JOURNAL OF BACTERIOLOGY, Mar. 1976, p. 1214-1216 Copyright X 1976 American Society for Microbiology
Vol. 125, No. 3 Printed in U.SA.
Effect of Mutations in Deoxyribonucleic Acid Repair Pathways on the Sensitivity of Escherichia coli K-12 Strains to Nitrofurantoin S. T. JENKINS* AND P. M. BENNETT University ofBristol, Department of Bacteriology, Medical School, University Walk, Bristol BS8 lTD, England
Received for publication 23 September 1975
Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of deoxyribonucleic acid lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains.
Members of the nitrofurantoin group of compounds are reduced in vivo to yield the active antibiotics (2, 4) that have been shown to bind to nucleic acids (4) and are known to be mutagenic (5). If the nitrofurans exert a lethal effect on bacterial cells by damaging deoxyribonucleic acid (DNA), it seems reasonable to expect that mutants that are deficient in DNA repair will be more susceptible to the action of these drugs than cells that are proficient in the repair of DNA. Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of DNA lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains. The strains used in this study are listed in Table 1. Our results (Table 1) show that mutations in the recA, recB, recC, or polA genes render the cell more sensitive to nitrofurantoin. The extent to which the cell is sensitized is dependent upon which mutation is introduced into the genome (Table 1). The acquisition of the recA13 mutation (or recA56) results in a 25- to 50-fold decrease in resistance to nitrofurantoin. On the other hand, introduction of the recC22 mutation leads to a decrease in resistance of little more than twofold. A comparison of the various rec- strains shows that sensitivity to nitrofurantoin increases as the cell's ability to mediate recombination decreases. Support for the conclusion that nitrofurantoin sensitivity can be correlated with the deficiency in recombination ability in these strains comes from the observation that UB2273, a rec+ revertant of JC6310, recovers recombination proficiency and resistance to nitrofurantoin simultaneously. Furthermore, the introduction of one of the indirect suppressors of the recB -C- mutations, sbcA or sbcB (3), into strains carrying the double lesion recB21 recC22 alleviates not only the
deficiency in recombination ability but also restores resistance to nitrofurantoin to a level characteristic of the parent strain. However, sensitivity to nitrofurantoin is not rigorously coupled to a deficiency in recombination ability, since mutants that have lesions in the polA gene but are still recombination proficient are more sensitive to nitrofurantoin than are the poLA+ strain from which they were derived (Table 1). However, all strains that show increased sensitivity to nitrofurantoin also display increased sensitivity to ultraviolet irradiation (Table 1). Since sensitivity to ultraviolet irradiation is indicative of an impaired DNA repair mechanism we conclude that the decrease in the efficiency of DNA repair, which results from the presence of mutations in the genes recA, recB, recC, or polA, is responsible for the increased sensitivity to nitrofurantoin. We have also shown that it is possible to detect cells carrying a recA mutation in a mixture of rec+ and recA - strains using the observed sensitivity of the recA- strains to nitrofurantoin. This has been accomplished experimentally by mixing cells of a streptomycinresistant recA - strain and cells of a nalidixic acid-resistant rec+ strain and replica plating colonies grown on nonselective agar, which had been inoculated from these mixtures, onto nutrient agar plates containing (per ml) 10 jug of nitrofurantoin, 100 jug of streptomycin, or 30 ,ig of nalidixic acid. All colonies that were resistant to nitrofurantoin were also resistant to nalidixic acid. Colonies that failed to grow in the presence of nitrofurantoin were resistant to streptomycin. Thus, colonies of the recAstrain failed to grow when replica plated onto nutrient agar containing nitrofurantoin (10 ,ig/ ml). The necessity of increasing the selective concentration of nitrofurantoin in the nutrient agar to 10 jig/ml presumably reflects an inocu-
1214
1215
NOTES
VOL. 125, 1976
TABLE 1. A comparison of some properties ofDNA repair-proficient and DNA repair-deficient mutant strains of E. coli K-12
Strain no.
