An Escherichia coli strain deficient for both exonuclease V and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity.     

Properties of uvrE mutants of Escherichia coli K12. I. Effects of UV irradiation on DNA metabolism

Escherichia coli K12 uvrE is a mutator strain which is highly sensitive to ultraviolet (UV) radiation.In an attempt to determine the underlying molecular basis for the UV sensitivity, we have compared a mutant and an isogenic wild type strain with regard to several metabolic responses to 254-nm radiation. The introduction of single-strand breaks into intracellular DNA after irradiation is normal. However, the rate of excision of pyrimidine dimers as well as of DNA degradation and final rejoining of the strand breaks is lower in the mutant as compared to the repair proficient strain.These data suggest that the uvrE gene product may be involved in a reaction between the incision and excision steps in the excision repair process.     

Effect of an Insertional Mutation in the cspA Gene Encoding the Major Cold-Shock Protein on Radiation Resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA ⁺). The radiation protective effect of this plasmid is abolished or drastically reduced in mutants recA13and rpoH15defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to -irradiation of E. coli mutants with an intermediate level of radiation resistance (Gam^r445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gam^r444 mutant. In the chromosome of E. coli strain with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of -rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. colicells to the lethal effect of -rays and UV light.     

Novel properties of the Thermus thermophilus RuvB protein, which promotes branch migration of Holliday junctions

Branch migration of Holliday junctions, which are central DNA intermediates in homologous recombination, is promoted by the RuvA-RuvB protein complex, and the junctions are resolved by the action of the RuvC protein in Escherichia coli. We report here the cloning of the ruvB gene from a thermophilic eubacterium, Thermus thermophilus HB8 (Tth), and the biochemical characterization of the gene product expressed in E. coli. The Tth ruvB gene could not complement the UV sensitivity of an E. coli ruvB deletion mutant and made the wild-type strain more sensitive to UV. In contrast to E. coli RuvB, whose ATPase activity is strongly enhanced by supercoiled DNA but only weakly enhanced by linear duplex DNA, the ATPase activity of Tth RuvB was efficiently and equally enhanced by supercoiled and linear duplex DNA. Tth RuvB hydrolyzed a broader range of nucleoside triphosphates than E. coli RuvB. In addition, Tth RuvB, in the absence of RuvA protein, promoted branch migration of a synthetic Holliday junction at 60°?C in an ATP-dependent manner. The protein, as judged by its ATPase activity, required ATP for thermostability. Since a RuvA protein has not yet been identified in T. thermophilus, we used E. coli RuvA to examine the effects of RuvA on the activities of Tth RuvB. E. coli RuvA greatly enhanced the ability of Tth RuvB to hydrolyze ATP in the presence of DNA and to promote branch migration of a synthetic Holliday junction at 37°?C. These results indicate the conservation of the RuvA-RuvB interaction in different bacterial species, and suggest the existence of a ruvA homolog in T. thermophilus. Although GTP and dGTP were efficiently hydrolyzed by Tth RuvB, these nucleoside triphosphates could not be utilized for branch migration in vitro, implying that the conformational change in RuvB brought about by ATP hydrolysis, which is necessary for driving the Holliday junction branch migration, cannot be accomplished by the hydrolysis of these nucleoside triphosphates.     

High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli

Background

Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.

Methodology/Principal Findings

E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m²) combination with different temperature (22, 28, 30, 32, and 37°C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.

Conclusions/Significance

These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.     

Analysis of the recJ gene and protein from Deinococcus radiodurans

The mechanism by which double-strand DNA breaks are repaired in the radiation-resistant bacterium Deinococcus radiodurans is not well understood. This organism lacks the RecBCD helicase/nuclease, which processes broken DNA ends in other bacteria. The RecF pathway is an alternative pathway for recombination and DNA repair in E. coli, when RecBCD is absent due to mutation, and D. radiodurans may rely on enzymes of this pathway for double-strand break repair. The RecJ exonuclease is thought to process broken DNA ends for the RecF pathway. We attempted to delete the recJ gene from D. radiodurans, using homologous recombination to replace the gene with a streptomycin-resistance cassette. We were unable to obtain a complete deletion mutant, in which the gene is deleted from all of the chromosome copies in this polyploid organism. Quantitative real-time PCR shows that the heterozygous mutants have a recJ gene copy that is ca. 10–30% that of the wild-type. Mutants with reduced recJ gene copy grow slowly and are more sensitive than wild-type to UV irradiation, gamma irradiation, and hydrogen peroxide. The mutants are as resistant as wild-type to methyl-methanesulfonate. The D. radiodurans RecJ protein was expressed in E. coli and purified under denaturing conditions. The re-folded protein has nuclease activity on single-stranded DNA with specificity similar to that of E. coli RecJ exonuclease.     

