A new mutation inEscherichia coli K12,isfA, which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli

The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1–2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K⁺ ions as the internal level of K⁺ ions is not modified significantly following glycine betaine accumulation.     

Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts

Summary We cloned the pem segment of plasmid R100 containing the two genes pemI and pemK, which are responsible for stable maintenance of R100 in dividing cells, into pHS1, a temperature-sensitive replication mutant of plasmid pSC101. We then examined the effect of the pem system on the maintenance of the resultant pem ⁺ plasmid pDOM17 in various Escherichia coli host strains upon inhibition of replication of the plasmid at a high temperature. We show that the pem ⁺ plasmid was maintained stably in the cell population and efficiently in the two hosts, km1213 (polA ^ts) and KP64 (recA), but less efficiently in others, such as W3110, C600, P3478 (polA), and SH2743 (sfiA sfiC); the rate of cell growth was reduced at or after the time when the copy number of pDOM17 was supposed to be 0 in all of the hosts examined. We also show that a large fraction of the non-viable pDOM17-free segregant cells was produced in the former two hosts, while a smaller fraction of such cells was produced in the latter hosts, in which cell division was inhibited for several generations. Based on these results and other observations, we point out that the pemK gene product has the function not to kill the plasmid-free segregant cells, but primarily to inhibit division of these segregants. Inhibition of cell division secondarily leads to death of the plasmid-free segregants very efficiently in the two particular hosts, resulting in an apparently more stable maintenance of the pem ⁺ plasmid in these two hosts than in others.     

DNA synthesis in an Escherichia coli dna B dnaC mutant.

Summary An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient strain dnaB dnaC transductants were discriminated from dnaB mutants by their inability to grow at 40° C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions.DNA synthesis was studied in strains containing dnaB, dnaC, or dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42° C of [³H]-thymidine pulselabeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42° C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein affects the dnaC function.     

Initiation of DNA replication in Escherichia coli

Summary When E. coli F⁺ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F⁺ dna-167 strain, but can do so in the F⁺ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

Altered SOS induction associated with mutations in recF, recO and recR

The SOS system of Escherichia coli aids survival following damage to DNA by promoting DNA repair while cell division is delayed. Induction of the SOS response is dependent on RecA and also on the product of recF. We show that normal induction also requires the products of recO and recR. SOS induction was monitored using a sfiA-lacZ fusion strain. Induction was delayed to a similar degree by mutation in recF, recO or recR. A similar effect was observed following overexpression of RecR from a recombinant recR ⁺plasmid. We show that the overexpression of RecR also reduces the UV resistance of a recBC sbcBC strain and of a sfiA strain, but not of a rec ⁺ sfiA ⁺strain. The implications of these data for the kinetics of DNA repair are discussed.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Effect of L-ethionine the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA⁺ Met⁺ RelA⁺ strain, nor in RecA⁺ Met⁻ RelA⁺ or RecA⁺ Met⁻ RelA⁻ mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42°C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42°C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA⁺ strain even if this compound is added to the cells several hours before irradiation.     

recA Gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites

Summary Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42° C was scored. Strains that failed to grow at 42° C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42° C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Physical mapping and characterization of the mitochondrial DNA and RNA sequences from mit- mutants defective in cytochrome oxidase peptide 1 (OXI 3).

Summary The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA ⁺ lexA ⁺-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication.The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3.Induced stable DNA replication was found to be considerably more resistant to UV irradiation than nromal replication both in a uvrA6 strain and a uvr ⁺ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.     

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae

Summary The induction of mitotic gene conversion and crossing-over inSaccharomyces cerevisiae diploid cells homozygous for thepso4-1 mutation was examined in comparison to the corresponding wild-type strain. Thepso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for thepso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of thePSO4 gene. On the other hand, thepso4-1 mutant is mutationally defective for all agents used. Therefore, thepso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that thePSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between thepso4-1 mutation and theRecA orLexA mutation ofEscherichia coli.     

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.     

Linear multimer formation of plasmid DNA in Escherichia coli hopE (recD) mutants

Summary The hopE mutants of Escherichia coli, which cannot stably maintain a mini-F plasmid during cell division, have mutations in the recD gene coding for subunit D of the RecBCD enzyme (exonuclease V). A large amount of linear multimer DNA of mini-F and pBR322 plasmids accumulates in these hopE mutants. The linear multimers of plasmid DNA in the hopE (recD) mutants accumulate in sbc ⁺ genetic backgrounds and this depends on the recA ⁺ gene function. Linear plasmid multimers also accumulated in a recBC xthA triple mutant, but not an isogenic xthA mutant or an isogenic recBC mutant. The recBC xthA mutant is defective in the conjugative type of recombination. Linear plasmid multimers were not detected in the recBC strain. We propose models to account for linear multimer formation of plasmids in various mutants.     

Prophage induction in Escherichia coli K12 cells deficient in DNA polymerase I.

Summary The induction of prophage by ultraviolet light has been measured inE. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater inpolA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than inpolA ⁺ polC single mutants. Similarly, thepolA1 mutation sensitizedtif-promoted lysogenic induction in apolA1 tif strain at 42°. In strains bearing thepolA12 mutation, which growth normally at 30°, induction of the prophage occured after the shift to 42°. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, to trigger the prophage induction process.     

cAMP inhibits bud growth in a yeast strain compromised for Ca2+ influx into the Golgi

Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to atpk ^w mutation) and bearing a lesion in a Golgi-localized Ca²⁺ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrestedtpk1 ^w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since thetpk1 ^w pmr1 double mutants retain viability. The growth defect of thetpk1 ^w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca²⁺. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of thepmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.     

Regulation and SOS induction of division inhibition in Escherichia coli K12

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

Overproduction of NAD+ and 5′-inosine monophosphate in the presence of 10 μM Mn2+ by a mutant of Corynebacterium ammoniagenes with thermosensitive nucleotide reduction (nrd ts) after temperature shift

Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction (nrd ^ts). The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures. No overproduction of NAD⁺ was detected in the control maintained at 27°C whereas NAD⁺ was accumulated extracellularily by strain CH31 at 37°C and at 40°C. As a result of the temperature shift, division-inhibited cells displayed only limited elongation. This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria. Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD⁺-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication. In contrast to the low extracellular accumulation of NAD⁺, at the non-permissive temperature of 35°C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells. The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 M Mn²⁺. Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.This publication is dedicated to Prof. Dr Hans G. Schlegel in honor of his 80th birthday.