Vol. 126, No. 1 Printed in U.SA.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 550-552 Copyright © 1976 American Society for Microbiology
Ultraviolet Light Inactivation and Photoreactivation of AS-1 Cyanophage in Anacystis nidulans YUKIO ASATO Biology Department, Southeastern Massachusetts University, North Dartmouth, Massachusetts 02747
Received for publication 10 September 1975
Black light effected photorecovery of AS-1 cyanophage and wild-type cells. However, only partial photoreactivation of AS-1 was observed in a partially photoreactivable mutant of Anacystis nidulans.
Photorecovery of ultraviolet (UV) radiation damage has been demonstrated in cyanobacteria (1, 8, 9) and cyanophage LPP-1 (9). Purified photoreactivation (PR) enzyme of Anacystis nidulans was reported to split pyrimidine dimers maximally at 436 nm (6). The UV absorption spectrum of purified enzyme showed a major protein peak at 275 nm and a small peak at 418 nm, but not at 436 nm (6). Upon further purification of the enzyme, a chromophore associated with the enzyme was revealed by its excitation (peak wavelength, 420 nm) and fluorescence (peak wavelength, 470 nm) spectra (3). These in vitro studies suggest that the PR enzymes play an important role in the photorecovery of UV-irradiated cells. Recently, mutants of the cyanobacterium A. nidulans have been reported to demonstrate a lower level of photorecovery from UV radiation, as compared with the wild type (WT) (1). Analysis of partially photoreactivable mutants could yield information of photorepair systems in cyanobacteria. The cyanophage AS-1, isolated by Safferman et al. (5), permits investigation on the effects of UV radiation on the phage deoxyribonucleic acid (DNA) within its unlcellular cyanobacterial host, A. nidulans. One could then test whether the partial PR characteristic of these mutants would result in partial PR of the cyanophage DNA. In this communication, I report the results of my studies on PR of AS-1 in the WT and in a partially photoreactivable mutant of A. nidulans. Early infection stages were analyzed according to the procedure of Safferman et al. (5), with some modification. A. nidulans 625 served as the host, and infection was performed at 32 C. Cells were grown on Dm medium (7) under white fluorescent bulbs (Sylvania, F40 CWX). After 1 h, 71% phage adsorption was detectable, although maximal adsorption occurs at 2 h. The adsorbed cell fraction increased from zero time, maintained a plateau from h 1 and 2 after infection, and then showed a subse-
quent decrease. This decrease is expected, since a fraction of the infected cells begins to lyse. The eclipse phase is approximately 1 h under these conditions. To analyze UV inactivation of cyanophage within the host cell, survival curves were determined on cyanophage alone, as compared with cyanophage DNA within the host. Dark-survival curves (in the absence of PR wavelengths) of free cyanophage and cyanophage within host cells are remarkably similar (see Fig. 1). Apparently little or no protection of cyanophage DNA is afforded by the host cell. For the dosage sufficient to inactivate 99% of phage, WT host cell inactivation is about 5%. This difference remains to be explained. The results demonstrate, however, that the PR of cyanophage DNA could be determined without involving other photorecovery systems in cyanobacteria. PR experiments were conducted, and the results are shown on Fig. 2. The black light (fluorescent bulb, Sylvania BLB 40) used here has a major peak at 350 nm and a minor peak at 436 nm. The partially photoreactivable mutant, uvs-67, was isolated as described elsewhere (1). Note that phage growth of the unirradiated phage-host complex does not occur under blacklight illumination (solid squares). Gold fluorescent light does not promote PR (open squares in Fig. 2A). The increases in plaque-forming units indicated for WT (solid circles) and for the partially photoreactivable mutant (open circles) are, therefore, ascribed to PR of UV-damaged phage DNA. Efficient PR of AS-1 DNA in WT cells and host cell DNA is observed (solid circles, Fig. 2A and B). In contrast, uvs-67 displays partial PR of cyanophage DNA, as well as partial PR of its own cellular DNA (open circles, Fig. 2A and B). Although not shown, partial PR of AS-1 was also obtained when AS-1 was irradiated before adsorption of host cells for determination of PR properties. For the UV dosage applied, the data suggest that the PR of cyanophage DNA involved pho-