Sensitivity of selected bacterial species to UV radiation

The effect of exposure of bacterial suspensions to UV radiation by means of the dose-response curves was assessed. The D₃₇ and D₁₀ values were used for subsequent statistical analysis of the results. The aim of this article is to evaluate the sensitivity to UV radiation of several microorganisms of different habitats (Rhizobium meliloti, Rhodobacter sphaeroides, Escherichia coli, and Deinococcus radiodurans), two mutants with nonfunctional SOS DNA repair system (R.meliloti recA ^- and E. coli recA ^-), and a mutant in the synthesis of carotenoids (R. sphaeroides crtD). The results reveal that D. radiodurans was an extremely resistant bacterium, R. meliloti was more resistant than R. sphaeroides, and E. coli was the most sensitive bacterium tested. The high sensitivity of recA ^- mutants was also verify. Moreover, it seems that the possession of pigments had no important effect in the sensitivity of R. sphaeroides to UV radiation.     

Solar and Temporal Effects on Escherichia coli Concentration at a Lake Michigan Swimming Beach

Studies on solar inactivation of Escherichia coli in freshwater and in situ have been limited. At 63rd St. Beach, Chicago, Ill., factors influencing the daily periodicity of culturable E. coli, particularly insolation, were examined. Water samples for E. coli analysis were collected twice daily between April and September 2000 three times a week along five transects in two depths of water. Hydrometeorological conditions were continuously logged: UV radiation, total insolation, wind speed and direction, wave height, and relative lake level. On 10 days, transects were sampled hourly from 0700 to 1500 h. The effect of sunlight on E. coli inactivation was evaluated with dark and transparent in situ mesocosms and ambient lake water. For the study, the number of E. coli samples collected (n) was 2,676. During sunny days, E. coli counts decreased exponentially with day length and exposure to insolation, but on cloudy days, E. coli inactivation was diminished; the E. coli decay rate was strongly influenced by initial concentration. In situ experiments confirmed that insolation primarily inactivated E. coli; UV radiation only marginally affected E. coli concentration. The relationship between insolation and E. coli density is complicated by relative lake level, wave height, and turbidity, all of which are often products of wind vector. Continuous importation and nighttime replenishment of E. coli were evident. These findings (i) suggest that solar inactivation is an important mechanism for natural reduction of indicator bacteria in large freshwater bodies and (ii) have implications for management strategies of nontidal waters and the use of E. coli as an indicator organism.     

The SbcCD complex of Deinococcus radiodurans contributes to radioresistance and DNA strand break repair in vivo and exhibits Mre11–Rad50 type activity in vitro

Deinococcus radiodurans lacks a homologue of the recB and recC genes, and the sbcA/B genes, of Escherichia coli. Thus, DNA strand break repair in Deinococcus proceeds by pathways that do not utilize these proteins. Unlike E. coli, the absence of recBC and sbcA/sbcB, and presence of only sbcC and sbcD in Deinococcus, indicates an enigmatic role of SbcCD in this bacterium. Studies on sbcCD mutation in Deinococcus showed nearly a 100-fold increase in gamma radiation sensitivity as compared to wild type. The mutant showed a higher rate of in vivo DNA degradation during the post-irradiation recovery period that corresponds to the RecA-dependent DSB repair phase. These cells showed a typical NotI pattern of DNA reassembly during the early phase of DSB repair, but were defective for the subsequent RecA-dependent phase II of DSB repair. Hydrogen peroxide had no effect on cell survival of the mutant. While its tolerance to higher doses of UVC and mitomycin C was significantly decreased as compared to wild type. Purified recombinant SbcCD proteins showed single-stranded endonuclease and 3′ → 5′ double-stranded DNA exonuclease activities similar to that of the Mre11–Rad50 complex, which is required for DNA strand break repair in higher organisms. These results suggested that the Mre11–Rad50 type nuclease activity of SbcCD proteins contributes to the radiation resistance of D. radiodurans perhaps by promoting the RecA-dependent DSB repair required for polyploid genome maturation.     

