The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

Joining of λ bacteriophage DNA moleculesin vitro using the gene product of the λint phage

The ability of cell extracts ofEscherichia coli 1100 prepared at different times after infection with bacteriophage λint 6 cI 857 or λ 857 to attach molecules of λ³²P-DNA to λ DNA adsorbed on a nitrocellulose membrane filter was compared. It was found that only with extracts from cells infected with bacteriophage λ cI 857 with an active int gene was it possible to attach molecules of λ³²P-DNA to λ DNA. This ability was reflected best in extracts prepared from cells 10 min after infection. A similar ability was observed also with extracts prepared from cells ofEscherichia coli 1100 (λ cI 857) after heat induction of the prophage. The maximum efficiency in this case was observed with cell extracts 15 min after the temperature rise.     

Induction of Phage Production in the Lysogenic Escherichia coli by Hydroxyurea

1) Hydroxyurea, a reversible DNA synthesis inhibitor, was used to study the mechanism of prophage λ induction in Escherichia coli K12. Induction of prophage was judged on two criteria: increase of phage-producing cells and loss of colony-forming ability of the cells. 2) Hydroxyurea induced an increase of phage-producing cells only in lysogenic strains known to be inducible with ultraviolet irradiation for prophage development and not in strains such as E. coli K12 (λind^–) or E. coli K12 recA (λ⁺). 3) When protein synthesis was inhibited, hydroxyurea did not increase phage-producing cells of lysogenic strains; it showed a bacteriocidal effect on lysogenic recA⁺ strains, but not on nonlysogenic strains. 4) The sensitivity of E. coli K12 recA to hydroxyurea was independent of whether or not the cells were lysogenic. 5) From the results it is suggested that certain steps leading to loss of colony-forming ability (i.e. prophage induction) do not require de novo protein synthesis but require the presence of the host recA⁺ gene.     

Mutagenic DNA repair in Escherichia coli XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.     

DNA repair in E. coli strains deficient in single-strand DNA binding protein

Summary Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb ^- recA^- and ssb ^- uvr^- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.     

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.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu

Summary Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants.By incubating the strains 302 and 328 at 42°C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.     

Effect of glutathione on UV induction of prophage lambda

The effect of glutathione (GSH) on the ultraviolet (UV) induction of lambda prophage was investigated in lysogenic Escherichia coli. The data showed that extracellular GSH could inhibit the UV induction of lambda prophage. The inhibitory rates were concentration dependent, and the maximal rate obtained was 94% with 3.0 M GSH. The effect was also measured in three different lambda lysogens: a wild-type strain (wt), an isogenic GSH-deficient strain, and an isogenic strain producing increased amounts of GSH. The result showed that when subjected to UV irradiation (254 nm, 60 J m⁻²), GSH-deficient strain was approximately fivefold more sensitive to be lysed than wt, whereas the strain with higher intracellular GSH levels was only 28% susceptible to be lysed. With electron spin resonance and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated lysogenic cells and the intensity of signals was influenced by GSH levels. These results indicate that GSH can significantly inhibit the UV induction of lambda prophage, and that this effect is correlated to its capacity to scavenge free radicals generated after UV irradiation.     

Chloramphenicol acetyltransferase fromPseudomonas aeruginosa—a new variant of the enzyme

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100–1,000 g/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are highly resistant to the drug. In an attempt to understand the resistance mechanism ofP. aeruginosa to chloramphenicol, CAT produced by this microorganism was examined and compared with other known variants of the enzyme which are usually associated with high-level resistance in other species. CAT ofP. aeruginosa was found to be synthesized constitutively and to have a molecular weight of 22,500 per protomer. The specific activity of the enzyme is 30-fold lower than that ofEscherichia coli type II CAT. The same specific activity was obtained for CAT produced by two isolates ofP. aeruginosa, one with high and one with low resistance. The elution patterns from affinity and hydrophobic chromatography,K _m 's for chloramphenicol, the high affinity of the enzyme for acetyl-CoA (2- to 6-fold greater than that of any other variant) and insensitivity to sulfhydryl reagents indicate that the properties of this enzyme are not identical with those of any of the known variants.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Increased stability of recombinant plasmids by Tn1000 insertion in chemostat cultures of recombinantEscherichia coli GT123

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.     

Inhibition of Shiga toxin-converting bacteriophage development by novel antioxidant compounds

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.     

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis

Summary Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage . But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.     

Toward a bacterial genome technology: integration of theEscherichia coli prophage lambda genome into theBacillus subtilis 168 chromosome

A novel approach to the cloning large DNAs in theBacillus subtilis chromosome was examined. AnEscherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome ofB. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In theB. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kbB. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in theB. subtilis chromosome and possible applications of this technique are discussed.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

W reactivation is inefficient in repair of the bacterial chromosome

Summary UV-inducible SOS processes associated with W reactivation of phage lambda were studied for their effect on repair of lambda prophage integrated in the bacterial chromosome. For this purpose, lambda cI857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30° to 42°. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30° after UV exposure W reactivation should be fully expressed and should also exert an effect on the bacterial chromosome and the prophage inside it. When heat-induction is delayed until the time at which W reactivation reaches its maximum, a considerable increase in phage survival might then be expected. The results presented in this report show, however, that the delayed induction had only a small effect on the survival of prophage in the wild-type strain (possibly attributable to excision repair) and no detectable effect on prophage in a uvrA strain. From these results we conclude that W reactivation is largely irrelevant to the repair of UV-damaged bacterial DNA.