Protective Roles of Bacterioruberin and Intracellular Kcl in the Resistance of Halobacterium Salinarium Against DNA-Damaging Agents
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Protective Roles of Bacterioruberin and Intracellular KCl in the Resistance of Halobacterium salinarium against DNA-damaging Agents HAMID REZA SHAHMOHAMMADI, EZAT ASGARANI, HIROAKI TERATO, TAKESHI SAITO, YOSHIHIKO OHYAMA, KUNIHIKO GEKKO, OSAMU YAMAMOTO and HIROSHI IDE* Graduate Department of Gene Science, Faculty of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8 526, Japan (Received, March 16, 1998) (Revision received, October 15, 1998) (Accepted, October 30, 1998) Halobacterium salinarium/Bacterioruberin/Salt effect! Radiation/DNA-damage Halobacteriumm salinarium, a member of the extremely halophilic archaebacteria, contains a C50-caro tenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protec tive roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC). The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spec tra of H. salinarium chromosomal DNA at various concentrations of KCI (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. How ever, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCI, presumably due to scavenging of free radicals. These results suggest that bacterioruberin and intracellular KC1 of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents. INTRODUCTION Halobacterium salinarium is an extremely halophilic archaebacterium. When grown aero bically, it produces large amounts of a red membrane consisting of C50-carotenoids called bacterioruberin' ). In our previous work', it has been shown that H. salinarium is highly resistant to the lethal effects of DNA-damaging agents including 60Co y-rays, ultraviolet light (UV), and *Corresponding author: Tel; +81-824-24-7457 , Fax; +81-824-24-7457, E-mail; [email protected] mitomycin-C (MMC). The molecular mechanisms responsible for this resistance is not clear. Photoreactivation of UV-induced pyrimidine dimers has been demonstrated halobacteria3), but the additional resistance of H. salinarium to ionizing radiation and MMC has led us to examine the molecular mechanisms involved in its high resistance to various types of DNA-damaging agents. Reactive oxygen species such as superoxide (02), hydrogen peroxide (H202),and hydroxyl radicals (. OH), are capable of damaging DNA, protein, and other cell components, generating a variety of so-called oxidative damage4`s'. Damage to DNA results in mutagenesisand cell death6,7). Carotenoids like /3-carotene are potent free radical scavengers, singlet oxygen quenchers, and lipid antioxidants' 12).Recently, we have found that the hydroxyl radical-scavenging ability of bacterioruberin is greater than that of /3-carotene' 3'. This also suggests that the high resistance of H. salinarium is at least partly due to the presence of a C50-carotenoid,namely bacterioruberin2) . In addition, halobacteria are characterized by their absolute requirement of very high levels of salts for growth 14). The intracellular potassium content of H. salinarium is about 5.3 M, greater than that of saturated KC115). It has been reported that the sensitivity of purified bacteriophage SP02c12 DNA to UV-inactivation decreases with increasing ionic strength16). In light of these unique features of H. salinarium, the present study was undertaken to examine the possible protective roles of bacterioruberin and concentrated cellular KCI of this bacterium against the lethal effects of DNA-damaging agents. We report here that the bacterioruberin-deficient mutant of H. salinarium is more sensitive to DNA-damaging agents such as ionizing radiation, H202 and UV, but not to MMC than the wild-type strain. Moreover, DNA irradiated by ionizing radiation exhibited less breakdown in the presence of either bacterioruberin or concentrated KC1 compared to in their absence. These results indicate that bacterioruberin and intracellular KCl of H. salinarium play important roles in the resistance of this organism against the lethal effects of oxidative DNA-damaging agents but not the cross-linking agent MMC. MATERIALS AND METHODS Bacteria and growth conditions The red pigmented wild-type Halobacterium salinarium NRC 34002, and a colourless mutant were used in this study. The wild-type strain was kindly provided by Dr. S. C. Kushwaha of the University of Ottawa, Canada. The colourless mutant was isolated in this work using MNNG mutagenesis as described below. Complex medium (CM) of Sehgal and Gibbons 17)was used throughout this experiment for growth of bacteria. The medium contained 200 g NaC1, 2 g KC1, 20 g MgSO4 . 