Mutagenesis in Escherichia coli by visible light.

Mutation to resistance to bacteriophage T5 in continuous cultures of Escherichia coli was induced by visible light (wavelength longer than 408 nanometers) and by black light (300 to 400 nanometers). Mutation rates more than 18 times greater than the spontaneous rate (no light) were obtained with moderate, nonlethal intensities of visible light. Mutation rates for both visible and black light were proportional to irradiance.

Photodynamic effects of dyes on bacteria. III. Mutagenesis by acridine orange and 500-nm monochromatic light in strains of Escherichia coli that differ in repair capability.

In the presence of acridine orange (AO) and monochromatic 500-nm light, the recombination-deficient strain of Escherichia coli, WP10 (recA), showed a 15-fold increase in mutation rate over the wild-type (WP2) strain. Under the same conditions, strain Bs--1 (uvrB lexA lon) showed a 5-fold increase in mutation rate over strain WP2. In contrast, the endonuclease-deficient, strain, WP2s (uvrA), showed a lower AO-500 nm mutation rate than wild-type. The extremely high mutation rate of the recA strain cannot be due to error-prone inducible SOS repair since the inducible recA + function is absent. Repair of the AO-500 nm-induced lesions is likely due to a recA+-dependent, error-free, recombination process. It is concluded that the high mutation rates with AO-500 nm light obtained in chemostat cultures of recA and lexA strains occur as a consequence of errors during semi-conservative DNA replication in the presence of unrepaired DNA lesions.

Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability.

Mutation to tryptophan independence after exposure to radiation at the monochromatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability. Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA), and WP6 (polA uvrA). Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100. These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair. Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire fluence range tested. In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6, and approximately 6.0 with strain WP2. Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr+ rec+ lex+ strains. It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2-4 x 10(5) J m-2; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system. The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.

Photodynamic effects of dyes on bacteria. V. Mutagenesis by acridine orange and 460-nm or 500-nm light in strains of Escherichia coli that differ in repair capability.

With acridine orange (AO) and monochromatic 460-nm light, the mutation rate of the wild-type strain of Escherichia coli (WP2) was 3-fold higher than that of the endonuclease-deficient strain WP2 (uvrA). In addition, the mutation rates of the recombination-deficient strains WP10 (recA) and Bs-1 (uvrB lexA) were also about 3-fold less than that of wild-type strain. This observation is in striking contrast to the earlier results with AO and 500-nm light in which strains WP10 and Bs-1 yielded mutation rates that were 12-fold and 5-fold greater, respectively, than the wild-type response. The relatively large decrease in mutation rate when the uvrA endonuclease was absent together with structural considerations in the binding of AO to DNA lead us to propose that the major lesions leading to mutations produced by 460-nm light in the presence of AO may be true DNa single-strand breaks and occur before DNA replication.

Biological effects of dyes on bacteria. VI. Mutation induction by acridine orange and methylene blue in the dark with special reference to Escherichia coli WP6 (polA1).

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2 (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 microM AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10(8) cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 microM AO increased the mutation rate in the dark 14-fold. We propose that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly suceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.

Photodynamic effects of dyes on bacteria. II. Genetic effects of broad-spectrum visible light in the presence of acridine dyes and methylene blue in chemostat cultures of Escherichia coli.

Photodynamic mutagenesis was studied in chemostat cultures of Escherichia coli B/r (TlR trp) exposed to one of six different acridine dyes or methylene blue. Mutation to phage T5 resistance was induced with a broad-spectrum fluorescent-light source. All of the agents tested were photomutagenic; acridine yellow was the most efficient sensitizer and quinacrine was the least efficient. Quinacrine also was moderately mutagenic in the dark, in contrast to the other agents tested, which were not significantly mutagenic in the dark at the low concentrations tested for photomutagenesis. The mutation rate with acridine orange was directly proportional to both fluence rate and dye concentration over the ranges tested. Photomutation rates with acridine orange, proflavine and methylene blue were independent of growth rate of the chemostat cultures. These results are consistent with photomutagenesis occurring as the result of photochemical damage to DNA-dye complexes, independent of cell expression was approximately 2.5 generations for each of the photomutagens tested. This short expression delay supports an earlier segregational model for expression of phage resistance. The following results suggest that photodynamic mutagenesis is due mainly to intercalated dye molecules: (1) both acridine and 9-aminoacridine are photodynamic mutagens; (2) acridine inhibits photomutagenesis with acridine orange; and (3) neither putrescine or spermine, which bind to DNA without intercalating, inhibited photomutagenesis by acridine orange or proflavine.

Lethal effects of pyrimidine dimers induced at 365 nm in strains of E. coli differing in repair capability.

Photoreactivation (PR) after 365-nm inactivation was measured in four strains of Escherichia coli differing in repair capability. Photoreactivation was observed in the recA strains K12 AB2480 and K12 AB2463 indicating a significant role of pyrimidine dimers in the lethal action of 365-nm radiation in these strains. Significant PR was not observed in the uvrA strain, K12 AB1886, or in the repair proficient strain, K12 AB1157, after 365-nm inactivation. Biological evidence indicated that stationary phase cells had not lost the capacity for photo-enzymatic repair after fluences of 365-nm radiation of 2 X 10(6) J/m-2 or less. It is proposed that pyrimidine dimers, although induced, are not significant 365-nm lethal lesions in uvrA and wild-type strains because of their efficient dark repair.

Harmful effects of near-ultraviolet radiation used for polymerization of a sealant and a composite resin.

An electronic device used to polymerize a sealant and a composite resin has been found to emit 365-nanometer radiation at levels sufficient to kill the bacterium Escherichia coli rapidly (greater than 14,000 ergs per square millimeter per second). Because of the absence of shielding on the probe, significant amounts of energy (up to 45% of that at the probe tip) were measured at the sides of the probe. These findings--supported by the well-documented findings of biological damage caused by near-ultraviolet radiation, including skin cancer, damage to the lens of the eye, and mutagenic effects--suggest that clinicians take appropriate precautions to avoid potential hazards to themselves and their patients.