Mutagenic and lethal action of polychromatic near-ultraviolet (325–400 nm) on Haemophilus influenzae in the presence of nitrogen

Mutation Research, 244 (1990) 141-145 Elsevier

141

MUTLET 0351

Mutagenic and lethal action of polychromatic near-ultraviolet (325-400 nm) on Haemophilusinfluenzaein the presence of nitrogen* Emiliano Cabrera-Ju/Lrez** and Mercedes Espinosa-Lara** Laboratorio de Genbtica Molecular, Departamento de Bioquimica, Escuela Naeional de Ciencias Biolbgicas, LP.N., Carpioy Plan de Ayala, 11340 Mexico City 17 (Mexico) (Accepted 18 December 1989)

Keywords: Near-UV light; Aerobic vs. anaerobic irradiation; Haemophilus influenzae

Summary The lethal effect of polychromatic near-UV light (325-400 nm) on Hearnophilus influenzae was 8 times higher under aerobic than anaerobic irradiation. This light increased the frequency of mutation to novobiocin resistance and ability to utilize protoporphyrin IX. The slope of mutagenic effect at low doses appeared greater for the aerobic than for the anaerobic group. We concluded that polychromatic near-UV mutation of H. influenzae under anaerobic irradiation was caused by direct oxygen-independent action on DNA.

We have shown that polychromatic near-UV light from 325 to 400 nm had lethal and mutagenic effects on H a e m o p h i l u s influenzae, in the presence o f air and the absence of exogenous photosensitizing compounds (Cabrera-Ju~irez and EspinosaCorrespondence: Dr. E. Cabrera-Ju~irez, Laboratorio de Gen6tica Molecular, Departamentode Bioquimica, Escuela Nacional de Ciencias Biol6gicas, I.P.N., Carpio y Plan de Ayala, 11340 Mexico City 17 (Mexico). *This investigation was sponsored by DGI-IPN (project: 83096), CONACYT (project: PCCBCNA-001042)and by the COSNET-Subsecretaria de Educaci6n e Investigaci6n Tecnol6gicas, S.E.P. (project 181/84), Mexico. **Fellow of the Direcci6n de Especializaci6n Docente e Investigaci6n Cientifica y Tecnol6gica-COFAA,I.P.N. Mexico.

Lara, 1974). This light caused the inactivation of transforming D N A from H. influenzae by 2 processes, one being dependent and another oxygenindependent (Cabrera-Jufirez, 1964). Thus we expected and found this light to have a relatively large lethal effect on H. influenzae cells under anaerobic irradiation, and a preliminary report of this finding has been published (Espinosa-Lara and CabreraJu~irez, 1983). It was surprising to find that monochromatic near-ultraviolet light did not mutate H. influenzae in the presence or absence of oxygen (Cabrera-Ju/trez and Setlow, 1979, 1980). In these studies, after irradiation the cultures were incubated until they reached about 0 . 5 - 1 . 0 x 10 9 cells/ml before plating, according to Kimball and Setlow (1972). We report now that, with the same

0165-7992/90/$ 03.50 © 1990Elsevier Science Publishers B.V. (Biomedical Division)

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procedure, polychromatic near-UV light mutagenized Haemophilus influenzae both in the presence and in the absence of oxygen.

Materials and methods

Microorganism H. influenzae Rd was used. Culture o f the cells to be irradiated H. influenzae was grown in brain heart infusion (Difco); hemin and oxidized nicotinamide adenine dinucleotide (NAD ÷ ), at final concentrations of 10 and 2 /zg/ml respectively (Goodgal and Herriott, 1961), were used as supplement. Growth was followed in a Klett-Summerson photocolorimeter using a red filter with a maximal transmittance at 660 nm. Under these conditions, 1 optical density unit is equal to 2 × 101° viable cells/ml. A 2-ml a m o u n t of cells was placed in a 250-ml Erlenmeyer flask containing 10 ml of growth medium. The bacterial culture was incubated at 37°C with gentle shaking until an optical density (OD) of 0.3 was obtained.

