Para-aminobenzoic acid inhibits a set of SOS functions in Escherichia coli K12

PABA - Vitamin H1 of group B, has obtained increasing fundamental interest as a very potent natural antimutagen after a series of our publications since 1979. In the first set of our experiments, we studied PABA in the assays with the alkylating agent N-methyl-N-nitrosourea (MNU). Mutagenic efficiency of this agent was suppressed up to 10-fold when PABA was administered into Escherichia coli cells concurrently with the mutagen or prior to the mutagenic treatment. NMR spectrometric and UV-spectrophotometric measurements did not reveal an interaction between the direct acting MNU and PABA, typical for some N-nitroso compounds and phenolics. PABA suppressed the error-prone DNA repair pathway induced by UV-irradiation. PABA decreased MNU-induced phage lambda lysogenic induction more than two orders of magnitude. PABA inhibited the thermal shift up to 400-fold in phage lambda from the permissive to non-permissive temperature in E. coli mutant tif-1 and decreased about two-fold W-reactivation of UV-damaged phage lambda. Chloramphenicol treatment of the cells just after the mutagenic treatment prevented the occurrence of PABA specific activity. The results suggest that PABA affects the SOS DNA repair pathway and the mutagenic response of E. coli. PABA appears to be an effective bioantimutagen reducing mutagenesis by modulating the error-prone DNA repair (SOS) response.     

W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.     

Roasting coffee beans produces compounds that induce prophage lambda in E. coli and are mutagenic in E. coli and S. typhimurium

Freshly brewed blended coffee, instant coffee and instant caffeine-free coffee induced prophage lambda in lysogenic E. coli K12, strain GY5027. Because coffee prepared from green beans by the same extraction method as used for freshly brewed blended coffee had no prophage-inducing activity, this activity may be attributed to compounds produced in the roasting process. Roasting also produced compounds that were mutagenic in S. typhimurium TA100 and E. coli WP2 uvrA/pKM101.     

Stimulation of mutations suppressing the loss of replication control by small alcohols

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.     

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.     

Crude tea extracts decrease the mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats

The effects of tea extracts and their ingredients, catechins and L-ascorbic acid (AsA), on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in vitro and in the stomachs of rats using E. coli WP2 and S. typhimurium TA100. The extracts of green tea and black tea leaves decreased the mutagenic activity of MNNG to E. coli WP2 in vitro in a desmutagenic manner. Catechins such as (-)-epigallocatechin from green tea leaves and the low-molecular-weight tannin fraction isolated from black tea extract with HP-20 resin also exhibited inhibitory effects against the mutagenic activity of MNNG. A desmutagenic effect of AsA on MNNG-induced mutagenicity was observed depending on the dose, though it was complicated. The effects were also demonstrated in the stomachs of rats by assaying the bacterial mutagenic in vitro; the tea extracts previously given orally to rats reduced the mutagenic activity of MNNG remarkably, though simultaneous administration showed less effect. The effectiveness of tea extracts for the decrease of MNNG-induced mutagenesis in vitro and in vivo suggests that the habitual drinking of tea may reduce the tumor-initiating potency of MNNG-type nitrosoureido compounds if they are formed in the stomach.     

UV-A induced DNA nicking activities of skin photosensitive drugs: phenothiazines, benzothiadiazines and afloqualone

Plasmid pBR 322 was subjected to UV-A irradiation in the presence of photosensitive drugs, i.e., phenothiazines [chlorpromazine hydrochloride (CPZ), promethazine hydrochloride (PMZ) and mequitazine (MQZ)], benzothiadiazines [penflutizide (PFZ), hydrochlorothiazide (HCT) and methyclothiazide (MCT)] and afloqualone (AQ). The distribution of the closed-circular and the open-circular form of the plasmid DNA was analyzed by means of neutral agarose gel electrophoresis. All the drugs used induced more or less DNA nicking to yield the open-circular form. The nicking activities of the phenothiazines were in the order: CPZ greater than PMZ greater than MQZ. CPZ elicited extensive degradation of the DNA by photosensitization. The nicking activities of the benzothiadiazines and AQ were much weaker than CPZ and PMZ.     

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.     

Mutagenicity of benzotrichloride and related compounds.