Source
Single-cell minimal inhibitory concentration to nitrofurantoin (Ag ml)a
Recombination frequency from Hfr mating relative to rec+ controlb
Resistance to ultraviolet irradiationc
+
JC3272
A. J. Clark (1)
5.0
100
JC6310
JC3272 recA56 (A. J. Clark, personal communication)
0.2
3.1 x 10-4
UB1752
JC3272 thyA (trimethoprim selection)
5.0
100
+
UB2241
UB1752 srl- (NTG induced) d
5.0
100
+
UB2257
JC3272 recA13 (thy+ recA- exconjugant of UB1752)
0.2
2.6 x 10-4
-
UB2261
JC3272 recB21 (thy+ recB- exconjugant of UB1752)
1.0
4.2 x 10-2
-
UB2262
JC3272 recC22 (thy+ recC- transductant of UB1752)
2.0
10-'
-
UB2269
JC3272 recB21 recC22 (thy+ srl+ recBrecC- exconjugant of UB2241)
0.5
8.2 x 10-3
-
UB2270
JC3272 recB21 recC22 sbcA21 (thy+ srl+ his+ trp+ recB- recC- sbcA- exconjugant of UB2241)
5.0
7.0 x 10-1
+
UB2271
JC3272 recB21 recC22 sbcB12 (his+ sbcB- transductant of UB2269)
5.0
7.8 x 10-1
+
UB2272
JC3272 polAl JC3272)
(rip poA- exconjugant of
1.0
8.0 x 10-1
JC6310 rec+ revertant (NTG induced)d + 100 5.0 UB2273 a Mid-logarithmic phase cultures diluted in saline to give approximately 20 colonies/plate. Plated on nutrient agar containing different concentrations of nitrofurantoin. Incubated at 37 C for 36 h. Single-cell minimal inhibitory concentration is defined as the first concentration of antibiotic that completely prevents single colony formation. bMid-logarithmic phase cultures of donor (HfrH) and recipient bacteria were mixed (ratio, 1:1) and incubated at 37 C, mating was interrupted, and dilutions were plated for donor, recipient, and recombinant cells that were able to utilize lactose as carbon source. Recombination frequency is calculated as number of lac+
transcipients/donor. c Approximately 107 cells were plated on nutrient agar plates and different parts of the plate were exposed to 0, 20, and 40 s of ultraviolet light from a Phillips 1TUV 15-W lamp at a distance of 50 cm. Sensitivity is defined as the absence of colony-forming ability after 40 s of irradiation. +, Resistance; -, sensitivity. d NTG, N-methyl-N-nitro-N'-nitrosoguanidine.
lum effect that becomes manifest when replica plating is carried out. This property, i.e., sensitivity to nitrofurantoin, may prove useful in recognition and isolation of recA mutants from mixed populations.
Programme Grant to M. H. Richmond for studies on the molecular and epidemiological aspects of bacterial plasmids. One of us, S. T. J., was in receipt of a Medical Research Council Scholarship for Training in Research Methods.
We wish to thank Smith, Kline and French Laboratories Ltd., Welwyn Garden City, Hertfordshire, England, for their kind gift of nitrofurantoin. This work was supported by a Medical Research Council
1. Achtman, M., N. Willets, and A. J. Clark. 1971. Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isola-
LITERATURE CITED
1216
NOTES
tion and characterization of transfer-deficient mutants. J. Bacteriol. 106:529-538. 2. Asnis, R. E. 1957. The reduction of furacin by cell free extracts of furacin-resistant and parent-susceptible strains of Escherichia coli. Arch. Biochem. Biophys. 66:208-216. 3. Barbour, S. D., H. Nagaishi, A. Templin, and A. J. Clark. 1970. Biochemical and genetic studies of re-
J. BACTERIOL. combination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec- mutations. Proc. Natl. Acad. Sci. U.S.A. 67:128-135. 4. McCalla, D. R., A. Reuvers, and C. Kaiser. 1970. Mode of action of nitrofurazone. J. Bacteriol. 104:1126-1134. 5. Tazima, Y., T. Kada, and A. Murakami. 1975. Mutagenicity of Nitrofuran derivatives, including furylfuramide, a food preservative. Mutat. Res. 32:55-80.