7H20, 2.3 mg FeC12• 4H2O, 3 g trisodium citrate, 10 g Bacto-yeast extract (Difco), 7.5 g Bacto-casamino acid (Difco) and 8.33 ml glycerol per liter of distilled water. The solutions containing the salts and the organic nutrients were autoclaved separately at 120°C for 10 min, allowed to cool and combined, then adjusted to pH 6.8, and finally autoclaved again at 120°C for 20 min. H. salinarium (wild-type) and the colourless mutant were grown with shaking at 37°C for 48 h in the fresh medium. Cells at logarithmic phase were collected by centrifugation at 1,200 x g for 10 min, washed three times, and then resuspended at a concentration of 107 cells/ ml. Twenty % NaCI solution in phosphate buffer (67 mM, pH 6.8) was used for cell washing and resuspension. When the medium was required in a solid form, 1.5% (w/v) Bacto-agar (Difco) was included. Escherichia coli B/r was a lab stock. Luria Bertani medium (LB) was used in this experi ment for growth of E. coli B/r. The medium contained 10 g NaCI, 10 g polypeptone and 5 g yeast extract (Difco) per liter of distilled water, and was adjusted to pH 6.8. Cells of E. coli B/r were grown up at 37°C for 3 h. Cells at the logarithmic phase were collected by centrifugation, washed, and then resuspended by the same method as above. Phosphate buffer (67 mM, pH 6.8) was used for cell washing and resuspension. Isolation of colourless mutant The wild-type strain of H. salinarium was grown in the complex medium to the logarithmic phase (107 cells/ml), collected by centrifugation, washed and resuspended in 20% NaCI solution containing 100 µg/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After shaking gently at 37°C for 1 h, the cells were collected by centrifugation, washed 3 times and resuspended in the complex medium. The cells were grown for 5 h at 37°C to allow the phenotypic expression of induced mutation, then plated on CM-agar plates after appropriate dilution. A colourless mutant was isolated after incubation at 37°C for one week. Isolation of chromosomal DNA DNA was isolated from H. salinarium by the modified method of Berns and Thomas's). Cells were washed twice with SSC (0.15 M NaCI, 0.015 M sodium citrate), resuspended in 27% sucrose in SSC, and lysed with 25% SDS (final concentration 1%) at 60°C for 10 min. Pronase was then added to a final concentration of 1 mg/ml, and the lysate was digested for 7 h at 37°C. The lysate was extracted with an equal volume of TE-saturated phenol by shaking gently at 60 rev./min for 20-30 min. After centrifugation at 1,200 x g for 10 min, the aqueous layer was dialyzed overnight against SSC. Then RNase A (DNase free) was added to a final concentration of 10 µg/ml, and the solution was incubated at 37°C for 30 min, and extracted with phenol twice. DNA was recovered by ethanol precipitation and dissolved in TE buffer (10 mM Tris-HCI, 1mM EDTA, pH 7.5). Extraction of bacterioruberin The wet cells of H. salinarium (ca. 13 g) were ground with 2 volumes of quartz powder in a porcelain mortar and extracted with ethanol. An equal volume of ether was added to the extract, and 15% NaCI aqueous solution was then added to separate the aqueous and organic layers. The organic layer was dried over anhydrous sodium sulfate, and concentrated to dryness under re duced pressure. A benzene solution (ca. 1 ml) of the crude extract was loaded on the silica gel column (0 10 mm x 450 mm, Merck silica gel 60). Pigments were eluted with acetone : benzene (42 : 58), and the fractions containing bacterioruberin were pooled and concentrated under re duced pressure. The bacterioruberin was dissolved in acetone and stored under N2 gas in the dark at -30°C. Measurement of circular dichroism (CD) spectra DNA solutions (40 p g/ml) were prepared in phosphate buffer (10 mM, pH 7) in either the absence or presence of KCl (0.1-3.5 M). CD spectra were recorded with a J-720 W spectropola rimeter (Jasco Inc.) using a 1 cm quartz cuvette at 25°C. y-Irradiation The cell suspension (3 ml, 107 cells/ml) was irradiated in a 5 ml glass tube with 60Co y-rays at a dose-rate of 3 Gy/min at 0°C. Colony counting was performed on CM-agar plates after incubation at 37°C for 7-8 days. UV-Irradiation The cell suspension (5 ml, 107 cells/ml) was UV-irradiated at 254 nm in an open petri dish (0 5 cm) with a Mitsubishi Electric 15-watt germicidal lamp (dose rate = 0.4 J/m 2/sec). The suspension was stirred with a magnetic stirrer during irradiation.