DNA synthesis in P1 infected E. coli mutants temperature-sensitive in DNA replication

Summary P1 DNA is synthesized in the E. coli ts dna mutants 165/70 (elongation defect) and 252 (initiation defect) at elevated temperatures. In strain 165/70, P1 infection at 41°C leads to phage production accompanied by a transient recovery of bacterial DNA synthesis. No phages are produced byt P1 DNA is still synthesized in strain 252 if infected after host DNA replication has come to a halt at 42°C.     

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12

Summary A mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.     

The fate of newly made DNA in Escherichia coli mutants thermosensitive in DNA synthesis

Summary E. coli mutants exist in which DNA synthesis is thermosensitive. In one class of these mutants DNA synthesis stops immediately if a critical temperature (42°C) is reached. When DNA replication in such mutants is followed by ³H thymidine incorporation at 33°C, it is found that 1. only the newly made DNA is degraded at 42°C, 2. the discontinuously replicated DNA is lost predominantly at 42°C, 3. 1–3% of the chromosomal DNA is rendered acid soluble at 42°C without concomitant loss of viability of the cells at 33°C.Replication of phage DNA is inhibited in the same mutant at 42°C. However, when DNA synthesis is followed in infected cells at 33°C it is found that 1. no degradation of specific DNA seems to occur at 42°C in the early phase of infection, 2. replicating DNA molecules in the late phase of infection are completed at 42°C before DNA synthesis comes to a halt.     

Temperature sensitive initiation of DNA synthesis in a mutant of Salmonella typhimurium

Summary A mutant (dna-1) of Salmonella typhimurium defective in DNA synthesis is described. DNA synthesis is stopped in this mutant at 42° after a residual synthesis amounting to about 50 to 60% of the total cellular DNA in minimal medium and about 120 to 200% in a medium enriched with amino acids. Reshift back to permissive temperature after the inhibition of DNA synthesis at 42° allows for recovery of DNA synthesis after a lag of about 30 min. Protein synthesis is required during that lag for the recovery of DNA synthesis at permissive temperature. The density transfer experiments indicate that in the mutant dna-1 chromosome termini are replicated normally at 42° while the initiation of new rounds of replication is inhibited although the mutation is probably leaky at this temperature. The mutant is hypersensitive to sodium deoxycholate at 42° which suggests alteration of the membrane structure.     

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe

Summary A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25°C but become blocked at DNA replication or mitosis when incubated at 36°C. Thirtynine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambigously assigned to either DNA replication or mitosis but were definitely required for one or the other.Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36°C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons.A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.     

Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Summary The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42° C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30° C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA ⁺, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42° to 30° C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42° C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA ⁺ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.A preliminary account of this work was presented at the Annual Meeting of the Molecular Biology Society of Japan in 1989.     

Evidence for an early regulatory function in phage P22

Summary A temperature sensitive mutant of P22 phage (ts X) was isolated and studied.This mutant seems to have a basic regulatory function: it is defective in an early function like the typical DNA^- mutant ts 12.1; it is unable to direct the phage DNA synthesis and does not lyse infected or induced cells.Unlike ts 12.1, the mutation ts X seems to involve a gene product necessary for the expression of any vegetative function, since no phage protein synthesis, no alteration of host DNA synthesis, and no cell killing can be observed under non-permissive conditions.The possible functional similarity between the N-cistron of the phage and the present X-cistron in P22 is discussed.     

Induction of prophage lambda in a mutant of E. coli K12 defective in initiation of DNA replication at high temperature

Summary Prophage is not induced when DNA synthesis ceases at 42°C in a mutant of E. coli which is unable to initiate rounds of DNA replication at high temperature. However, induction occurs when the cells are UV irradiated after completion of rounds of replication at 42°C. Evidence is presented that the uvr functions, necessary for dimer excision, are not required for this induction, and that the UV irradiation itself does not provoke net host DNA synthesis under these conditions. We conclude that prophage induction can result from irradiation damage in chromosomes that are unable to replicate.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12.

Summary A strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.     

