Vol. 127, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 1022-1023 Copyright © 1976 American Society for Microbiology
Role of Rec Pathways on Sensitivity of Escherichia coli to Near-Ultraviolet and Visible Light K. GOPALAKRISHNA AND S. K. BHATTACHARJEE* Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received for publication 28 April 1976
In Escherichia coli lack of the RecRC or RecF pathway is found to cause sensitivity to near-ultraviolet and visible light. Resistance to this light is restored in the RecBC-defective strain carrying either the sbcB (Rec+) or xonA (Rec-) mutation. The sensitivity, therefore, is not found to correlate with the degree of recombination proficiency as measured by genetic crosses.
It had been observed earlier that near-ultraviolet (near-UV) and visible light (VL) in the wavelength region generally used for photoreactivation of shortwave UV (254 nm)-induced damage can itself be lethal to bacteria (3). Our studies on a eukaryotic organism, Amoeba proteus, have shown that the site of damage by such light is the nucleus and that a repair system, apparently functional only in certain phase of its life cycle, can repair such damage (1). The observation that Rec mutants of Escherichia coli are also sensitive to VL suggested that wherever the site of damage is, the recombination function is probably essential for recovery after VL exposure in prokaryotes (3). In investigations with Rec mutants of E. coli K12, Clark has shown that there are at least three different pathways of recombination (2). In studying such mutants, we have investigated the role of two of these pathways in repair of the damage by VL. No attempt has been made to investigate the site or the nature of damage. All the strains used are isogenic and listed in Table 1. The cultures were grown to exponential phase in nutrient broth at 37°C, washed, and resuspended in fresh nutrient broth (0.5 x 106 to 2.0 x 10" cells/ml) immediately before irradiation. The light source was a "Philips" 400-W white fluorescent lamp. Spectral characteristics of the lamp and the glass filter employed wer& as reported earlier (1). The intensity of light used for exposure was 2.3 x 105 J/m2 per s. To eliminate as much as possible of the radiation below 300 nm, two glass filters were interposed between the light source and the bacteria. Under these conditions AB2470-C showed 32% survival after irradiation with 6.8 x 105 J/m2 of visible light. This is the same survival shown by AB2470-C after irradiation by only 10.5 J/m2 of light at 254 nm. Hence, it
had to be shown that 1.5 x 10-3% of the energy from the visible light source was not in the far-UV region even though two glass filters were present. This was shown by determining the sensitivity of AB1885 (a uvrB mutant) under the same conditions. This strain is more sensitive to far-UV (254 nm) than is strain AB2470-C, as seen by its 0.9% survival after irradiation by 10.5 J/m2 of light at 254 nm. AB1885 is, however, totally insensitive to up to 12.3 x 105 J/m2 of the doubly filtered VL. By comparing the survival curves of AB1885, it can be estimated that less than 8 x 10-5% of the energy of the doubly filtered light is due to far-UV irradiation. After the exposure, the cultures were plated on nutrient agar plates and then incubated in the dark at 37°C. Colony counts were taken after about 40 h of incubation. The ratio of survivors after and before the exposure was plotted as function of dose. Of all the Rec mutants investigated here, the recA strain AB2463 was most sensitive to VL (Fig. 1). It is known that the recA mutation shows effects such as excessive deoxyribonucleic acid (DNA) degradation, both spontaneous and following shortwave UV exposure (5, 8), uncoupling of cell division from 'DNA synthesis (6), and high UV sensitivity (5), besides lowering the recombinational proficiency (2). Therefore, to look into the possible role of recombination function in repair of VL-induced damage, strains with blocks in RecBC and RecF pathways were considered. Strain AB2470-C, lacking the RecBC pathway, was found to be sensitive to VL. Its recombinational proficiency is only about 2% of wild type (2). In JC7623, the recombinational proficiency is restored to the order of wild-type level by an additional mutation in the sbcB gene. This also restored the VL resistance. It appeared, therefore, that the capacity to form viable recombinants was di-
1022
NOTES
VOL. 127, 1976 Strain
AB1157 AB2463 AB2470-C JC7623 JC9239 JC8218
TABLE 1. Strains used Phenotype Genotype Rec+ VLr Rec- VLS recA13 Rec- VLS recB21 recC22 Rec+ VLr recB21 recC22 sbcB15 recF143 Rec+ VLS Rec- VLr recB21 recC22 xonAl
1023
Source P. Howard-Flanders P. Howard-Flanders J. Tomizawa A. J. Clark A. J. Clark A. J. Clark
strain lacks the RecF pathway and is phenotypically Rec+, having a wild-type level of recombinational proficiency. It therefore seems that a certain function of the recF gene product is very significant in repair of VL-induced damage even in the presence of the RecBC pathway. This is in contrast to its non-essential role in producing viable recombinants in genetic crosses (4). We, therefore, conclude that certain steps in the RecBC and RecF pathways are involved in repair of VL-induced damage. However, all the steps necessary to produce viable recombinant progeny in genetic crosses are not needed for repair of VL-induced damage.
z 0
L.)
n
z
CZ
(I
We wish to thank S. K. Mahajan for his critical review of the manuscript.
