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Protective Roles of Bacterioruberin and Intracellular KCl in the Resistance of 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. such as superoxide (02), hydrogen peroxide (H202),and hydroxyl radicals (. OH), are capable of damaging DNA, , 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 . 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. 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. Subsequent experiments were performed under yellow light. Colony counting on the agar plate was performed as described for y-irradiation.

Treatment with MMC Mitomycin-C solutions (100 p g/ml) were prepared for each set of experiments by dissolv ing MMC in 20% NaCl. Various volumes of MMC solution were added to cell suspensions (107 cells/ml, 10 ml) in light-proof tubes (MMC is unstable under light). The final concentration of MMC was 1-5 p g/ml. These tubes were left standing for 1 h in an ice-water bath, centrifuged, washed with 20% NaCl three times to remove MMC, and then resuspended in 10 ml of NaCl solution. Colony counting on the agar plate was performed as described for y-irradiation.

Treatment with H202 Hydrogen peroxide (final concentration 10-50 mM) was added to 5 ml of freshly prepared cell suspension in 20% NaCl. After incubation at 37°C for lh, cells were collected and plated at appropriate dilutions on CM-agar plates to determine cell survival.

Gel electrophoresis of DNA The solution of calf thymus DNA (200 pg/ml) was prepared in phosphate buffer (1 mM, pH 7), then irradiated with 60Coy-rays in the absence or presence of KCI (2 M). Following irradia tion, the DNA solution was dialyzed against phosphate buffer (1 mM, pH 7) at 4°C for 16 h. To elucidate the effect of bacterioruberin, DNA solutions containing bacterioruberin (10-5M) and a detergent (Brij 56, 2.9 x 10-4M) were prepared in phosphate buffer (1 mM, pH 7) and irradiated with y-rays. The DNA solutions containing Brij and a SH compound (L-cysteine or reduced glutathione, both 10-5 M) were irradiated under the same conditions. In the control reactions, DNA was irradiated in the presence of Brij alone. The irradiated sample was electrophoresed on a 0.7% agarose gel in TAE buffer (40 mM Tris-acetate, 1mM EDTA, pH 8) to analyze double strand-breaks, or in alkaline buffer (50 mM NaOH, 1 mM EDTA) to analyze single strand-breaks. RESULTS

Figure 1 shows the survival curves of the wild-type and bacterioruberin-deficient (colourless) mutant of H. salinarium exposed to ionizing radiation, hydrogen peroxide, UV, and MMC. The

Figure 1. Sensitivity of the bacterioruberin-deficient mutant of Halobacterium salinarium (0), its red-pigmented wild type (•), and Escherichia coli B/r (A) to y-rays (A), hydrogen peroxide (B), UV (C) and mitomycin-C (D). The data is based on five independent experiments. D37 values calculated from the survival curves are summarized in Table 1. The experimental points and standard deviations in Figure 1 were based on five independent experiments. For comparison, data for E. coli B/r treated in a similar manner are included in Figure 1 and Table 1. The colourless mutant of H. salinarium was more sensitive to both y-rays and hydrogen peroxide than the wild-type (Figure 1 A, B). According to the reduction in D37 values (Table 1), the mutant was about 2.5 times more sensitive than the wild-type to y-rays and hydrogen peroxide. However, the mutant was still more resistant than E. coli B/r for either agent. The red-colour of the wild-type was gradually bleached upon exposure to hydrogen peroxide, suggesting degrada tion of bacterioruberin. Comparison of the sensitivities of the mutant and the wild-type of H. salinarium to UV radiation are shown in Figure 1C. UV induces damage to DNA mostly by production of pyrimi dine dimers. As seen in this figure, the colourless mutant was more sensitive to the lethal effect of UV light than the wild-type. The difference in the relative sensitivity between the wild-type and mutant cells was greater than those for ionizing radiation and hydrogen peroxide described above (Table 1), indicating that the protective function of bacterioruberin is more efficient in UV radia tion. The sensitivity of the mutant and the wild-type of H. salinarium to MMC is shown in Figure 1D. The mutant had a level of resistance equivalent to that of the wild-type against MMC, but was 4 times more resistant than E. coli B/r (Table 1). MMC inhibits DNA synthesis by producing cross-links in DNA. Therefore, the comparable resistance of the mutant and wild-type cells against MMC suggests that bacterioruberin does not protect DNA from cross-link formation. The protective role of bacterioruberin was also examined by analyzing strand-breaks of DNA induced by y-rays (Figure 2). DNA double strand-breaks and single strand-breaks induced by ionizing radiation were analyzed by neutral (Figure 2A) and alkaline (Figure 2B) agarose gel electrophoresis, respectively. Bacterioruberin effectively reduced both double (lanes 8 to 11 in Figure 2A) and single (lanes 8 to 11 in Figure 2B) strand-breaks. For comparison, the protective effects of L-cysteine and reduced glutathione, known as typical radical scavengers in cells, were also analyzed by neutral agarose gel electrophoresis (Figure 2C). In contrast to bacterioruberin, L-cysteine and reduced glutathione at the same concentrations as bacterioruberin did not reduce double strand-breaks significantly (lanes 8 to 11 and 13 to 16 in Figure 2C). Therefore, the effec

