JOURNAL OF BACTERIOLOGY, Feb. 1975. p. 735-736 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 2
Printed in U.S.A.
N OT E S Ultraviolet Light Sensitivity of Escherichia coli K-12 Strains Carrying ruv Mutations in Combination with uvrA or lon Mutant Alleles HIROAKI IYEHARA
AND
NOZOMU OTSUJI*
Faculty of Pharmaceutical Sciences, Kyushu University, Maedashi, Higashi-ku, Fukuoka 812, Japan Received for publication 30 October 1974
Strains of Escherichia coli K-12 have been prepared that carry the ruv mutation in combination with lon or uvrA mutant alleles. The ruv uvrA - double mutant is more sensitive to ultraviolet light than the uvrA single mutant, whereas the strain with ruv and Ion mutations shows the same sensitivity to ultraviolet light as the ruv- single mutant.
The ruv- mutants of Escherichia coli K-12 are sensitive to ultraviolet (UV) light and to gamma ray irradiation. They have normal ability for recombination (Rec+) and host cell reactivation (Hcr+), but form multinucleate filaments after irradiation with low doses of UV or by treatment with deoxyribonucleic acid (DNA) synthesis inhibitors (9). These properties of the mutants are very similar to those of the lonmutant of E. coli, except that the lon- mutant, but not the ruv-, forms mucoid colonies (1, 6, 7, 9, 10). These two genes are located at a different position on the E.coli chromosome; the lon gene is located close to the lac gene and the ruv is near the his gene (2, 5, 6, 9). It is known that wild-type strains of E. coli contain mechanisms that repair pyrimidine dimer produced in DNA by UV irradiation. UV-sensitive uv,-A --, uvrB- and uvrC- mutants of E. coli are deficient in their capacity for excision of such DNA damage (4). The objective of this work was to study the possible functional relationship between the ruv gene product and the uvr or lon gene products by comparing UV sensitivity of ruv- uvrA- or ruv- lon- double mutants with that of the
and H136 (AB1157 ruvB-) with strain BE5459 (Hfr lon-) which transfers the lon gene as a proximal marker. Recombinants that formed mucoid colonies on minimal agar lacking proline were isolated. The presence of the lon mutation in strains BE5580 and BE5581 was demonstrated by transducing NS31-11 (proC-; requirement for proline) with P1 phage grown on the test cultures, selecting for proC+ transductants. The frequency of the lon- marker among the proC+ transductants from the respective crosses was found to be approximately the same as that obtained with P1 grown on the proC+ lon- parent strain (2). The presence of the ruvmutation was confirmed by P1 transduction using the double mutant as donor and strain BE5284 (eda-; utilization of sodium glucuronate) as a recipient. The eda+ transductants were found to have the ruv mutation at the expected frequencies of about 20% (Iyehara and Otsuji, unpublished results). To test UV sensitivity of the mutants, bacteria grown to a logarithmic phase were collected by centrifugation, washed, and resuspended in M buffer (8) at a concentration of about 3 x 108 cells per ml. Samples were irradiated at a dose rate of 1.5 or corresponding single mutant. 3.4 ergs/mm2 per s. Survival was determined on The ruv- uvrA- mutants, BE5518 and complete broth agar (8) with incubation at 37 C BE5525, were prepared by crossing strain for about 20 h. AB1886 (AB1157 uvrA-) with strains BE5468 As seen in Fig. 1, strains with mutation in the (Hfr ruvA-) and BE5471 (Hfr ruvB-) which ruv and the uvrA genes are much more sensitive transfer the ruv gene as a proximal marker, and to UV than the uvrA - single mutant. A UV dose selection was made for receiving his+. The ruv- to reduce survivors to 37% for double mutants lon- mutants, BE5580 and BE5581, were pre- was about 1 erg/mm 2, whereas the dose of about pared by crossing strains H124 (AB1157 ruvA -) 10 ergs/mm2 was required to yield the same ,35
736
NOTES
J. BACTERIOL.
with mutation in the ruv and the lon genes show the same sensitivity to UV as the ruv- single mutants. In a previous paper we assumed that the ruv- as well as the lon- mutant is defective in some step in septum formation after inhibition of DNA synthesis, since they have normal ability for excision repair of pyrimidine dimers on DNA, but form multinucleate filaments after low doses of UV irradiation. The experiments presented in this paper indicate that the ruv and the lon gene products may influence some step in the same pathway of recovery from DNA damage induced in DNA that may be related to control of cell division of E. coli.