Irradiation with polychromatic near- UV light The fresh culture at an OD of 0.3 was centrifuged to eliminate the complex medium, and the bacterial cells were then washed twice and suspended at a concentration of 3 × 109 cells/ml in a solution containing 0.1 M sodium chloride, 0.01 M phosphate buffer, and 0.02% Tween 80 at p H 7.0 (Cabrera-Ju~irez and Herriott, 1963). The bacterial suspension was irradiated with a 15-W General

Electric bulb (F. 15T8/BLB) with a range of emission of 313-400 nm and a m a x i m u m at 355 nm. A 1 cm thick layer of 1.3% naphthalene solution in ethanol was used as filter to absorb almost all the radiation below 325 nm (Cabrera-Ju~irez and Espinosa-Lara, 1974). The bacterial suspension in a 3-ml volume was in a 15 × 150 m m tube, which was inside another test tube that contained the naphthalene filter. The suspension was 5 cm from the radiation source; the temperature was 4°C. Air or nitrogen was bubbled through the suspension 10 min before and during irradiation. To eliminate most of the oxygen contamination, the nitrogen was put through pyrogallol before going to the sample. Irradiation in the presence of air or nitrogen was performed at the same time. The intensity was 4.6 J / m Z / s and was measured with a dosimeter Model J-221 (Ultraviolet Products). Samples of 0.3 ml were taken before and after various irradiation doses and kept at 4°C until all the samples were obtained.

Measurement o f survival and mutation Part of each sample (0.1 ml) was diluted with 10%0 growth medium in 0.15 M NaCI and plated immediately after the last sample was taken. The survival is given as percent of unirradiated sample. For mutation assays, 8 ml of growth medium was added to the remaining 0.2 ml of the samples, and

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Fig. 4. Mutation frequency for novobiocin resistance as a function of dose of polychromatic near-UV light. Irradiation in nitrogen. 2 ml o f irradiated cells were grown to an OD of 0.5 before plating.

the mixture was grown to an OD of 0.5 before plating; in other experiments 2 ml of the sample was used instead of 0.2 ml (Cabrera-Ju~irez and Setlow, 1979). The assay for novobiocin (1/zg/ml) resistance mutants and for those able to utilize 1 #g/ml protoporphyrin IX was peformed as described before (Cabrera-Ju~irez and Espinosa-Lara, 1974; Carbrera-Ju~trez and Setlow, 1979). Growth medium containing hemin but not antibiotic was used for plating of total viable cells.

greater for the aerobic than for the anaerobic group. Similar results were found for the increase of the mutation frequency for ability to utilize protoporphyrin IX (Fig. 3). The results shown in Figs. 2 and 3 were obtained when 0.2 ml of irradiated cells were used to grow to an OD of 0.5 before plating. Those obtained using 2.0 ml of irradiated cells under nitrogen are presented in Fig. 4. It is clear that the broad near-UV irradiation also increased the mutation frequency for novobiocin resistance. Fig. 5 shows the growth curves of the irradiated cells under nitrogen, using 0.2-ml (A) or 2.0-ml samples (B) into 8 ml of growth medium. It can be seen that the growth was a little faster with 2.0-ml than with 0.2-ml samples.

Results

Lethal action o f polychromatic near-UV light on H. influenzae Fig. 1 shows the loss of viability after irradiation in the presence of nitrogen or air. The inactivation in air showed a shoulder, which was not apparent when the irradiation was under nitrogen. Comparison of the 1/e doses during aerobic and anaerobic irradiation shows that the lethal effect was 8 times higher under aerobic than under anaerobic conditions.

Mutagenic effect o f polychromatic near-UV light on H. influenzae The polychromatic near-UV irradiation increased the mutation frequency for novobiocin resistance (Fig. 2). The slope at low doses appears

Discussion

Polychromatic near-UV light (325-400 nm) has been shown to be lethal for H. influenzae under aerobic irradiation and in the absence of exogenous photodynamic compounds (Cabrera-Ju~irez and Espinosa-Lara, 1974). We have shown here that irradiation under anaerobic conditions also had a substantial lethal effect. The lethal action of polychromatic near-UV is thus apparently caused by 2 mechanisms, one that requires oxygen and perhaps endocellular photodynamic compounds

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and another that is oxygen-independent. These 2 mechanisms were also shown for the near-UV light inactivation of transforming D N A from H. influenzae (Cabrera-Ju~irez, 1964; Cabrera-Ju~irez et al., 1976). The oxygen-dependent lethal effect seems predominant at 365 nm during aerobic irradiation both in H. influenzae (Cabrera-Ju~irez and Setlow, 1980) and in E. coli (Webb and Brown, 1979), and may be due to the formation of single-strand breaks and pyrimidine dimers (Cabrera-Ju~irez and Setlow, 1980; Webb and Brown, 1982). In contrast, with 334-nm light (Cabrera-Ju~irez and Setlow, 1980) and polychromatic near-UV, lethality appears more the result of an oxygenindependent mechanism, and may involve the formation of a thymine-containing near-UV photoproduct also produced in purified transforming D N A (Cabrera-Ju~irez and Setlow, 1977). It has been shown that monochromatic near-UV light mutates transforming D N A from H. influenzae under aerobic conditions both in vitro (Cabrera-Ju~irez and Setlow, 1976) and in vivo (Cabrero-Ju~irez and Setlow, 1981), and that polychromatic near-UV light mutates transforming D N A in vitro under aerobic and anaerobic conditions (Chapital-Blanno and Cabrera-Ju~irez, 1985). We conclude that this direct oxygen-independent mutation of DNA is responsible for the polychromatic near-UV mutation of 1-1. influenzae