Vol. 125, No. 3 Printed in U.SA.
Effect of Mutations in Deoxyribonucleic Acid Repair Pathways on the Sensitivity of Escherichia coli K-12 Strains to Nitrofurantoin S. T. JENKINS* AND P. M. BENNETT University ofBristol, Department of Bacteriology, Medical School, University Walk, Bristol BS8 lTD, England
Received for publication 23 September 1975
Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of deoxyribonucleic acid lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains.
Members of the nitrofurantoin group of compounds are reduced in vivo to yield the active antibiotics (2, 4) that have been shown to bind to nucleic acids (4) and are known to be mutagenic (5). If the nitrofurans exert a lethal effect on bacterial cells by damaging deoxyribonucleic acid (DNA), it seems reasonable to expect that mutants that are deficient in DNA repair will be more susceptible to the action of these drugs than cells that are proficient in the repair of DNA. Escherichia coli K-12 strains that carry mutations in one or more genes coding for proteins involved in repair of DNA lesions are more sensitive to the antibiotic nitrofurantoin than are the nonmutant parent strains. The strains used in this study are listed in Table 1. Our results (Table 1) show that mutations in the recA, recB, recC, or polA genes render the cell more sensitive to nitrofurantoin. The extent to which the cell is sensitized is dependent upon which mutation is introduced into the genome (Table 1). The acquisition of the recA13 mutation (or recA56) results in a 25- to 50-fold decrease in resistance to nitrofurantoin. On the other hand, introduction of the recC22 mutation leads to a decrease in resistance of little more than twofold. A comparison of the various rec- strains shows that sensitivity to nitrofurantoin increases as the cell's ability to mediate recombination decreases. Support for the conclusion that nitrofurantoin sensitivity can be correlated with the deficiency in recombination ability in these strains comes from the observation that UB2273, a rec+ revertant of JC6310, recovers recombination proficiency and resistance to nitrofurantoin simultaneously. Furthermore, the introduction of one of the indirect suppressors of the recB -C- mutations, sbcA or sbcB (3), into strains carrying the double lesion recB21 recC22 alleviates not only the
deficiency in recombination ability but also restores resistance to nitrofurantoin to a level characteristic of the parent strain. However, sensitivity to nitrofurantoin is not rigorously coupled to a deficiency in recombination ability, since mutants that have lesions in the polA gene but are still recombination proficient are more sensitive to nitrofurantoin than are the poLA+ strain from which they were derived (Table 1). However, all strains that show increased sensitivity to nitrofurantoin also display increased sensitivity to ultraviolet irradiation (Table 1). Since sensitivity to ultraviolet irradiation is indicative of an impaired DNA repair mechanism we conclude that the decrease in the efficiency of DNA repair, which results from the presence of mutations in the genes recA, recB, recC, or polA, is responsible for the increased sensitivity to nitrofurantoin. We have also shown that it is possible to detect cells carrying a recA mutation in a mixture of rec+ and recA - strains using the observed sensitivity of the recA- strains to nitrofurantoin. This has been accomplished experimentally by mixing cells of a streptomycinresistant recA - strain and cells of a nalidixic acid-resistant rec+ strain and replica plating colonies grown on nonselective agar, which had been inoculated from these mixtures, onto nutrient agar plates containing (per ml) 10 jug of nitrofurantoin, 100 jug of streptomycin, or 30 ,ig of nalidixic acid. All colonies that were resistant to nitrofurantoin were also resistant to nalidixic acid. Colonies that failed to grow in the presence of nitrofurantoin were resistant to streptomycin. Thus, colonies of the recAstrain failed to grow when replica plated onto nutrient agar containing nitrofurantoin (10 ,ig/ ml). The necessity of increasing the selective concentration of nitrofurantoin in the nutrient agar to 10 jig/ml presumably reflects an inocu-
1214
1215
NOTES
VOL. 125, 1976
TABLE 1. A comparison of some properties ofDNA repair-proficient and DNA repair-deficient mutant strains of E. coli K-12
Strain no.
