistent with the view that heat shock-induced repression of the amp gene expression is due to inactivation of a supernatant factor(s) required for effective expression of the amp gene.     

Leakage of periplasmic enzymes from lipopolysaccharide-defective mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium with defects in the heptose region of the lipopolysaccharide (LPS) molecule (heptose-deficient, chemotype Re) leak periplasmic enzymes (acid phosphatase (EC 3.1.3.2), cyclic phosphodiesterase, ribonuclease I (EC 3.1.4.22), and phosphoglucose isomerase (EC 5.3.1.9) (PGI is at least partially periplasmic in E. coli and S. typhimurium; see below)) and do not leak an internal enzyme (glucose-6-phosphate dehydrogenase) into the growth medium. The extent of this leakage is markedly increased at higher temperature (42 degrees C). Leakage of periplasmic enzymes from the strains lacking units distal to heptose I in the LPS molecule (chemotype Rd2) occurs only at 42 degrees C, and not at 30 or 37 degrees C. The extent of leakage of these enzymes from smooth strain and mutants of other LPS chemotypes (Rc, Rd1) is not significant, and is not influenced by growth temperatures. The kinetics of leakage of periplasmic enzymes after shift to 42 degrees C in nutrient broth reveal an accelerated release into the medium from heptose-deficient strains of cyclic phosphodiesterase and ribonuclease I after 30 min at 42 degrees C, and phosphoglucose isomerase after 60 min at 42 degrees C; at 30 degrees C the rate of release of cyclic phosphodiesterase and ribonuclease I is relatively slower. After 60 min at 42 degrees C in nutrient broth, growth of these strains has either slowed down or stopped. In L-broth, which permits the growth of the heptose-deficient strain (SA1377) at 42 degrees C, leakage of cyclic phosphodiesterase and phosphoglucose isomerase occurs, whereas there is no detectable leakage of these enzymes from the isogenic smooth strain (SA1355). Thus, leakage of the periplasmic enzymes from the heptose-deficient strain occurs with or without growth. Mg2+ (0.75 mM), sodium chloride (50 mM), and sucrose (100 mM) in nutrient broth at 42 degrees C prevent the leakage of these enzymes. The shedding of LPS from the heptose-deficient as well as the smooth strains is enhanced by high temperature (42 degrees C), whereas considerable leakage of protein occurs only in the heptose-deficient strain at 42 degrees C and not in the smooth strain. The smooth and heptose-deficient strains are equally sensitive to osmotic shock although a significant proportion of acid phosphatase and cyclic phosphodiesterase activities from the heptose-deficient cells grown at 42 degrees C comes off in the Tris-NaCl wash step suggesting a rather loose attachment of these enzymes onto the cell surface.     

Nuclear division in the ameboflagellate Adelphamoeba galeacystis.

Nuclear division is synchronized cultures of the ameoboflagellate Adelphamoeba galecystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stsins is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronizationof amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.     

ENVIRONMENTAL FACTORS AFFECTING RESISTANCE TO DESICCATION IN THE DIATOM STAURONEIS ANCEPS

Several environmental parameters influence the ability of Stauroneis anceps Ehr. to survive periods of dehydration to equilibrium with atmospheric humidity. Cells grown in soil-water are better able to survive desiccation than cells grown in defined medium and cells from older cultures survive better than those from young cultures. Since active culture growth and cell division do not hinder survival, the factor responsible for increased survival in older cultures may be the accumulation of secreted metabolites in the medium. There is no survival when drying occurs in artificial substrata with particles 50 microns or less in diameter. Survival occurs when the particulate matter is 100 microns or larger. Slow drying seems to enhance survival, perhaps by allowing cells to interact longer with environmental organic substances conferring some degree of protection. Desiccated cells are better able to withstand temperature extremes than are vegetative cells in an aqueous environment. Dry cells survived longer than 9 days at 60 C and longer than 8 hr at 80 C but normal cultures were unable to survive 1 hr at 60 C. Temperatures of —15 to —20 C were also sustained more consistently by desiccated cells. Cells stored at vapor pressure deficits of 11.9 mm Hg or higher survived longer than 16 months but storage at 5.9 mm Hg or lower reduced survival time.     

Response of Lactobacillus helveticus PR4 to Heat Stress during Propagation in Cheese Whey with a Gradient of Decreasing Temperatures

The heat stress response was studied in Lactobacillus helveticus PR4 during propagation in cheese whey with a gradient of naturally decreasing temperature (55 to 20°C). Growth under a gradient of decreasing temperature was compared to growth at a constant temperature of 42°C. Proteinase, peptidase, and acidification activities of L. helveticus PR4 were found to be higher in cells harvested when 40°C was reached by a gradient of decreasing temperature than in cells grown at constant temperature of 42°C. When cells grown under a temperature gradient were harvested after an initial exposure of 35 min to 55°C followed by decreases in temperature to 40 (3 h), 30 (5 h 30 min), or 20°C (13 h 30 min) and were then compared with cells grown for the same time at a constant temperature of 42°C, a frequently transient induction of the levels of expression of 48 proteins was found by two-dimensional electrophoresis analysis. Expression of most of these proteins increased following cooling from 55 to 40°C (3 h). Sixteen of these proteins were subjected to N-terminal and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. They were identified as stress proteins (e.g., DnaK and GroEL), glycolysis-related machinery (e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase), and other regulatory proteins or factors (e.g., DNA-binding protein II and ATP-dependent protease). Most of these proteins have been found to play a role in the mechanisms of heat stress adaptation in other bacteria.