Table 1. Relative sensitivity of wild type (W) and bacterioruberin-defi cient mutant (M) of H. salinarium, and Escherichia coli B/r (E) against DNA damaging agents. Figure 2. Effects of bacterioruberin, cysteine and glutathione on DNA strand break formation by y-rays. (A) Neutral agarose gel electrophoresis (double strand break analysis) of DNA y-irradiated in the absence or presence of bacterioruberin. (B) Alkaline agarose gel electrophoresis (single strand break analysis) of DNA y-irradiated in the absence or presence of bacterioruberin. (C) Neutral agarose gel electrophoresis of DNA y-irradiated in the absence or presence of L-cysteine and reduced glutathione. Lane I is the marker (k DNA Hind III digest). Irradiation doses are indicated on the top of gels. tive concentration range of the SH compounds to show any protective effects appeared to be much higher than bacterioruberin. Since hydroxyl radicals are primarily responsible species for DNA strand breaks in y-irradiation, these results indicate that bacterioruberin is a more effective radical scavenger than L-cysteine or reduced glutathione. These observations provide further evidence that bacterioruberin efficiently protects H. salinarium against damage induced by reac tive oxygen species. High salt conditions can induce DNA structural transitions, for example B to C or Z'9'201.In order to examine whether or not such conformational changes occur under the high salt condi tions present in H. salinarium cells, the chromosomal DNA of H. salinarium was isolated and its CD spectra were measured in the presence of KC1 (Figure 3). As seen in the figure, the presence of KCl up to 3.5 M did not induce any global structural changes. The CD spectra exhibited a positive cotton peak around 265 nm, a negative peak at 240-245 nm, and a crossover point around 253 nm, indicating that DNA remained in the B-form. These results suggest that confor mational changes of DNA under high salt conditions are not responsible for the resistance of H. salinarium to y-rays, UV, and H202. It has been reported that the inactivation efficiency of bacteriophage SP02c12 DNA by UV decreases with increasing ionic strength 16). To determine whether the intracellular salts of H. salinarium exert any protective effects on radiation-induced DNA strand breaks, DNA was irra diated with 60Co y-rays in the absence or presence of concentrated KCl (Figure 4), and analyzed by agarose gel electrophoresis. As seen in the figure, double strand-breaks (lanes 8 to 11 in

Figure 3. CD spectra of H. salinarium chromosomal DNA in the absence or presence of KC1 (0.1, 2, 3.5 M). Figure 4. Effects of KCI on DNA strand break formation by y-rays. (A) Neutral agarose gel electrophoresis (double strand break analysis) of DNA y-irradiated in the absence or presence of KCI. (B) Alkaline agarose gel electrophoresis (single strand break analysis) of DNA y-irradiated in the absence or presence of KCI. Lane I is the marker (.L DNA Hind III digest). Irradiation doses are indicated on the top of gels.

Figure 4A), and single strand-breaks (lanes 8 to 11 in Figure 4B) were clearly reduced by the presence of KC1, indicating that concentrated KCI has protective effects against DNA damage induced by ionizing radiation.