0D z
5.1 z
0
.4
UL.
This work was supported by a grant from the Ministry of
Education, Japan.
LITERATURE CITED 1. Adler, H. I., and A. A. Hardigree. 1964. Analysis of a gene controlling cell division and sensitivity to radiation in
Escherichia coli. J. Bacteriol. 87:720-726. 2. Donch, J., and J. Greenberg. 1968. Genetic analysis of Ion mutants of strain K-12 of Escherichia coli. Mol. Gen.
50
100 150 200 (ERGS/ MM 2 ) FIG. 1. Survival of double mutants after UVirradiation. Symbols: 0, wild (AB1157); +, uvrA- (AB1886); A, ruvA- (HI24); 0, ruvB- (HI36); E)0, Ion(AB1899, BE5578); V, ruvA - uvrA (BE5518);.*, ruvB- uvrA- (BE5525); A, ruvA- lon- (BE5580); *, ruvB- lon- (BE5581). AB1157 (F- thr-1,leu-6,proA2,his-4,thi-1,argE-3,lac Y-1, galK-2,ara-14,xyl-5,mtl1,-tsx-33,strA-31,sup-37), AB1886 (AB1157 uvrA -), HI24 (AB1157 ruvA-), HI36 (AB1157 ruvB-), AB1899 (AB1157 lon-, BE5578 (AB1157proA+ lon-, BE5518 (AB1886 his+ ruvA-), BE5525 (AB1886 his+ ruvB-), BE5580 (HI24 proA+ Ion-), BE5581 (HI36 proA+ Ion-). 0
UV DOSE
survival of the uvrA strain. Green et aL. (3) and Howard-Flanders et al. (5) reported that a lonuvr- double mutant is more sensitive to UV than is the corresponding single mutant. These results indicate that the ruv and the lon genes affect UV sensitivity through a mechanism independent of that of the uvr gene. In contrast to the ruv uvrA - double mutant, introduction of the lon - mutation into the ruv strain did not increase UV sensitivity. Strains
Genet. 103:105-115. 3. Green, M. H. L., J. Donch, and J. Greenberg. 1969. The effect of a uvr mutation on rescue of UV irradiated Ion strains of Escherichia coli. Photochem. Photobiol. 10:397-405. 4. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. Radiat. Res. 6 (Suppl):156-184. 5. Howard-Flanders, P., E. Simson, and L. Theriot. 1964. The excision of thymine dimers from DNA, filament formation and sensitivity to ultraviolet light in Escherichia coli K-12. Mutat. Res. 1:219-226. 6. Howard-Flanders, P., E. Simson, and L. Theriot. 1966. A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics
49:237-246. 7. Kantor, G. J., and R. A. Deering. 1968. Effect of nalidixic acid and hydroxyurea on division ability of Escherichia coli fil+ and lon- strains. J. Bacteriol. 95:520-530. 8. Otsuji, N. 1968. Properties of mitomycin C-sensitive mutants of Escherichia coli K-12. J. Bacteriol. 95:540-545. 9. Otsuji, N., H. Iyehara, and Y. Hideshima. 1974. Isolation and characterization of Escherichia coli ruv mutant which forms nonseptate filaments after low doses of ultraviolet light irradiation. J. Bacteriol. 117:337-344. 10. Walker, J. R., and A. B. Pardee. 1968. Evidence for a relationship between deoxyribonucleic acid metabolism and septum formation in Escherichia coli. J. Bacteriol. 95:123-131.