VL DOSE 5 2 IN UNITS OF 10OJ/m FIG. 1. Survival of E. coli after exposure to UV and VL.
near-
rectly correlated to the resistance to VL. It was also noted earlier by Eisenstark in Salmonella and E. coli that phenotypically Rec- cells were sensitive to such light (3). However, we happened to observe certain anamolies while working with some of the Rec mutants. E. coli JC8218, which has mutation in the xonA gene in addition to recB recC mutations, was resistant to VL. This strain is phenotypically Rec- as determined by genetic crosses, having the same order of recombinational proficiency as that of the recB recC strain (7). Strain JC9239 was found to be sensitive to VL. This
LITERATURE CITED 1. Chatterjee, S., and S. K. Bhattacharjee. 1975. Effect of near ultraviolet and visible light on Amoeba. J. Cell Sci. 19:117-126. 2. Clark, A. J. 1971. Pathways of genetic recombination in bacteria. Recent Adv. Microbiol. 31:257-265. 3. Eisenstark, A. 1970. Sensitivity ofSalmonella typhimurium recombinationless (rec) mutants to visible light. Mutat. Res. 10:1-6. 4. Horii, Z., and A. J. Clark. 1973. Genetic analysis of the RecF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J. Mol. Biol. 80:327-344. 5. Howard-Flanders, P., and L. Theriot. 1966. Mutants of E. coli K-12 defective in DNA repair and in genetic recombination. Genetics 53:1137-1150. 6. Inouye, M. 1971. Plieotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid synthesis. J. Bacteriol. 106:539542. 7. Kushner, S. R., H. Nagaishi, and A. J. Clark. 1972. Indirect suppression of recB and recC mutations by exonuclease I deficiency. Proc. Natl. Acad. Sci. U.S.A. 69:1366-1370. 8. Willets, N. S., and A. J. Clark. 1969. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J. Bacteriol. 100:231-239.
JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 1022-1023 Copyright © 1976 American Society for Microbiology
Role of Rec Pathways on Sensitivity of Escherichia coli to Near-Ultraviolet and Visible Light K. GOPALAKRISHNA AND S. K. BHATTACHARJEE* Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay 400 085, India Received for publication 28 April 1976
In Escherichia coli lack of the RecRC or RecF pathway is found to cause sensitivity to near-ultraviolet and visible light. Resistance to this light is restored in the RecBC-defective strain carrying either the sbcB (Rec+) or xonA (Rec-) mutation. The sensitivity, therefore, is not found to correlate with the degree of recombination proficiency as measured by genetic crosses.
It had been observed earlier that near-ultraviolet (near-UV) and visible light (VL) in the wavelength region generally used for photoreactivation of shortwave UV (254 nm)-induced damage can itself be lethal to bacteria (3). Our studies on a eukaryotic organism, Amoeba proteus, have shown that the site of damage by such light is the nucleus and that a repair system, apparently functional only in certain phase of its life cycle, can repair such damage (1). The observation that Rec mutants of Escherichia coli are also sensitive to VL suggested that wherever the site of damage is, the recombination function is probably essential for recovery after VL exposure in prokaryotes (3). In investigations with Rec mutants of E. coli K12, Clark has shown that there are at least three different pathways of recombination (2). In studying such mutants, we have investigated the role of two of these pathways in repair of the damage by VL. No attempt has been made to investigate the site or the nature of damage. All the strains used are isogenic and listed in Table 1. The cultures were grown to exponential phase in nutrient broth at 37°C, washed, and resuspended in fresh nutrient broth (0.5 x 106 to 2.0 x 10" cells/ml) immediately before irradiation. The light source was a "Philips" 400-W white fluorescent lamp. Spectral characteristics of the lamp and the glass filter employed wer& as reported earlier (1). The intensity of light used for exposure was 2.3 x 105 J/m2 per s. To eliminate as much as possible of the radiation below 300 nm, two glass filters were interposed between the light source and the bacteria. Under these conditions AB2470-C showed 32% survival after irradiation with 6.8 x 105 J/m2 of visible light. This is the same survival shown by AB2470-C after irradiation by only 10.5 J/m2 of light at 254 nm. Hence, it