under anaerobic irradiation. The question why cells are not mutagenized by monochromatic near-UV light (Cabrera-Ju~irez and Setlow, 1979, 1980), whereas mutation was found with polychromatic near-UV under aerobic (Cabrera-Ju~irez and Espinosa-Lara, 1974) or anaerobic irradiation, cannot be answered by different amounts of growth before testing for mutation, because the increase of the mutation frequency for novobiocin resistance (Fig. 4) was obtained under the same conditions of growing in which monochromatic light did not cause mutation (Cabrera-Ju~irez and Setlow, 1979, 1980). We suggest 2 possible explanations: (a) the mixture of wavelenghts from 325 to 400 nm in the polychromatic near-UV had some synergistic effect (Cabrera-Ju~irez and Setlow, 1979), and (b) with monochromatic near-UV light the lethal lesions appear at lower doses than the mutagenic ones (Cabrera-Ju~irez and Setlow, 1981) and these last cannot be expressed in the dead cells. The finding that the transforming D N A was mutated in vivo by monochromatic near-UV light (Cabrera-Ju~irez and Setlow, 1981) and our present results are in accord with the second explanation but do not eliminate the first possibility.

References Cabrera-Juhrez, E. (1964) 'Black light' inactivation of transforming deoxyribonucleicacid from Haemophilus influenzae, J. Bacteriol., 87, 771-778. Cabrera-Ju~trez, E., and M. Espinosa-Lara (1974) Lethal and mutagenic action of black light (325-400 nm) on Haemophilus influenzae in the presence of air, J. Bacteriol., 117, 960-964. Cabrera-Ju~rez, E., and R.M. Herriott (1963) Ultraviolet irradiation of native and denatured transforming deoxyribonucleic acid from Haemophilus influenzae, J. Bacteriol., 85,671-675. Cabrera-Ju~rez, E., and J.K. Setlow (1976) Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum, Mutation Res., 35, 199-206. Cabrera-Ju~rez, E., and J.K. Setlow (1977) Formation of thymine photoproduct in transforming DNA by near ultraviolet irradiation, Biochim. Biophys. Acta, 475, 315-322.

145 Cabrera-Ju~ez, E., and J.K. Setlow (1979) Action spectrum for lethality of near UV light on Haemophilus influenzae and lack of mutation, Mutation Res., 62, 1-6. Cabrera-Ju~irez, E., and J.K. Setlow (1980) Repair and action spectrum of oxygen-independent lethality of near UV light on Haemophilus influenzae and lack of mutation, Mutation Res., 72, 49-55. Cabrera-Ju~irez, E., and J.K. Setlow (1981) Near-ultraviolet mutation of transforming DNA irradiated in vivo, Mutation Res., 83, 301-306. Cabrera-Ju/trez, E., J.K. Setlow, P.A. Swenson and M.J. Peak (1976) Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histidine and repair, Photochem. Photobiol., 23, 309-313. Chapital-Blanno, L., and E. Cabrera-Ju~irez (1985) Nueva informaci6n gen6tica en el ~icido desoxirribonucleico por acci6n directa de la luz cercana al ultravioleta, Rev. Lat.-Am. Microbiol., 27, 267-274.

Espinosa-Lara, M., and E. Cabrera-Ju~irez (1983) Irradiaci6n anaerobica de Haemophilus influenzae con luz policromfitica cercana al ultravioleta (325-400 nm), Rev. Lat.-Am. Microbiol., 25,271-274. Goodgal, S.H., and R.M. Herriott (1961) Studies on transformation of Haemophilus influenzae. I. Competence, J. Gen. Physiol,, 44, 1201-1227. Kimball, R.F., and J.K. Setlow (1972) Mutations induced in Haemophilus influenzae by transformation with nitrosoguanidine-treated DNA, Mutation Res., 14, 137-146. Webb, R.B., and M.S. Brown (1979) Action spectra for oxygendependent and independent inactivation of Escherichia coli WP2s from 254 to 460 nm, Photochem. Photobiol., 29, 407-409. Webb, R.B., and M.S. Brown (1982) Genetic damage in Escherichia coli KI2 AB2480 by broad-spectrum nearultraviolet radiation, Science, 215,991-993. Communicated by R.J. Preston