DISCUSSION

We have previously reported that H. salinarium, an extremely halophilic archaebacterium, is highly resistant to the lethal effects of DNA-damaging agents''. The aim of the present inves tigation was: 1) to confirm that the C5()-carotenoid of H. salinarium, namely bacterioruberin, plays a protective role in this organism as an antioxidant against the lethal effects of oxidative DNA-damaging agents, 2) to examine whether or not concentrated salts in H. salinarium cells cause any conformational changes in DNA, and 3) to characterize the effect of intracellular KG of this organism against oxygen free radicals. Our results show that the bacterioruberin-deficient (colourless) mutant of H. salinarium was more sensitive than its red-pigmented wild-type to the all DNA-damaging agents tested except MMC, an agent which inhibits DNA synthesis by forming cross-links in DNA. MMC exerts genotoxic effects by a mechanism quite different from those of oxidative DNA-damaging agents. Hydrogen peroxide, and reactive oxygen species generated by ionizing radiation can oxi dize cell components such as and DNA. The reaction between bacterioruberin and hy drogen peroxide appears to reduce the oxidative action of this agent, since hydrogen peroxide bleached the red-colour of the wild-type strain. Similar results were reported for the other red pigmented radioresistant bacterium, radiodurans2". Although hydrogen peroxide is not itself a free radical, it generates hydroxyl radicals, the most toxic species of the reactive oxygen species. Thus, the increased sensitivity of the colourless mutant to 'y-rays and hydrogen peroxide could be partly the result of its lack of protective bacterioruberin. In , important regions of DNA, such as ori, ter, replication region, are associated with the cell mem brane. Therefore, bacterioruberin associated with the cell membrane may suppress damage to the membrane associated regions of DNA and their close vicinity, when oxygen radicals are gener ated on the surface or in the constituents of the cell membrane. It is also noted that the antioxidation effect of bacterioruberin on the cell membrane suppresses the formation of genotoxic malondialdehyde that is derived from membrane peroxidation. In the recent work13),we have shown that bacterioruberin (which contains 13 conjugated double bonds) scavenges hydroxyl radicals much more efficiently than /3-carotene (which contains 11 conjugated double bonds). Therefore, the highly conjugated double bonds of bacterioruberin act as a very effective oxida tion protector. In agreement with this, the present results also demonstrate that bacterioruberin confers resistance against oxidative DNA-damaging agents such as ionizing radiation and hydro gen peroxide in vivo. The protective effects of bacterioruberin were more evident for UV-radiation than for ioniz ing radiation and hydrogen peroxide (Table 1). One possible explanation for this efficient protec tion against UV is that bacterioruberin may have an extra protective mechanism for UV-radia tion, for instance, UV-energy absorbant. Carotenoid pigments might also aid in the recovery from UV damage by supplying energy indirectly to the photoreactivating enzyme for the reversal of thymine dimers3,12'22) The conformation of DNA could be changed from B to other forms in concentrated salts so that the accessibility of DNA-damaging agents to the target regions of DNA may be reduced. One can suppose that the resistance of H. salinarium might be the result of the structural changes in DNA. However, the CD spectra of H. salinarium chromosomal DNA (Figure 3) revealed that DNA conformation was virtually independent of KCl concentrations. DNA was in the right handed B-form under these conditions. Therefore, the high resistance of H. salinarium against DNA-damaging agents is not due to alterations in the DNA structure. Gel electrophoretic analysis of DNA irradiated by ionizing radiation (Figures 2 and 4) showed that DNA strand-breaks were notably reduced in the presence of bacterioruberin or con centrated KCI, possibly due to the free radical scavenging effects of bacterioruberin and KCI. The protection effects of bacterioruberin found in this study are in good agreement with our previous work 13),which demonstrated that bacterioruberin protects radiation-induced degrada tion of thymine. Bacterioruberin constitutes the major red pigment not only in H. salinarium" but also in Rubrobacter radiotolerans, an extremely radioresistant bacterium23). In aqueous solutions, hydroxyl radicals produced by ionizing radiation are primarily responsible for the induction of DNA damage, such as strand breaks. When DNA solutions are y-irradiated in the presence of concentrated KCI, hydroxyl radicals (. OH) generated by radiolysis of water react with chloride anions (Cl-), yielding hydroxyl anions (OH-) and chlorine atoms (CI •) via electron transfer 24).The chlorine atoms further react with chloride anions yielding chloride radicals (C12 ') which are much less reactive to DNA than hydroxyl radicals24).Consequently, radiation-induced DNA strand-breaks were significantly suppressed by the presence of concentrated KCI. In conclusion, the present results suggest that bacterioruberin and intracellular concentrated salts of H. salinarium protect this organism against DNA damaging agents, although it may not be the sole mechanism for the resistance of this bacterium against DNA-damaging agents. The additional protective role of DNA repair presumably present in this organism remains to be elucidated.

REFERENCES

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