Vol. 121, No. 2
Printed in U.S.A.
N OT E S Ultraviolet Light Sensitivity of Escherichia coli K-12 Strains Carrying ruv Mutations in Combination with uvrA or lon Mutant Alleles HIROAKI IYEHARA
AND
NOZOMU OTSUJI*
Faculty of Pharmaceutical Sciences, Kyushu University, Maedashi, Higashi-ku, Fukuoka 812, Japan Received for publication 30 October 1974
Strains of Escherichia coli K-12 have been prepared that carry the ruv mutation in combination with lon or uvrA mutant alleles. The ruv uvrA - double mutant is more sensitive to ultraviolet light than the uvrA single mutant, whereas the strain with ruv and Ion mutations shows the same sensitivity to ultraviolet light as the ruv- single mutant.
The ruv- mutants of Escherichia coli K-12 are sensitive to ultraviolet (UV) light and to gamma ray irradiation. They have normal ability for recombination (Rec+) and host cell reactivation (Hcr+), but form multinucleate filaments after irradiation with low doses of UV or by treatment with deoxyribonucleic acid (DNA) synthesis inhibitors (9). These properties of the mutants are very similar to those of the lonmutant of E. coli, except that the lon- mutant, but not the ruv-, forms mucoid colonies (1, 6, 7, 9, 10). These two genes are located at a different position on the E.coli chromosome; the lon gene is located close to the lac gene and the ruv is near the his gene (2, 5, 6, 9). It is known that wild-type strains of E. coli contain mechanisms that repair pyrimidine dimer produced in DNA by UV irradiation. UV-sensitive uv,-A --, uvrB- and uvrC- mutants of E. coli are deficient in their capacity for excision of such DNA damage (4). The objective of this work was to study the possible functional relationship between the ruv gene product and the uvr or lon gene products by comparing UV sensitivity of ruv- uvrA- or ruv- lon- double mutants with that of the
and H136 (AB1157 ruvB-) with strain BE5459 (Hfr lon-) which transfers the lon gene as a proximal marker. Recombinants that formed mucoid colonies on minimal agar lacking proline were isolated. The presence of the lon mutation in strains BE5580 and BE5581 was demonstrated by transducing NS31-11 (proC-; requirement for proline) with P1 phage grown on the test cultures, selecting for proC+ transductants. The frequency of the lon- marker among the proC+ transductants from the respective crosses was found to be approximately the same as that obtained with P1 grown on the proC+ lon- parent strain (2). The presence of the ruvmutation was confirmed by P1 transduction using the double mutant as donor and strain BE5284 (eda-; utilization of sodium glucuronate) as a recipient. The eda+ transductants were found to have the ruv mutation at the expected frequencies of about 20% (Iyehara and Otsuji, unpublished results). To test UV sensitivity of the mutants, bacteria grown to a logarithmic phase were collected by centrifugation, washed, and resuspended in M buffer (8) at a concentration of about 3 x 108 cells per ml. Samples were irradiated at a dose rate of 1.5 or corresponding single mutant. 3.4 ergs/mm2 per s. Survival was determined on The ruv- uvrA- mutants, BE5518 and complete broth agar (8) with incubation at 37 C BE5525, were prepared by crossing strain for about 20 h. AB1886 (AB1157 uvrA-) with strains BE5468 As seen in Fig. 1, strains with mutation in the (Hfr ruvA-) and BE5471 (Hfr ruvB-) which ruv and the uvrA genes are much more sensitive transfer the ruv gene as a proximal marker, and to UV than the uvrA - single mutant. A UV dose selection was made for receiving his+. The ruv- to reduce survivors to 37% for double mutants lon- mutants, BE5580 and BE5581, were pre- was about 1 erg/mm 2, whereas the dose of about pared by crossing strains H124 (AB1157 ruvA -) 10 ergs/mm2 was required to yield the same ,35
736
NOTES
J. BACTERIOL.
with mutation in the ruv and the lon genes show the same sensitivity to UV as the ruv- single mutants. In a previous paper we assumed that the ruv- as well as the lon- mutant is defective in some step in septum formation after inhibition of DNA synthesis, since they have normal ability for excision repair of pyrimidine dimers on DNA, but form multinucleate filaments after low doses of UV irradiation. The experiments presented in this paper indicate that the ruv and the lon gene products may influence some step in the same pathway of recovery from DNA damage induced in DNA that may be related to control of cell division of E. coli.
