Increased superoxide radical production evokes inducible DNA repair in Escherichia coli

Paraquat induced the SOS response in Escherichia coli. This was measured in terms of acquired resistance towards UV lethality in a wild-type strain and in terms of appearance of beta-galactosidase activity in a din::Mu d(Ap lac) fusion strain. However measured, the induction of the SOS response by paraquat was entirely dioxygen-dependent; whereas induction of the SOS response by mitomycin C was independent of the presence of dioxygen. As expected, recA(Def) and lexA(Ind-) isogenic strains did not show the SOS response. It appears likely that O-2, whose intracellular production is increased by paraquat, leads to DNA damage which in turn induces the SOS response.     

Characterization of stress-responsive behavior in Pseudomonas aeruginosa PAO: isolation of Tn3-lacZYA fusions with novel damage-inducible (din) promoters.

Although the pervasive soil and water microorganism Pseudomonas aeruginosa demonstrates heightened sensitivity to UV radiation, this species possesses a recA gene that, based on structural and functional properties, could mediate a DNA damage-responsive regulon similar to the SOS regulon of Escherichia coli. To determine whether P. aeruginosa encodes such stress-inducible genes, the response of P. aeruginosa to DNA-damaging agents including far-UV radiation (UVC) and the quinolone antimicrobial agent norfloxacin was investigated by monitoring the expression of fusions linking P. aeruginosa promoters to a beta-galactosidase reporter gene. These fusions were obtained by Tn3-HoHoI insertional mutagenesis of a P. aeruginosa genomic library. Eight different damage-inducible (din) gene fusions were isolated which lack homology to the P. aeruginosa recA gene. Expression of the three gene fusions studied, dinA::lacZYA, dinB::lacZYA, and dinC::lacZYA, increased following UVC and quinolone exposure but not following heat shock. Similar to E. coli SOS genes, the din genes were induced to different extents and with dissimilar kinetics following UVC irradiation.     

Micro-BIA, a colorimetric microtiter assay of lambda prophage induction

Escherichia coli that are lysogenic for a lambda-lacZ fusion phage produce beta-galactosidase, product of the lacZ gene, upon induction of the prophage by DNA-damaging agents. The miniaturization of a quantitative, colorimetric beta-galactosidase (prophage) induction assay (BIA) is presented. Induction assays are performed in microtiter wells with the aid of multichannel pipetting devices. Results are shown with screening strain BR513 (uvrB delta envA) and a strain, BR339 (uvrB delta lexA3ind-) which exhibits enhanced induction. A method developed for strain BR339 utilizes bacteria stored frozen in log phase, permeabilized in vitro, and used immediately; with this method, 2 consecutive assays may be completed in 1 working day. Mutagens utilized for the model studies included 4NQO, ENNG, daunorubicin, bleomycin, acetoxy-AAF, B[a]P, DMBA, and DEN (the last three in the presence of liver S9). Induced levels of beta-galactosidase were monitored using a vertical light path photometer that measured color absorbance in each microtiter well. Alternatively, color intensity could be determined by using a color chart prepared for this assay. These values were then plotted to generate dose-response curves. Considerable savings in labor and materials are achieved with the method described, one which may be used as a screen for DNA-damaging chemicals. Automated equipment and computers may be used to advantage with this assay, but they are not required.     

Induction of the SOS-like system in Rec-mutants of Bacillus subtilis]

Influence of the recE1, recB2, recB3, recB19, recF15, recF18, recL16, recM13 and recM27 mutations of the induction of the SOS-like system component, i. e. the RecE protein of Bacillus subtilis was studied by RIA-dot-blot method in UV-irradiated or treated by nalidixic acid cells. These agents caused a significant increase in the wild type (rec+) cells but did not stimulate the RecE synthesis in the rec mutants tested. The two exceptions were recB2 and recF18 mutants treated by nalidixic acid. The tsi23 mutation caused thermoinduction of phi 105 bacteriophage in the rec+ genetic background while no prophage particles were induced in the recE, recF, recL, recM mutants. The data suggest that the genetic damage of several rec genes including recB, recE, recF, recL and recM can block induction of the SOS-like system of Bacillus subtilis.     