Regulation of replication of λ phage and λ plasmid DNAs at low temperature

It was previously demonstrated that while lysogenic development of bacteriophage λ in Escherichia coli proceeds normally at low temperature (20–25° C), lytic development is blocked under these conditions owing to the increased stability of the phage CII protein. This effect was proposed to be responsible for the increased stimulation of the p _E promoter, which interferes with expression of the replication genes, leading to inhibition of phage DNA synthesis. Here we demonstrate that the burst size of phage λcIb2, which is incapable of lysogenic development, increases gradually over the temperature range from 20 to 37° C, while no phage progeny are observed at 20° C. Contrary to previous reports, it is possible to demonstrate that p _E promoter activation by CII may be more efficient at lower temperature. Using density-shift experiments, we found that phage DNA replication is completely blocked at 20° C. Phage growth was also inhibited in cells overexpressing cII, which confirms that CII is responsible for inhibition of phage DNA replication. Unexpectedly, we found that replication of plasmids derived from bacteriophage λ is neither inhibited at 20° C nor in cells overexpressing cII. We propose a model to explanation the differences in replication observed between λ phage and λ plasmid DNA at low temperature. Received: 30 December 1997 / Accepted: 25 February 1998     

Size distribution and molecular polarity of nascent DNA in a temperature-sensitive dna G mutant of Escherichia coli

Summary In the dna G t.s. strain BT 308, made lysogenic for the phage , nascent DNA was labelled by short pulses of ³H-thymidine, isolated and separated as a function of size by alkaline sucrose gradient sedimentation. The molecular polarity of the labelled DNA was then determined by hybridization to the separated strands of DNA.At 30° C, strand r DNA, made in the direction opposite that of fork movement, is synthesized in the form of short pieces. The first observable consequences of a shift to 42° C are the preferential inhibition of strand r synthesis and the small amount of strand r DNA which is made is recovered in long pieces of DNA rather than in short fragments. This indicates that the t.s. product, in strain BT 308, may be involved in the synthesis of the strand growing in the direction opposite that of replication fork movement.Newly synthesized strand l DNA, made in the same direction as replication fork movement, is found in long pieces in wild-type bacteria; it is found in pieces of intermediate size in strain BT 308 at 30° C as well as at 42° C. This indicates additional differences in the replication machinery between strain BT 308 and wild-type bacteria.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

Strand-specific DNA degradation in a mutant of Escherichia coli

Summary A dna B mutant of Escherichia coli which is thermosensitive for DNA synthesis at 42° C degrades DNA at the restrictive temperature. The degradation specifically affects newly synthesized DNA, begins at the replication forks and proceeds toward the replication origin, and is limited to 10–15% of one chromosome. The parameters of DNA degradation, as well as DNA-DNA annealing experiments on newly synthesized DNA which is resistant to degradation, indicate a specific strand of newly synthesized DNA is degraded.     

Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coli K-12

Summary The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30°C, whereas at 42°C DNA replication ceased after the DNA content increased only 40–45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42°C were isolated from the double mutant. The suppressor mutant retained all other recA ^– characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA ⁺ dependency of stable DNA replication. It is suggested that the recA ⁺ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.     

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.     

Alteration of the rate of cell division independent of the rate of DNA synthesis in a mutant of Salmonella typhimurium

Summary Salmonella typhimurium strain IIG has a temperature—sensitive DNA synthesis initiation apparatus and completes rounds of DNA replication when shifted to 38°. At this temperature there is a period of apparently normal division followed by a second phase in which DNA-less cells are produced. The rate of division in this second phase can be markedly increased if a culture growing in MM is shifted to nutrient broth at the time of the temperature shift. The extra divisions induced by the nutritional shift are not due to extra replication forks being introduced by this process nor to the rapid growth of ts ⁺ revertants. It is concluded that in this strain at 38°, the rate of division can be increased without altering the rate of DNA synthesis. The extra divisions induced by the shift-up do not take place for about 90 min. The possible occurrence of such a period between the triggering of division and the division event in normal cells is discussed.     

Role of DNA replication in the induction and turn-off of the SOS response in Escherichia coli

Summary We have studied the role of DNA replication in turnon and turn-off of the SOS response in Escherichia coli using a recA::lac fusion to measure levels of recA expression.An active replication fork does not seem to be necessary for mitomycin C induced recA expression: a dnaA43 initiation defective mutant, which does not induce the SOS response at non-permissive temperature, remains mitomycin C inducible after the period of residual DNA synthesis. This induction seems to be dnaC dependent since in a dnaC325 mutant recA expression not only is not induced at 42° C but becomes mitomycin C non-inducible after the period of residual synthesis.Unscheduled halts in DNA replication, generally considered the primary inducing event, are not sufficient to induce the SOS response: no increase in recA expression was observed in dnaG(Ts) mutants cultivated at non-permissive temperature. The replication fork is nonetheless involved in induction, as seen by the increased spontaneous level of recA expression in these strains at permissive temperature.Turn-off of SOS functions can be extremely rapid: induction of recA expression by thymine starvation is reversed within 10 min after restoration of normal DNA replication. We conclude that the factors involved in induction-activated RecA (protease) and the activating molecule (effector)-do not persist in the presence of normal DNA replication.Abbreviations Ts thermosensitive - SDS sodium dodecyl sulfate - Ap ampicillin - UV ultraviolet - X-Gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

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.