0D z
5.1 z
0
.4
UL.
This work was supported by a grant from the Ministry of
Education, Japan.
LITERATURE CITED 1. Adler, H. I., and A. A. Hardigree. 1964. Analysis of a gene controlling cell division and sensitivity to radiation in
Escherichia coli. J. Bacteriol. 87:720-726. 2. Donch, J., and J. Greenberg. 1968. Genetic analysis of Ion mutants of strain K-12 of Escherichia coli. Mol. Gen.
50
100 150 200 (ERGS/ MM 2 ) FIG. 1. Survival of double mutants after UVirradiation. Symbols: 0, wild (AB1157); +, uvrA- (AB1886); A, ruvA- (HI24); 0, ruvB- (HI36); E)0, Ion(AB1899, BE5578); V, ruvA - uvrA (BE5518);.*, ruvB- uvrA- (BE5525); A, ruvA- lon- (BE5580); *, ruvB- lon- (BE5581). AB1157 (F- thr-1,leu-6,proA2,his-4,thi-1,argE-3,lac Y-1, galK-2,ara-14,xyl-5,mtl1,-tsx-33,strA-31,sup-37), AB1886 (AB1157 uvrA -), HI24 (AB1157 ruvA-), HI36 (AB1157 ruvB-), AB1899 (AB1157 lon-, BE5578 (AB1157proA+ lon-, BE5518 (AB1886 his+ ruvA-), BE5525 (AB1886 his+ ruvB-), BE5580 (HI24 proA+ Ion-), BE5581 (HI36 proA+ Ion-). 0
UV DOSE
survival of the uvrA strain. Green et aL. (3) and Howard-Flanders et al. (5) reported that a lonuvr- double mutant is more sensitive to UV than is the corresponding single mutant. These results indicate that the ruv and the lon genes affect UV sensitivity through a mechanism independent of that of the uvr gene. In contrast to the ruv uvrA - double mutant, introduction of the lon - mutation into the ruv strain did not increase UV sensitivity. Strains
Genet. 103:105-115. 3. Green, M. H. L., J. Donch, and J. Greenberg. 1969. The effect of a uvr mutation on rescue of UV irradiated Ion strains of Escherichia coli. Photochem. Photobiol. 10:397-405. 4. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. Radiat. Res. 6 (Suppl):156-184. 5. Howard-Flanders, P., E. Simson, and L. Theriot. 1964. The excision of thymine dimers from DNA, filament formation and sensitivity to ultraviolet light in Escherichia coli K-12. Mutat. Res. 1:219-226. 6. Howard-Flanders, P., E. Simson, and L. Theriot. 1966. A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics
49:237-246. 7. Kantor, G. J., and R. A. Deering. 1968. Effect of nalidixic acid and hydroxyurea on division ability of Escherichia coli fil+ and lon- strains. J. Bacteriol. 95:520-530. 8. Otsuji, N. 1968. Properties of mitomycin C-sensitive mutants of Escherichia coli K-12. J. Bacteriol. 95:540-545. 9. Otsuji, N., H. Iyehara, and Y. Hideshima. 1974. Isolation and characterization of Escherichia coli ruv mutant which forms nonseptate filaments after low doses of ultraviolet light irradiation. J. Bacteriol. 117:337-344. 10. Walker, J. R., and A. B. Pardee. 1968. Evidence for a relationship between deoxyribonucleic acid metabolism and septum formation in Escherichia coli. J. Bacteriol. 95:123-131.
