J. Mol. Hiol.
(1991)
219,
Isolation
393-398
and Characterization of Visible Light-sensitive Mutants of Escherichia coli K12
Kazumasa
Miyamoto, Kenji Nakahigashi, and Hachiro Inokuchi?
Koichi
Nishimura
Department of Biophysics Faculty of Science, Kyoto University Sakyo-lcu, Kyoto 606-01, Japan (Received
18 December
1990; accepted 23 ,January
1991)
Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that carried the visA +/GA+ diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB. These mutant strains of bacteria are killed by illumination with visible light. The effective wavelength of the light is around 460 nm. The nucleotide sequence of the visA gene was determined. As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme. A possible mechanism for the killing of the visA mutant bacteria is discussed. Keywords: light-sensitive mutant; Escherichia coli K12: hemH; protoporphyrin 1,X: protoporphyria
Recently, we came upon a novel phenomenon whereby, when a particular vector carried a specific fragment of the Escherichia coli genome, the reporter gene was expressed in the dark but not in the light (Nakahigashi et al., 1990). Although the specific* fragment was certainly associated with the light-sensitive phenomenon, the promoter activity of the fragment was very weak. Therefore, we attempted to isolate a mutant that showed a much clearer light-sensitive phenotype, expecting to isolate promoter mutants in the specific fragment inserted in the plasmid vector. We were fortunate in that a candidate mutant was obtained spontaneously. During the course of our experiments, however, we found that this mutant had no relationship to the light-sensitive promoter and the mutant, bacteria were killed by illumination with visible light. We now report the isolation and characterization of a new type of mutant of E. coli that is sensitive to visible light (which is lethal). In addition to our spontaneous mutant, we screened cultures for similar mutants by the classical penicillin-screening ? Author addressed.
to whom
all correspondence
should
be
method after chemical mutagenesis with nitrosoguanidine (Miller, 1972). Figure 1 shows the killing profile of the mutants under ordinary fluorescent light. The varying degrees of sensitivity may reflect the extent of the functional defect. All the mutant bacteria form slightly smaller colonies than the wild-type. It should be noted that the most sensitive mutant (VS103) forms red colonies on Luria broth plates when incubated at 42°C in the dark. We determined the effective wavelength of the light on the visA1 mutant strain VSlOl by the use of five different interference filters (maximum transmission wavelengths: 403, 434, 460, 4765 and 522 nm). It was clear that light with a wavelength of 460 nm was most effective in killing the mutant bacteria (data not, shown). In order to analyze the gene involved in this sensitivity to light, we initially isolated revertants of mut)ant strain VSlOl that were resistant to visible light. Genomic DNA from one of the revertants was prepared and digested with the restriction enzyme EcoRI. The fragments from the digest were cloned into phage vector, Igt.X (Thomas et al., 1974). Then, a phage clone that was able, upon infection, to complement the growth of VSlOl in
K.
394
et al.
Miqlnmoto
IOr> 1 2 2
IO-
IO-
IO-
I
I
I
60
120
180
I
240
I
300
I
360
Tome (mm) Figure 1. Effects of visible light on 4 mutants in the uisA gene of Esch~richia coli: VSIO1 (a). VSlO2 (A), VSIO~ (m) and VS104 ( x ). Each is derived from strain CA274 (Russell et al., 1970). VSlOl is a spontaneous mutant, but the other mutants were induced by treatment with nitrosoguanidine. The mutants were screened as follows: the mutagc~nixetl bacteria (in 5 ml of Luria broth) were cultivated at 37°C in t,he dark in test tubes cyjvrred wit,h aluminium foil Ilntil the logarithmic phase of growth, the tubes were then illuminated at a position 15 rm below z.?fluorescent lamps (Hitachi, Sunlight FL40SSD/37-G, 40 W). The illumination conditions (approx. 7500 lux) and the incubation t,emperature of :
(1991)
219,
Isolation
393-398
and Characterization of Visible Light-sensitive Mutants of Escherichia coli K12
Kazumasa
Miyamoto, Kenji Nakahigashi, and Hachiro Inokuchi?