VOL. 126, 1976
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TIME, sec FIG. 1. Inactivation of AS-1 cyanophage. To determine inactivation offree cyanophage, 5 ml of minimal medium containing 107 plaque-forming units per ml was transferred into 55-mm-diameter petri dish. The absorbance of the suspension at 260 nm was 1.08. The suspension was irradiated with a germicidal lamp (General Electric, G8T5) at a distance of 20 cm. The dose rate measured by a photovoltaic cell (ultraviolet meter, model J-225, Ultraviolet Products, Inc.) was 10.2 Wm-2 for wavelengths 230 through 270 nm, with peak sensitivity of 250 nm. At 2-s intervals, samples were removed and plated with 0.1 ml of WT Annacystis (108 cells) on Dm agar plates. To determine the effect of UV irradiation on cyanophage DNA within the host cell, AS-1 phage was permitted to infect Anacystis for 40 min. The host cell concentration and multiplicity of infection were 107 per ml and 0.1, respectively. The mixture was centrifuged twice at 3,000 rpm for 10 min. Five milliliters ofphosphate buffer (0.01 M K2HPO4, pH 8) was added to the pellet, mixed, and transferred to a petri dish. The absorbance of the infected cell suspension at 260 nm was 1.7. UV irradiation, sampling, and plating were done as above. All plates were placed under gold fluorescent light (GE, F40, GO) at room temperature, and plaques were counted after 5 to 7 days. Open circles, inactivation of free cyanophage. Solid circles, inactivation of cyanophage-host cell complex. Solid squares, inactivation of Anacystis.
torepair of pyrimidine dimers and that a common enzyme is responsible for the PR of UVdamaged DNA of the host cells and cyanophage. Biochemical analysis of pyrimidine photorepair in determining the partial PR characteristic of these mutants is quite difficult (unpublished data) because of the low-level incorporation of labeled pyrimidines (4, 2). Two or more different interpretations could account for the partial PR of uvs-67. (i) The mutant is a "leaky" mutant, i.e., the enzyme retains some activity. (ii) A mutation has altered the active site of the PR enzyme. (iii) There is more than one distinct class of photorepair enzymes, and
FIG. 2. PR of AS-1 cyanophage and host cells. (A) WT (solid circles) and uvs-67 (open circles) were infected at a multiplicity of infection of0.1 for 40 min and then centrifuged twice to remove unadsorbed phage. The phage-host complexes in phosphate buffer (absorbance at 260 nm, 1.7) were irradiated for 5 s to obtain approximately 1% AS-1 survivors. The suspension was immediately exposed to black light (Sylvania, BLB 40) at a distance of 15 cm. PFU, Plaqueforming units. Controls: (a) non-irradiated WTphage complexes exposed to black light (darkened squares). (b) irradiated WT-phage complexes exposed to gold light (open squares). Plating and incubation of WT and uvs-67 were described in Fig. 1. (B) UV irradiation and PR of WT and uvs-67 were done as described previously (2). Solid circles, PR of WT. Open circles, PR of uvs-67.
uvs-67 has lost the activity in one of them. It is not possible to distinguish these possibilities with the data that are presently available. I thank Rose McQuaid and Bill Auerbach for their technical assistances in these experiments.
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Anacystis nidulans and Synechococcus cedrorum. Virology 47:105-113. 6. Saito, N., and H. Werbin. 1970. Purification of bluegreen algal deoxyribonucleic acid photoreactivating enzyme. An enzyme requiring light as a physical cofactor to perform its catalytic functions. Biochemistry 9:2610-2620. 7. Van Baalen, C. 1965. Quantitative surface plating of
J. BACTERIOL. coccoid blue-green algae. J. Phycol. 1:19-22. 8. Van Baalen, C. 1968. The effects of ultraviolet irradiation on a coccoid blue-green alga: survival, photosynthesis and photoreactivation. Plant Physiol. 43:16891695. 9. Wu, J. H., R. A. Lewin, and H. Werbin. 1967. Photoreactivation of uv-irradiated blue-green algal virus LPP-1. Virology 31:657-664.