Reinitiation of chromosome replication in a thermosensitive DNA mutant of Escherichia coli. II. Synchronization of chromosome replication after temperature shifts

A short incubation at the non-permissive temperature, 10 to 15 minutes at 40 °C, suffices to induce chromosome reinitiation in CRT 266, a thermosensitive DNA mutant of Escherichia coli. In order to acquire the potentiality to reinitiate chromosome replication, protein synthesis is necessary, both during the 40 °C incubation and also during the first 15 minutes after returning to 30 °C.     

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants

The genetic aspects of ori C replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dna A cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dna A integrated at the att B locus in dna A null background was exchanged with an incoming plasmid bearing a mutagenized dna A gene. Next, the mutants that were defective for growth at 30°C, a non-permissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dna A gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dna A expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dna Acos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dna A promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

Temperature-induced Changes in the Synthesis of Unsaturated Fatty Acids by Acanthamoeba castellanii

ABSTRACT. Major fatty acid components of Acanthamoeba castellanii lipids extracted after growth at 30°C include myristate, palmitate, stearate and the polyunsaturates linoleate, eicosadienoate, eicosatrienoate and arachidonate, with oleate as the sole major monounsaturated fatty acid. By comparison, growth at 15°C gave increased linoleate, eicosatrienoate and arachidonate, but decreased oleate and palmitate. When the growth temperature was shifted downwards from 30°C to 15°C, increased lipid unsaturation occurred over a period of 24 h; thus decreases of oleate and eicosadienoate were accompanied by increases in linoleate, eicosatrienoate, arachidonate and eicosapentaenoate. An upwards shift from 15°C to 30°C gave negligible alterations in fatty acid composition over a similar period. At 15°C organisms rapidly use [1-¹⁴C] acetate for de novo fatty acid synthesis; stearate is converted via oleate to further desaturation and chain elongation products. Similar short term experiments at 30°C indicate only de novo synthesis and Δ9-desaturation; synthesis of polyunsaturates was a much slower process. Rapid incorporation of [1-¹⁴C] oleate at 30°C was not accompanied by metabolic conversion over two hours, whereas at 15°C n-6 desaturation to linoleate was observed. Temperature shift of organisms from 15°C to 30°C in the presence of [1-¹⁴C] acetate revealed that over half of the fatty acids in newly-synthesised lipids were saturated, but the proportions of unsaturated fatty acids increased with time until the total polyenoate components reached 17% after 22 h. A shift of temperature in the reverse direction gave a corresponding figure of 60% for polyunsaturated fatty acids. These results emphasize the importance of n-6 desaturation in the low temperature adaptation of Acanthamoeba castellanii .     

DNA replication in Escherichia coli 15T- growing at 20 degrees C

Escherichia coli 15T⁻ grows slowly in succinate or aspartate-M9 media. In both media, a gap in DNA replication is observed at 37 °C which is either not present at 20 °C or of very much shorter duration than at 37 °C. However, dichotomous replication is not observed in glucose M9 at 20 °C. The results suggest that initiation of replication in glucose is different from that in aspartate or succinate cultures.     

DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10

A hypermodified base (Y-Thy) replaces 20% of the thymine (Thy) in mature DNA of Bacillus subtilis phage SP10. Two noncomplementing hypermodification-defective (hmd) mutants are described. At 30°C, hmd phage carried out a normal program, but at temperatures of ≥37°C, the infection process was nonproductive. When cells were infected at 37°C with hmd phage, DNA synthesis started at its usual time (12 min), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under hmd conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodified nascent strands. The reduced levels of DNA synthesis observed under hmd conditions were accompanied by weak expression of late genes. A sucrose gradient analysis of SP10 hmd⁺ replicating DNA intermediates was made. Two intermediates, called VG and F, were identified. VF consisted of condensed DNA complexed to protein; VF also contained negatively supercoiled domains covalently joined to relaxed regions. F was composed of linear concatenates from which mature DNA was cleaved. None of those intermediates was evident in cells infected at 37°C with hmd phage. Shiftup experiments were performed wherein cells infected with hmd phage at 30°C were shifted to 37°C at a time when replication was well under way. DNA synthesis stopped or declined manyfold 10 min after shiftup. The hmd DNA made after shiftup was conserved as a form sedimentationally equivalent to the F intermediate, but little mature DNA was evident. It is proposed that Y-Thy is required for replication and DNA maturation because certain key proteins involved with these processes interact preferentially with hypermodified DNA.     

Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15°C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28°C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28° and 15°C for 6 d but there was no growth at 10°C after 50 d. At 28°C aflatoxins were detected at a concentration of 132 μg/g after 13 d, the highest level obtained. In cheese paste at 28° and 15°C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10°C the growth was heavy, but aflatoxins were not detected.     

Influence of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers: does otolith growth cease at low temperatures?

The influences of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers were investigated using individuals reared at 5, 10, 15, 20, 25 and 30° C and in fed or unfed conditions at salinity 32 after their otoliths were marked with alizarin complexone (ALC). To eliminate the difficulty of observing the edges of otoliths with optical (OM) or scanning electron (SEM) microscopes, three to 10 individuals were sampled from each tank at 10, 20 and 30 days during the experiment and reared for an additional 10 days at 25° C after their otoliths were marked a second time. Otolith growth and the number of increments were measured using both OM and SEM. Most A. japonica commenced feeding after 10 days at 20–30° C or after 20 days at 15° C, but no feeding occurred at 5 and 10° C. No otolith growth occurred at 5 and 10° C except in two individuals with minimal increment deposition at 10° C. Otolith growth was proportional to water temperature within 15–25° C and not different between 25 and 30° C. At 15, 25 and 30° C, the mean otolith growth rate in fed conditions was higher than in unfed conditions. The number of increments per day was significantly different among water temperatures (0·00–0·01 day⁻¹ at 5 and 10° C, 0·43–0·48 day⁻¹ at 15° C and 0·94–1·07 day⁻¹ at 20–30° C). These results indicated that otolith growth in A. japonica glass eels and elvers was affected by temperature and ceased at ≤10° C under experimental conditions. Hence, future studies analysing the otoliths of wild-caught A. japonica glass eels and elvers need to carefully consider the water temperatures potentially experienced by the juveniles in the wild.     

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (k_pol) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K_d for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10⁻⁴) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10⁻³ at 72 °C which is four-fold higher than that of 37 °C.     

Diapause in a Glasgow strain of the flour moth, Ephestia kuehniella

ABSTRACT. The incidence of diapause in Ephestia kuehniella Zeller from an unhealed granary in Scotland was influenced by both photoperiod and temperature. At 25°C, nearly 50% of larvae entered diapause when reared in continuous darkness (DD) and up to 30% did so in short photoperiods. Little diapause was detected around LD 14:10 but a second, smaller peak of about 20% occurred at LD 16:8 and LD 18:6, falling away again to nearly zero in continuous light. More larvae entered diapause when reared continuously at 15 or 20°C than at 25 and 30°C. However, when larvae reared from hatch at 25°C in LD 16:8 were transferred after 1 week to 15°C in LD 9:15, almost twice as many entered diapause as did those reared at 15°C throughout. The sensitive phase for diapause induction occurred near the start of the final instar. The mean duration of diapause was between 2 and 3 months in most photoperiods at 20 and 25°C, and was shorter at 15°C. However, in DD at 25°C, it lasted about 7 months. Termination of diapause was hastened in larvae reared at 25°C in DD by transferring them to LD 14:10, and also by chilling them at 7.5°C for 6 weeks before returning to 25°C in DD. In an unhealed store in southern England, viable adults emerged from May to July and originated from larvae which terminated diapause relatively late. It would appear from the results of transferring larvae back to the laboratory at various times during the winter that some phases of diapause development were completed quite early after exposure to low temperatures, although no further development took place in the store until temperatures rose again in April.     

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

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.     