The effect of gamma-irradiation on mu DNA transposition and gene expression

Mobile genetic elements are a ubiquitous presence in the genomes of all well-studied organisms. The effect of genomic stress on the status and transposition of these elements has not, as yet, been extensively characterized. We have been using temperate, transposable bacteriophage Mu as a model system to examine the behavior of mobile genetic elements and have previously shown that many DNA-damaging agents did not induce a Mu prophage to enter the lytic cycle of multiple rounds of DNA transposition. To extend these results and to examine the possibility that they were a reflection of damage to the DNA substrate for Mu transposition, we have constructed a mini-Mu plasmid, pMD12, which contains the early region of Mu, flanked by both extremities required for transposition in cis, and the beginning of the transposase gene A fused in frame to the lacZ gene. This A'-lacZ fusion protein maintains beta-galactosidase enzymatic activity under the control of the expression of the Mu transposase A gene and thus, the capacity for Mu transposition can be easily monitored by assaying for beta-galactosidase. By measuring the amount of beta-galactosidase after various doses of gamma-irradiation, we found that doses of up to 75 krad had no effect on the expression of the Mu transposase gene A. This was confirmed by the lack of induction of a Mu prophage in strains containing a chromosomally inserted Mu genome. Although the plaque-forming units per colony-forming unit of strain CSH67, containing a chromosomally inserted lambda prophage, increased approximately 100-fold from 0 to 75 krad, no stimulation of induction of prophage Mu lytic growth was observed. We also found that plasmid pMD12 did not transpose and chromosomally associate upon gamma-irradiation. This supports the assertion that DNA-damaging agents, including gamma-rays, do not induce the transposition of prokaryotic mobile genetic elements.     

Induction of SOS responses in Escherichia coli by Panfuran-S, 3-di(hydroxymethyl)amino-6-(5-nitro-2-furylethenyl)-1,2,4-triazine

The inducibility of SOS responses by Panfuran-S, which has been used as an antimicrobial medicine in Japan, was studied in Escherichia coli cells having different DNA-repair capacities for UV lesions. Panfuran-S induced mutations at high frequencies in uvrA and the wild-type strains, and significant killing effects of Panfuran-S were detected in DNA-repair-deficient strains, uvrA and recA. The effective prophage induction was detected in two kinds of lambda-lysogenized cells treated with Panfuran-S. The expression of the umuC+ gene was apparently induced in uvrA and the wild-type strains, but not induced in lexA and recA strains. In particular, high inducibility of the gene expression was detected in uvrA strain as compared with the wild-type strain. From these results, we conclude that Panfuran-S is a DNA-damaging agent and may induce the error-prone SOS responses.     

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.     

Two Lactococcus lactis genes, including lacX, cooperate to trigger an SOS response in a recA-negative background.

A 4.3-kb EcoRI fragment from a Lactococcus lactis genomic library alleviates the methyl methanesulfonate, mitomycin C, and UV sensitivities of an Escherichia coli recA mutant (M. Novel, X. F. Huang, and G. Novel, FEMS Microbiol. Lett. 72:309-314, 1990). It complements recA1 and delta recA mutations but not recA13. Three proteins (with molecular masses of 20, 35, and 23 kDa) were produced from this fragment in a T7-directed system, and three corresponding genes were detected by DNA sequencing, namely, ISS1CH;lacX, which is the distal gene of the lac operon; and a third open reading frame, named lacN, which encodes 211 amino acids. Mutations produced in either lacX or in lacN resulted in the loss of the resistance to DNA-damaging agents. Thus, these two genes appeared to be involved in this activity. Introduction of pUCB214 carrying the 4.3-kb fragment into a lexA+ delta recA306 sfiA::lacZ strain resulted in UV-inducible synthesis of beta-galactosidase. A uvrA strain or a lexA (Ind-) strain containing pUCB214 did not support any DNA repair. However, a lexA (Def-) strain carrying pUCB214 could partly repair UV damage. We discuss possible targets for LacX and LacN products, and we speculate that LacX and LacN may constitute a two-component regulatory system that is able to respond to SOS signals, and then to act in the SOS response, bypassing the RecA-activated function.     

A bacterial strain for detecting agents that produce free radical-mediated DNA strand breaks

In an E. coli strain carrying two mutations, one in the dnaC gene involved in initiation of DNA replication and another in the uvrB gene which affects the excision-repair system, it has been shown that the SOS response cannot be induced by UV. This is probably due to the absence of any inducing signal (Salles and Defais, 1984). The capacity to induce the SOS network was followed using RecA protein amplification as a probe. When breaks were produced in DNA, RecA protein induction was restored. We describe here a strain in which both RecA protein and beta-galactosidase from a sfiA::lacZ fusion can be measured simultaneously in the same bacterial extract. In conditions in which no replication proceeds, this strain can be used to detect the ability of chemicals to produce free radical-mediated DNA breaks in vivo.     

SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.     

The SOS-function-inducing activity of chemical mutagens in Escherichia coli

The SOS-function-inducing activities of 42 chemical mutagens were investigated in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ37 . To correct for the inhibitory effects of test chemicals on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Most of the mutagens reported to be mutagenic to the Ames' Salmonella tester strains showed the SOS-function-inducing activity. The inducible SOS repair may be responsible for not only base-change mutations but also frameshift mutations. However, 9-aminoacridine, ethidium bromide and 4-nitro-o-phenylenediamine did not induce the SOS function, suggesting that the mutagenesis induced by these mutagens may occur independently of SOS repair. Present results support the SOS mutagenesis model that error-prone SOS repair plays an important role in mutagenesis induced by most chemical mutagens.     

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.     

Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens

The umu operon in Escherichia coli is responsible for chemical and radiation mutagenesis, and the expression of the operon itself is inducible by these DNA-damaging agents. The principle of the umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A plasmid (pSK1002) carrying a fused gene umuC'-'lacZ was introduced into Salmonella typhimurium TA1535. The strain TA1535/pSK1002 enabled us to monitor the levels of umu operon expression by measuring the beta-galactosidase activity in the cells produced by the fusion gene. Using this strain, a simple, inexpensive, and sensitive system, the umu-test, for the screening of environmental mutagens and carcinogens was developed. 38 chemicals with different structures and modes of action, including 31 known animal carcinogens, were examined by the test to evaluate the system. The threshold sensitivity of the umu-test was approximately equal to that of the Ames test for chemicals genotoxic in both tests. By the umu-test, using the single tester strain, we detect many types of DNA-damaging agents for which the Ames test requires several tester strains. Furthermore, the umu-test provides a potential practical advantage for the screening of various environmental samples containing amino acids and nutrients such as urine, serum and foods.     

The induction of SOS function in Escherichia coli K-12/PQ37 by 4-nitroquinoline oxide (4-NQO) and fecapentaenes-12 and -14 is bile salt sensitive: implications for colon carcinogenesis

The response of Escherichia coli to genotoxic agents involves the triggering of a complex system of genes known as the SOS response. In E. coli PQ37, a test organism used for the assessment of genotoxicity, lacZ, the beta-galactosidase gene is placed under the control of sfiA, one of the SOS genes through an operon fusion. The induction of beta-galactosidase activity, when the organism is exposed to genotoxic agents, is an indirect measure of the genotoxic activity of the test compound. Incubation of E. coli PQ37 with either 4-nitroquinoline oxide (4-NQO) or one of the fecal mutagens, fecapentaene-12 or -14 (F-12 or F-14) in the presence of sodium taurocholate or sodium deoxycholate resulted in a significant enhancement of induction of beta-galactosidase activity. The molecular mechanisms of 4-NQO-induced mutagenesis in E. coli are similar to those of the effects of UV light in which both replication-dependent and repair-dependent pathways of mutagenesis exist. Since E. coli PQ37 is excision-repair-deficient, alternate pathways are involved in this system. Bile salts by themselves do not trigger the SOS response, and hence their role in enhancing the SOS-inducing potency of mutagens may involve the potentiation of the cleavage-inactivation of lexA (repressor of SOS) by the protein product of the SOS-controlled gene, recA. The potentiating effect of bile salts on the fecal mutagens, F-12 and F-14, has implications in their suspected role in colon carcinogenesis associated with high-fat, low-fiber diets.     

Characterization of an inducible oxidative stress system in Bacillus subtilis.

Exponentially growing cells of Bacillus subtilis demonstrated inducible protection against killing by hydrogen peroxide when prechallenged with a nonlethal dose of this oxidative agent. Cells deficient in a functional recE+ gene product were as much as 100 times more sensitive to the H2O2 but still exhibited an inducible protective response. Exposure to hydrogen peroxide also induced the recE(+)-dependent DNA damage-inducible (din) genes, the resident prophage, and the product of the recE+ gene itself. Thus hydrogen peroxide is capable of inducing the SOS-like or SOB system of B. subtilis. However, the induction of this DNA repair system by other DNA-damaging agents is not sufficient to activate the protective response to hydrogen peroxide. Therefore, at least one more regulatory network (besides the SOB system) that responds to oxidative stress must exist. Furthermore, the data presented indicate that a functional catalase gene is necessary for this protective response.     

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.