Source
Single-cell minimal inhibitory concentration to nitrofurantoin (Ag ml)a
Recombination frequency from Hfr mating relative to rec+ controlb
Resistance to ultraviolet irradiationc
+
JC3272
A. J. Clark (1)
5.0
100
JC6310
JC3272 recA56 (A. J. Clark, personal communication)
0.2
3.1 x 10-4
UB1752
JC3272 thyA (trimethoprim selection)
5.0
100
+
UB2241
UB1752 srl- (NTG induced) d
5.0
100
+
UB2257
JC3272 recA13 (thy+ recA- exconjugant of UB1752)
0.2
2.6 x 10-4
-
UB2261
JC3272 recB21 (thy+ recB- exconjugant of UB1752)
1.0
4.2 x 10-2
-
UB2262
JC3272 recC22 (thy+ recC- transductant of UB1752)
2.0
10-'
-
UB2269
JC3272 recB21 recC22 (thy+ srl+ recBrecC- exconjugant of UB2241)
0.5
8.2 x 10-3
-
UB2270
JC3272 recB21 recC22 sbcA21 (thy+ srl+ his+ trp+ recB- recC- sbcA- exconjugant of UB2241)
5.0
7.0 x 10-1
+
UB2271
JC3272 recB21 recC22 sbcB12 (his+ sbcB- transductant of UB2269)
5.0
7.8 x 10-1
+
UB2272
JC3272 polAl JC3272)
(rip poA- exconjugant of
1.0
8.0 x 10-1
JC6310 rec+ revertant (NTG induced)d + 100 5.0 UB2273 a Mid-logarithmic phase cultures diluted in saline to give approximately 20 colonies/plate. Plated on nutrient agar containing different concentrations of nitrofurantoin. Incubated at 37 C for 36 h. Single-cell minimal inhibitory concentration is defined as the first concentration of antibiotic that completely prevents single colony formation. bMid-logarithmic phase cultures of donor (HfrH) and recipient bacteria were mixed (ratio, 1:1) and incubated at 37 C, mating was interrupted, and dilutions were plated for donor, recipient, and recombinant cells that were able to utilize lactose as carbon source. Recombination frequency is calculated as number of lac+
transcipients/donor. c Approximately 107 cells were plated on nutrient agar plates and different parts of the plate were exposed to 0, 20, and 40 s of ultraviolet light from a Phillips 1TUV 15-W lamp at a distance of 50 cm. Sensitivity is defined as the absence of colony-forming ability after 40 s of irradiation. +, Resistance; -, sensitivity. d NTG, N-methyl-N-nitro-N'-nitrosoguanidine.
lum effect that becomes manifest when replica plating is carried out. This property, i.e., sensitivity to nitrofurantoin, may prove useful in recognition and isolation of recA mutants from mixed populations.
Programme Grant to M. H. Richmond for studies on the molecular and epidemiological aspects of bacterial plasmids. One of us, S. T. J., was in receipt of a Medical Research Council Scholarship for Training in Research Methods.
We wish to thank Smith, Kline and French Laboratories Ltd., Welwyn Garden City, Hertfordshire, England, for their kind gift of nitrofurantoin. This work was supported by a Medical Research Council
1. Achtman, M., N. Willets, and A. J. Clark. 1971. Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isola-
LITERATURE CITED
1216
NOTES
tion and characterization of transfer-deficient mutants. J. Bacteriol. 106:529-538. 2. Asnis, R. E. 1957. The reduction of furacin by cell free extracts of furacin-resistant and parent-susceptible strains of Escherichia coli. Arch. Biochem. Biophys. 66:208-216. 3. Barbour, S. D., H. Nagaishi, A. Templin, and A. J. Clark. 1970. Biochemical and genetic studies of re-
J. BACTERIOL. combination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec- mutations. Proc. Natl. Acad. Sci. U.S.A. 67:128-135. 4. McCalla, D. R., A. Reuvers, and C. Kaiser. 1970. Mode of action of nitrofurazone. J. Bacteriol. 104:1126-1134. 5. Tazima, Y., T. Kada, and A. Murakami. 1975. Mutagenicity of Nitrofuran derivatives, including furylfuramide, a food preservative. Mutat. Res. 32:55-80.