Benzotrichloride (BTC), benzal chloride (BDC), benzyl chloride (BC) and benzoyl chloride (BOC) were surveyed for their mutagenicity in microbial systems such as rec-assay using Bacillus subtilis and reversion assays using E. coli WP2 and Ames Salmonella TA strains with or without metabolic activation in vitro. BTC and BDC required metabolic activation for their mutagenic activities in several strains of E. coli and Salmonella. The mutagenic metabolites of these compounds may not have been produced by hydrolysis. BC was weakly mutagenic without metabolic activation. Only BOC exhibited no mutagenic activity in the detection procedures used. The mutagenic metabolite of BTC might be very unstable under our experimental conditions. The strain E. coli WP2 try hcr was more sensitive than E. coli B/r WP2 try (hcr+) with regard to the mutagenicity of BTC.     

Induced mutagenesis of plasmid as well as chromosomal genes inserted into plasmid DNA. II. The mutagenic action of chemical factors

To proceed the works on induced mutagenesis in plasmids, mutagenic effects of chemicals on the DNA of RSF2124 plasmid mediating colicine E1 biosynthesis and resistance to ampicillin, were studied. After exposure to mutagens, plasmid DNA was used to transform Escherichia coli C600 rk-mk-cells. The lethal effect was estimated from inactivation of the ampicillin marker, the mutagenic effect being measured by the appearance of mutants unable to synthesize colicine (Col-). The reaction of the plasmid DNA with a mutagen was stopped by 10-fold dilutions of aliquots in TEN buffer, followed by dialysis in 10 mH CaCl2 for 24 h. To select the most efficient mutagens for plasmid DNA, the compounds were predominantly tested which are known to be effective in other systems (transforming and transfecting DNA, microbial viruses). An a result, all chemicals tested by their activity were classified into 4 groups: inducing more than 100 fold increase (hydroxylamine, O-methylhydroxylamine); inducing 10 fold increase (UV-irradiation, lambda = 254 nm; W-mutagenesis, gamma-irradiation, nitrous acid, mitomycin C); inducing less than 10fold increase (indirect UV-mutagenesis, nitrous acid, beta-chloroethyldiethylamine hydrochloride, nitrosoguanidine); no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, O-beta-diethylhydroxylamine).     

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

Specificity of the lethal and mutagenic actions of pico-second laser pulses of 532-nm wavelength

A study was made of the lethal effect of pulse laser (second harmonic Nd+3:YAG laser of 532 nm, pulse length 3.3.10(-11) s, peak intensity from 4.10(12) to 1.10(14) W/m2) on HeLa cells at the phases of active and stationary growth, and lethal and mutagenic effects of this radiation on E. coli cells. As was shown, HeLa cells at both growth phases and E. coli cells exhibited low sensitivity to laser radiation at lambda = 532 nm.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

The lethal and mutagenic action of 3H incorporated into the 2' position of the deoxyribose in the DNA of the extracellular phage lambda]

The lethal and mutagenic effects of 3H decay in 2' position of deoxyribose residues in DNA of extracellular lambda phage were studied, [2'-3H]-deoxyadenosine (3H-dA) or [2'-3H]-thymidine (3H-dT) being used as labelled DNA precursors. As estimated by the efficiency of the lethal and mutagenic actions of 3H decay in position 2' was significantly lower than that of the decay in the incorporated 3H-pyrimidines. The genetic effects of 3H decay in 2' position may be attributed to the radiation effect of beta-particles on DNA. In UV-irradiated E. coli cells, with the induced SOS repair, the mutagenic effect of 3H-dA in phage lambda is significantly higher than that of 3H-dT. This is perhaps related to the formation in DNA of AP-sites, resulting from 3H-decay in 2' position, and to the predominant incorporation of adenosine residues opposite to AP-sites during SOS repair.     

DNA sequence changes in mutations induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ and urvA cells

Summary Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr ⁺ and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr ⁺ cells. This conclusion is confirmed by analysis of published results for genes in both uvr ⁺ and uvr ^– cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cl repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr ⁺ cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Lack of additive effect in mutagenesis of E. coli by UV-light and ethylene oxide

The interaction of UV-irradiation and ethylene oxide (EtO) on forward mutation frequencies in the lacI gene of E. coli strains 3835 and 3951 and on the frequency of leu+ revertants in E. coli WU36-10-89 were studied. Pre-exposure to low doses of UV-light with the following treatment by low and intermediate doses of EtO showed lack of additive effect in the mutagenic response in all strains studied. The number of mutants actually obtained in the respective experiments was much lower than the additive model predicted.     

Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS)

Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration. IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man). Truncation of IIC(Man) renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).