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive ( ts ) mutants. Six of these ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 28 and tsm 30) failed to replicate in mice and were avirulent. Five mutants ( tsm 14, tsm 18, tsm 19, tsm 25 and tsm27 ) were to similar virulence to the parenthal wild-type ( wt ) virus, five ( tsm 7, tsm 15, tsm 24, tsm31 ) were 12–100 fold less virulent, five ( tsm 8, tsm 12, tsm 16, tsm 23 and tsm 29) were 150–1500 fold less virulent and four ( tsm 10, tsm 11, tsm 17 and tsm 21) were between 2,000 and 85,000 fold less virulent than wt . One mutant ( tsm 28) did not plaque or replicate at 39°C while 5 other mutants ( tsm 7, tsm 9, tsm 23, tsm 24 and tsm 27) also failed to plaque at 39°C but only failed to replicate or replicated poorly at 40°C. A further two mutants ( tsm 10 and tsm 13) were able to plaque and replicate at 39°C but not 40°C. Six other mutants ( tsm 14, tsm 15, tsm 16, tsm21 , tsm 22 and tsm 30) failed to form plaques at 40°C and were severely restricted in their replication at 40°C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.     

Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: Stable DNA replication

After inhibiting DNA synthesis in Escherichia coli, repeated cycles of chromosome replication can occur in the absence of protein synthesis. This “stable” replication requires the products of all of the known dna genes.Stable replication results from inhibiting DNA synthesis by treatment with naladixic acid, cytosine arabinoside or hydroxyurea; or by placing dnaB, dnaE or dnaG mutants at non-permissive temperatures. It also follows a “shift-up” into rich medium in which RNA and protein are synthesized more rapidly than DNA. Paradoxically, stable replication is induced also by treatment with concentrations of streptolydigin which do not inhibit DNA replication but temporarily and partially inhibit RNA and protein synthesis. During all of these treatments, some protein synthesis must occur.Stable replication is not immediately expressed after a short period of thymine starvation or streptolydigin treatment, but requires a subsequent period of protein synthesis. Once established, however, the stable replication state is permanent and will persist in the absence of protein synthesis or during normal growth.After stable replication has been determined by a period of DNA inhibition, it is possible to inactivate replication by heating dnaA, B, C, E and G temperature-sensitive mutants. However, resynthesis of these gene products in the presence of thymine and at the permissive temperature restores stable replication activity. Since restoration of activity can occur under normal growth conditions which do not induce stable replication, it was concluded that the dnaA, B, C, E and G gene products do not directly determine the stabilized character of the replication fork.A model is presented which attempts to explain the ability of different treatments to induce stable replication.     

Dormancy and germination of olive embryos as affected by temperature

Olive seeds do not germinate promptly when placed under favourable conditions, which is a problem in raising young plants for breeding or experimental purposes. In a series of experiments an investigation of the role of temperature in the germination of olive embryos was conducted. Naked, unchilled olive embryos ( Olea europaea L. cv. Chalkidikis), cultured in vitro at 20°C, had a germination capacity of 73%, whereas that of embryos which had previously been chilled at 10°C for 2 or more weeks reached 96%. Intact seeds did not germinate at 20°C unless they had previously been subjected to 10°C for 3 or 4 weeks. Embryos chilled while in the intact seed and excised just before transfer to 20°C, reacted in a similar way to naked embryos, but reached their maximum germination capacity after 4 weeks at 10°C. Under constant temperature conditions the highest germination percentage of embryos was observed at 10 and 15°C and the highest germination rate at 15°C, while a moderate capacity and rate of germination occurred at 20°C, and a very low percentage and rate at 25 and 30°C. Prechilling at 10°C did not affect germination at 15°C, but improved the percentage and the rate of germination at 20, 25 and 30°C. The germination percentages of embryos chilled for 1 or 2 weeks at 10°C and then transferred to 25°C were lower than those of similarly chilled embryos transferred to 20°C. The chilling effect could not be reversed at 25°C when the embryos had been chilled for 3 or more weeks. The results show that olive seeds exhibit a state of dormancy that is caused by factors residing partly in the endosperm and partly within the embryo.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute