Site-specific mutagenesis in cells with normal DNA repair systems: transitions produced from DNA carrying a single O6-alkylguanine.

This paper describes a systematic study of transition frequencies produced in vivo when a homologous series of O6-alkylguanine residues located at a preselected position in gene G of phi X174 form I' DNA (double-stranded, circular, covalently-closed, relaxed) is transfected into spheroplasts from two strains of Escherichia coli having normal DNA repair systems. Mutant frequencies were measured as percent of total phage produced by single bursts. The results are: (A) Synthetic DNA without any alkyl group gave a transition frequency of 0.02%. (B) In E. coli AB1157, the frequencies fall into two groups depending on the alkyl group: methyl and ethyl, 8-11%; n-propyl and n-butyl approximately 0.9%. (C) The average transition frequencies were higher in AB1157 than in C600. These data demonstrate that a single O6-alkylguanine residue can produce a specific transition at significant frequencies in cells with normal repair systems and that the mutant frequency depends upon the nature of the alkyl group and the cell type.     

Enhanced survival of gamma-irradiated Escherichia coli following pretreatment with dithiothreitol

Survival of three strains of Escherichia coli K12 was studied with respect to radiation protection by dithiothreitol (DTT). The three strains compared were AB2462 recA, AB2470 rec21 and their DNA repair-competent prototype, AB1157. The strains were incubated in 10 mmol dm-3 DTT for 60 min and allowed an expression period for SOS functions to appear which may have been induced by DTT. Following the expression period the DTT-incubated cells and incubated control cells were irradiated. When AB1157 cells were pretreated with chloramphenicol (200 micrograms cm-3) for a period of 30 min prior to addition of the induction media no increase in survival was seen. When catalase (0.1 mg cm-3) was added to the AB1157 cells prior to the induction media a decrease in the degree of induction was noted with an enhancement ratio (ER) of 0.893 (ER-1 = 1.12). Furthermore, DTT-treated AB2462 and AB2470 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with or without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a dose modification factor (DMF) of 1.7 with DTT present and 1.3 with pretreatment; (2) the rec mutants showed no change in survival at any dose with a DMF of approximately 1.0. Results indicate that, using our protocol, inducible repair is of more importance than free radical scavenging by DTT. Furthermore, DTT-treated AB2462 demonstrated no increase in survival when compared to control cells. In radiation experiments on either strain of E. coli with and without DTT present during irradiation, the following were observed: (1) survival of AB1157 was enhanced with a DMF of 1.7 with DTT present during irradiation and 1.3 with only pretreatment; (2) the recA and recB mutants showed no change in cell survival at any dose with a DMF of approximately 1.0. Results indicate that, using our pretreatment protocol, inducible repair is of more importance in protection than free radical scavenging by DTT.     

Difference in frequency spectrum of extremely-low-frequency effects on the genome conformational state of AB1157 and EMG2 E. coli cells

The effect of weak extremely-low-frequency (ELF) magnetic fields (sinusoidal, 30 μT amplitude) on the genome conformational state (GCS) of E. coli mutant and wild type cells was studied by using the method of anomalous viscosity time dependency (AVTD) in the 6–37 Hz frequency range. We confirmed the existence of three resonance frequencies of 8.9, 15.5, and 29.4 Hz when mutant cells of K12 AB1157 strain were exposed. In the same frequency range, the wild type K12 EMG2 cells displayed only two effective windows, with resonance frequencies of 8.3 and 27 Hz. The resonance frequencies differed significantly (P < .001–.000001) in the strains studied, whereas other resonance parameters did not. It was concluded that mutations in the AB1157 strain resulted in a significant rearrangement in the ELF action spectrum, including the appearance of a new resonance. © 1996 Wiley-Liss, Inc.     

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.     

Enhancement by cysteamine of N-methyl-N-nitrosourea mutagenesis in E. coli

Cysteamine (MEA) is comutagenic to methylnitrosourea (MNU) in E. coli AB 1157 but not in the nonadaptable mutant derivative ada-6 of that strain. The comutagenic action of MEA was eliminated by cysteine at low concentrations, which also lowered mutation frequencies in AB1157 but not in ada-6. In model experiments it was shown that cysteine counteracted the inhibition by MEA of beta-galactosidase induction in both bacterium strains. The comutagenic action of MEA is interpreted as being due to an inhibition of induction of methyltransferase during treatment with MNU.     

Reduced restriction of phage lambda in Escherichia coli K-12 cells after gamma-irradiation

In gamma-irradiated cells of Escherichia coli K-12 restriction alleviation of an unmodified phage lambda is only observed in AB1157 strain. No restriction alleviation by gamma-rays is registered in AB1157 mutants (rec A and ssb-1).     

Induction of error-free DNA repair in Escherichia coli by thiamine deprivation

Incubation of Escherichia coli AB1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine prototrophy which are abolished in its uvrA mutant, but only delayed in lon- and recA- cells. The response of the lexA3 mutant resembles that of the parental cells. These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process. They may represent a general response to non-mutagenic stress in these cells.     

Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.     

Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444]

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.     

Dithiothreitol pretreatment and inducible repair in UV-irradiated Escherichia coli K12 cells

The UV radiation survival of several Escherichia coli K12 strains was measured after pretreatment of the cells with dithiothreitol (DTT). In DNA repair-competent cells (AB1157), UV survival was enhanced (ER = 1.2) after pretreating cells for 1.0 h using 10 mmol dm-3 DTT and then incubating the cells for 1.5 h in buffer before UV irradiation. Similar experiments using the excision repair mutant, AB1886uvrA6, or the recombination repair and SOS-deficient mutant, AB2462recA, strains did not show enhanced UV survival. None of the E. coli strains tested were protected against UV killing by simultaneous treatment with DTT (10 mmol dm-3). These results, and the fact that incubation in chloramphenicol removed the wild-type response in DTT-pretreated, UV-irradiated cells, suggest that the observed UV radioprotection was a result of inducible enzymatic repair processes such as recA-dependent repair. The proposed stimulus for inducible repair in these cells is DNA damage caused by intracellular hydroxyl radicals arising from thiol oxidation. The involvement of oxygen radicals in the induction pathway is supported by results that showed superoxide dismutase and catalase could inhibit a portion (one-third) of the inducible repair.     

The variation in photoreactivating enzyme activity as a function of the stage of growth of three K12 strains of Escherichia coli

An investigation of the photoreactivity of the UV-sensitive double mutant K12 AB2480 has indicated that the number of active photoreactivating enzyme molecules is profoundly affected by the physiological state of the cells. This is shown both by the change in repair response of UV-irradiated cells to a single intense flash of light and also by the rate of photoreactivation under conditions of continuous illumination. The cells were found to possess a minimum of active enzyme molecules in exponential phase and a maximum in the late stationary phase. Evidence that this phenomenon is not due to a change in the photoreactivable sector of UV-induced damage is also presented. Further evidence supporting these conclusions is presented with data of the excision-deficient AB1886, and the resistant AB1157 strains.     

Morphological characteristics and ganglioside composition of substratum adhesion sites in a hepatoma cell line (CMH5123 cells) during different phases of growth

This study compares the ganglioside composition of tissue culture substrate-attached material (SAM) with that of cell bodies in a line of transformed hepatocytes derived from the minimal deviation Morris hepatoma 5123 c (CMH5123 cells). We examined both confluent cultures (late-phase cultures) and cells which were allowed to attach for only 3 h (early-phase cultures). We also determined to what extent ganglioside compositions of SAM and cell bodies from early- and late-phase cultures of CMH5123 cells are affected by the block of complex ganglioside biosynthesis induced by treatment with chelating agents (EGTA + EDTA). The morphological characteristics of SAM were monitored by scanning electron microscopy during the different steps of this study. In early-phase cultures, SAM was composed of fragments of filopodia and small vesicles probably representing newly formed substratum adhesion sites. In contrast, SAM of late-phase cultures was made up of large pools of membranous material resulting from the breakage of thick retraction fibers connecting the cell body with broad, mature adhesion sites. SAM of early-phase cultures yielded ganglioside profiles with a higher content of GM1 and GD1 a than those of cell bodies, while in late-phase cultures there was no difference between SAM and cell body gangliosides. When cells were grown in the presence of chelating agents, SAM of early-phase cultures was composed of vesicles and filopodial fragments similar to those found in early-phase cultures grown in regular media; these morphological features also appeared in SAM of confluent cultures (in contrast to the membranous material characteristic of late-phase cultures grown in regular media). In early-phase cultures grown in the presence of chelating agents, gangliosides of SAM were enriched in complex homologs relative to their content in cell bodies. These ganglioside characteristics were also found in SAM of confluent cultures grown in the presence of chelating agents, reflecting the presence of newly formed adhesion sites. On the basis of these results, we may conclude that the molecular assembly of newly formed adhesion sites implies the preferential distribution of several surface components involved in cell adhesion, including complex gangliosides.     

Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49

Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells. In the latter case mutations are called adaptive or stationary-phase mutations. High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III. The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E. coli cells. Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C. At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased. Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C. Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures. Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

DNA damage, prophage induction and mutation by furazolidone

Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that furazolidone treatment of Vibrio cholerae cells produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the reaction obeyed a first order relation. Sensitivities of the Escherichia coli strains to the lethal action of the drug were in the order: AB 2480(uvr- rec-) greater than AB 2463(rec-) greater than AB 1886(uvr-) greater than AB 1157(repair proficient) or AB 4401(wild type). Furazolidone was 'Rec test' positive, produced dose-dependent prophage induction in E. coli cells and also dose-dependent streptomycin-resistance forward mutation in V. cholerae cells. The quantitative aspect and also the mode of furazolidone action on DNA were discussed.     

Inhibition of cell division in hupA hupB mutant bacteria lacking HU protein.

Escherichia coli hupA hypB double mutants that lack HU protein have severe cellular defects in cell division, DNA folding, and DNA partitioning. Here we show that the sfiA11 mutation, which alters the SfiA cell division inhibitor, reduces filamentation and production of anucleate cells in AB1157 hupA hupB strains. However, lexA3(Ind-) and sfiB(ftsZ)114 mutations, which normally counteract the effect of the SfiA inhibitor, could not restore a normal morphology to hupA hupB mutant bacteria. The LexA repressor, which controls the expression of the sfiA gene, was present in hupA hupB mutant bacteria in concentrations half of those of the parent bacteria, but this decrease was independent of the specific cleavage of the LexA repressor by activated RecA protein. One possibility to account for the filamentous morphology of hupA hupB mutant bacteria is that the lack of HU protein alters the expression of specific genes, such as lexA and fts cell division genes.