<Emphasis Type="BoldItalic">In situ</Emphasis> real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe <Emphasis Type="BoldItalic">Deinococcus radiodurans</Emphasis>

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.     

Properties of a UV-sensitive Neurospora strain defective in pyrimidine dimer excision

The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains.Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene.These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.     

Rescue by chloramphenicol and rifampicin of UV-irradiated lon, exrA and recA mutants of Escherichia coli K12

The effect of temporary treatment with chloramphenicol or rifampicin on the survival of UV irradiated cells of selected Escherichia coli K12 radiation sensitive mutants was examined. Increased survival resulted for both exrA and recA mutants, and also for the unsuppressed lon mutant, but cells of the parent strain and the recB mutant were not rescued. This contrasts with our earlier finding that after exposure of the bacteria to γ-rays, chloramphenicol treatment rescued the exrA and lon mutants but not the recA mutant. We now report that an exrA recA double mutant was rescued by chlramphenicol after UV radiation, but not after anaerobic ionizing radiation. Inclusion of inhibitors of uvrA governed repair, caffeine and 8-methoxypsoralen (8-MOP), in the incubation medium containing chloramphenicol, did not reduce the rescue of the exrA or recA mutants, although caffeine eliminated rescue of the lon mutant, which was itself unaffected by 8-MOP. However it is concluded that chlormaphenicol rescue of the exrA and recA mutants after UV radiation was not entirely independent of the excision-repair process, since the uvrA recA and uvrA exrA double mutants were not rescued by this treatment.     

Photoreactivation, Excision, and Strand-Rejoining Repair in R Factor-Containing Minicells of Escherchia coli K-12

Chromosomeless “minicells” are formed by misplaced cell fissions near the polar extremities of an Escherichia coli K-12 mutant strain. Resistance (R)-factor deoxyribonucleic acid (DNA) can be introduced into minicells by segregation from an R⁺ (R64-11) derivative of the original mutant. We have assessed the ability of R⁺ minicells to correct defects produced in their plasmid DNA by ultraviolet (UV) and gamma radiations. Minicells harboring plasmid DNA, in comparison with their repair-proficient minicell-producing parents, possess (i) an equal competence to rejoin single-strand breaks induced in DNA by gamma rays, (ii) a reduced capacity for the photoenzymatic repair of UV-induced pyrimidine dimers, and (iii) a total inability to excise dimers, apparently owing to a deficiency in UV-specific endonuclease activity responsible for mediating the initial incision step in excision repair. Assuming that the DNA repair properties of R⁺ minicells reflect the concentration of repair enzymes located in the plasmid-containing polar caps of entire cells, these findings suggest that: (i) the enzymes responsible for rejoining single-strand breaks are distributed throughout the cell; (ii) photoreactivating enzyme molecules tend to be concentrated near bacterial DNA and to a lesser extent near plasmid DNA; and (iii) UV-specific endonuclease molecules are primarily confined to the central region of the E. coli cell and, thus, seldom segregate with R-factor DNA into minicells.     

Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12

Summary DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme. The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ. A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant. Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV. Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD ⁺ strain. The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.     

Effects of Ionizing Radiation on Synchronous Cultures of Escherichia coli B/r

The sensitivity of Escherichia coli B/r to X-irradiation is correlated with the replication cycle of deoxyribonucleic acid (DNA). The sensitivity to X-irradiation in the wild type can be attributed to the presence of nuclear targets plus DNA repair mechanisms. The effects of nuclear targets are observed in the recombination-deficient (rec−) mutant B/r, but the sensitivity reflected by changes in the slope of killing curves is absent. A study of different growth conditions indicates that maximal resistance to X rays occurs toward the middle of the division cycle. Evidence is offered that branched chromosomes respond as one-hit targets to X-irradiation. The killing effects of heavy-ion bombardment on E. coli are due primarily to ionizing radiation.     