had to be shown that 1.5 x 10-3% of the energy from the visible light source was not in the far-UV region even though two glass filters were present. This was shown by determining the sensitivity of AB1885 (a uvrB mutant) under the same conditions. This strain is more sensitive to far-UV (254 nm) than is strain AB2470-C, as seen by its 0.9% survival after irradiation by 10.5 J/m2 of light at 254 nm. AB1885 is, however, totally insensitive to up to 12.3 x 105 J/m2 of the doubly filtered VL. By comparing the survival curves of AB1885, it can be estimated that less than 8 x 10-5% of the energy of the doubly filtered light is due to far-UV irradiation. After the exposure, the cultures were plated on nutrient agar plates and then incubated in the dark at 37°C. Colony counts were taken after about 40 h of incubation. The ratio of survivors after and before the exposure was plotted as function of dose. Of all the Rec mutants investigated here, the recA strain AB2463 was most sensitive to VL (Fig. 1). It is known that the recA mutation shows effects such as excessive deoxyribonucleic acid (DNA) degradation, both spontaneous and following shortwave UV exposure (5, 8), uncoupling of cell division from 'DNA synthesis (6), and high UV sensitivity (5), besides lowering the recombinational proficiency (2). Therefore, to look into the possible role of recombination function in repair of VL-induced damage, strains with blocks in RecBC and RecF pathways were considered. Strain AB2470-C, lacking the RecBC pathway, was found to be sensitive to VL. Its recombinational proficiency is only about 2% of wild type (2). In JC7623, the recombinational proficiency is restored to the order of wild-type level by an additional mutation in the sbcB gene. This also restored the VL resistance. It appeared, therefore, that the capacity to form viable recombinants was di-
1022
NOTES
VOL. 127, 1976 Strain
AB1157 AB2463 AB2470-C JC7623 JC9239 JC8218
TABLE 1. Strains used Phenotype Genotype Rec+ VLr Rec- VLS recA13 Rec- VLS recB21 recC22 Rec+ VLr recB21 recC22 sbcB15 recF143 Rec+ VLS Rec- VLr recB21 recC22 xonAl
1023
Source P. Howard-Flanders P. Howard-Flanders J. Tomizawa A. J. Clark A. J. Clark A. J. Clark
strain lacks the RecF pathway and is phenotypically Rec+, having a wild-type level of recombinational proficiency. It therefore seems that a certain function of the recF gene product is very significant in repair of VL-induced damage even in the presence of the RecBC pathway. This is in contrast to its non-essential role in producing viable recombinants in genetic crosses (4). We, therefore, conclude that certain steps in the RecBC and RecF pathways are involved in repair of VL-induced damage. However, all the steps necessary to produce viable recombinant progeny in genetic crosses are not needed for repair of VL-induced damage.
z 0
L.)
n
z
CZ
(I
We wish to thank S. K. Mahajan for his critical review of the manuscript.
VL DOSE 5 2 IN UNITS OF 10OJ/m FIG. 1. Survival of E. coli after exposure to UV and VL.
near-
rectly correlated to the resistance to VL. It was also noted earlier by Eisenstark in Salmonella and E. coli that phenotypically Rec- cells were sensitive to such light (3). However, we happened to observe certain anamolies while working with some of the Rec mutants. E. coli JC8218, which has mutation in the xonA gene in addition to recB recC mutations, was resistant to VL. This strain is phenotypically Rec- as determined by genetic crosses, having the same order of recombinational proficiency as that of the recB recC strain (7). Strain JC9239 was found to be sensitive to VL. This
LITERATURE CITED 1. Chatterjee, S., and S. K. Bhattacharjee. 1975. Effect of near ultraviolet and visible light on Amoeba. J. Cell Sci. 19:117-126. 2. Clark, A. J. 1971. Pathways of genetic recombination in bacteria. Recent Adv. Microbiol. 31:257-265. 3. Eisenstark, A. 1970. Sensitivity ofSalmonella typhimurium recombinationless (rec) mutants to visible light. Mutat. Res. 10:1-6. 4. Horii, Z., and A. J. Clark. 1973. Genetic analysis of the RecF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J. Mol. Biol. 80:327-344. 5. Howard-Flanders, P., and L. Theriot. 1966. Mutants of E. coli K-12 defective in DNA repair and in genetic recombination. Genetics 53:1137-1150. 6. Inouye, M. 1971. Plieotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid synthesis. J. Bacteriol. 106:539542. 7. Kushner, S. R., H. Nagaishi, and A. J. Clark. 1972. Indirect suppression of recB and recC mutations by exonuclease I deficiency. Proc. Natl. Acad. Sci. U.S.A. 69:1366-1370. 8. Willets, N. S., and A. J. Clark. 1969. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J. Bacteriol. 100:231-239.