Koichi
Nishimura
Department of Biophysics Faculty of Science, Kyoto University Sakyo-lcu, Kyoto 606-01, Japan (Received
18 December
1990; accepted 23 ,January
1991)
Six mutants of Escherichia coli K12 that are sensitive to visible light have been isolated. Five of them, including an amber mutant, are defective in a gene that maps near 11 minutes on the linkage map of the chromosome, and this gene has been designated visA. The sixth mutant, which was isolated from bacteria that carried the visA +/GA+ diploid allele, is defective in a gene that maps near 63 minutes on the linkage map, which has been designated visB. These mutant strains of bacteria are killed by illumination with visible light. The effective wavelength of the light is around 460 nm. The nucleotide sequence of the visA gene was determined. As a result of a search for homologous products, we found that visA may be identical to hemH, the structural gene for ferrochelatase which catalyzes a final step in the biosynthesis of heme. A possible mechanism for the killing of the visA mutant bacteria is discussed. Keywords: light-sensitive mutant; Escherichia coli K12: hemH; protoporphyrin 1,X: protoporphyria
Recently, we came upon a novel phenomenon whereby, when a particular vector carried a specific fragment of the Escherichia coli genome, the reporter gene was expressed in the dark but not in the light (Nakahigashi et al., 1990). Although the specific* fragment was certainly associated with the light-sensitive phenomenon, the promoter activity of the fragment was very weak. Therefore, we attempted to isolate a mutant that showed a much clearer light-sensitive phenotype, expecting to isolate promoter mutants in the specific fragment inserted in the plasmid vector. We were fortunate in that a candidate mutant was obtained spontaneously. During the course of our experiments, however, we found that this mutant had no relationship to the light-sensitive promoter and the mutant, bacteria were killed by illumination with visible light. We now report the isolation and characterization of a new type of mutant of E. coli that is sensitive to visible light (which is lethal). In addition to our spontaneous mutant, we screened cultures for similar mutants by the classical penicillin-screening ? Author addressed.
to whom
all correspondence
should
be
method after chemical mutagenesis with nitrosoguanidine (Miller, 1972). Figure 1 shows the killing profile of the mutants under ordinary fluorescent light. The varying degrees of sensitivity may reflect the extent of the functional defect. All the mutant bacteria form slightly smaller colonies than the wild-type. It should be noted that the most sensitive mutant (VS103) forms red colonies on Luria broth plates when incubated at 42°C in the dark. We determined the effective wavelength of the light on the visA1 mutant strain VSlOl by the use of five different interference filters (maximum transmission wavelengths: 403, 434, 460, 4765 and 522 nm). It was clear that light with a wavelength of 460 nm was most effective in killing the mutant bacteria (data not, shown). In order to analyze the gene involved in this sensitivity to light, we initially isolated revertants of mut)ant strain VSlOl that were resistant to visible light. Genomic DNA from one of the revertants was prepared and digested with the restriction enzyme EcoRI. The fragments from the digest were cloned into phage vector, Igt.X (Thomas et al., 1974). Then, a phage clone that was able, upon infection, to complement the growth of VSlOl in
K.
394
et al.
Miqlnmoto
IOr> 1 2 2
IO-
IO-
IO-
I
I
I
60
120
180
I
240
I
300
I
360
Tome (mm) Figure 1. Effects of visible light on 4 mutants in the uisA gene of Esch~richia coli: VSIO1 (a). VSlO2 (A), VSIO~ (m) and VS104 ( x ). Each is derived from strain CA274 (Russell et al., 1970). VSlOl is a spontaneous mutant, but the other mutants were induced by treatment with nitrosoguanidine. The mutants were screened as follows: the mutagc~nixetl bacteria (in 5 ml of Luria broth) were cultivated at 37°C in t,he dark in test tubes cyjvrred wit,h aluminium foil Ilntil the logarithmic phase of growth, the tubes were then illuminated at a position 15 rm below z.?fluorescent lamps (Hitachi, Sunlight FL40SSD/37-G, 40 W). The illumination conditions (approx. 7500 lux) and the incubation t,emperature of :