Overexpression of Escherichia coli nucleotide excision repair genes after cisplatin-induced damage

Cisplatin is currently used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. It is a commonly used chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA. Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) were submitted to increasing concentrations of cisplatin, and the results revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and cho mutants remain as the wild type strain. The time required for both gene expression turn-off and return to normal weight DNA in wild-type E. coli was not accomplished even after 4 h post-treatment with cisplatin, while the same process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a heavily damaging action of cisplatin can be seen not only by persistent nicks on genomic DNA, but also by NER gene expression exceeding manifold that seen after equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB gene expression from its putative upstream promoter P3 in an SOS-independent manner.     

Reactivating effect of Escherichia coli exometabolites on UV-irradiated cells

Wild-type and mutant (AB 1157 and K-12) strains of Escherichia coli were shown to synthesize the logarithmic growth phase, exometabolites reactivating UV-irradiated cells of producer strains. The exometabolites of the strain K-12 were of protein nature and had a molecular weight of no more than 10 kDa. The reactivating activity of these exometabolites was inversely related to bacterial survival and slightly increased under the influence of stress factors. The reactivating factor of Luteococcus casei had a cross-reactivating and protective effect on UV-irradiated cells of E. coli strain K-12. Due to activation of the reactivating factor after UV irradiation and heating, the cross-protective effect increased more than threefold. The reactivating effect remained unchanged under these conditions. The protein exometabolites of E. coli did not induce cross-stress response in L. casei.     

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function

Summary The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between argB and purD loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and -rays and incorporates little or no ³H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of ³H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42° C before their transfer to 30° C results in higher survival as compared to their incubation at 30° C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.     

The structural gene for the ribosomal protein S18 in Escherichia coli. I. Genetic studies on a mutant having an alteration in the protein S18

A mutant of Escherichia coli strain CR341, originally isolated as a temperature-sensitive mutant, was found to have an altered 30 S ribosomal protein (S18) in addition to and independently of temperature sensitivity. Protein S18 from the mutant strain differs in electrophoretic mobility in polyacrylamide gel electrophoresis at pH 4.5 from protein S18 of the parental origin. The mutation responsible for the alteration in S18 is different from two other mutations in the mutant strain which give the temperature-sensitive phenotype. The gene involved in the S18 alteration is located in a region between 76 and 88 minutes on the E. coli genetic map; the location is outside the str-spc region at 64 minutes, where several known ribosomal protein genes are located. An episome covering the loci rha (76 min) through pyr B (84 min) was introduced into the mutant. The resultant merodiploid strains were shown to produce both the normal and the mutant forms of S18. The results support the conclusion described in the accompanying paper (Kahan et al., 1973) that the mutation studied is in the structural gene for S18.     

Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase

Two Escherichia coli K12 mutants defective in 3-methyladenine-DNA glycosylase have been isolated following mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine. The mutants, which are of independent origin and have been designated tag-1 and tag-2, contain greatly reduced amounts of 3-methyladenine-DNA glycosylase activity in cell-free extracts. The defect in the tag-1 strain is observed at 43 °C but not at 30 °C, and a partially purified enzyme from this strain is unusually heat-labile, indicating that the defect in the tag-1 strain is due to a mutation in the structural gene for 3-methyladenine-DNA glycosylase.We have shown that 3-methyladenine-DNA glycosylase is responsible for the rapid removal of 3-methyladenine from the DNA of E. coli cells treated with monofunctional alkylating agents. The active release of this base is greatly impaired in the mutant strains. Both tag mutant strains are abnormally sensitive to killing by monofunctional alkylating agents and are defective in the host cell reactivation of methyl methanesulphonate-treated bacteriophage A. The tag mutation does not confer an increased sensitivity to ultraviolet or X-irradiation, and host cell reactivation of irradiated λ is normal in these strains. Further, there was no increase in the rate of spontaneous mutation in a tag strain.Three-factor transductional crosses with nalA and nrdA have shown that the tag-2 mutation is located at 47.2 minutes on the map of the E. coli K12 chromosome. In the mapping experiments, the tag-1 mutation behaved differently and appeared to be located at 43 to 46 minutes, in a closely situated but non-adjacent gene. Possible implications of the non-identity of the tag-1 and tag-2 mutations are discussed.