Role of glutathione peroxidase against oxidative stress in yeast: phenotypic character of lipid hydroperoxide-sensitive mutants derived from Hansenula mrakii

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 75, No. 3, 229-231. 1993

Role of Glutathione Peroxidase against Oxidative Stress in Yeast: Phenotypic Character of Lipid Hydroperoxide-Sensitive Mutants Derived from Hansenula mrakii Y O S H I H A R U INOUE,* L I N H - T H U O C TRAN, ~'~D AKIRA KIMURA Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan Received 24 September 1992/Accepted 16 December 1992 Wild type cells of Hansenula mrakii IFO 0895 were highly resistant to the oxidative stress caused by lipid hydroperoxide. The resistance was due to a glutathione peroxidase (GSHPx) which was induced when the yeast was cultured in a medium containing lipid hydroperoxide (Inoue, Y. et al., Agric. Biol. Chem., 54, 3289-3293, 1990). In order to investigate the role of GSHPx, two mutants sensitive to lipid hydroperoxide were isolated. The phenotypes of the mutants were temperature-dependent; i.e., the mutants could grow at 28°C, but not at 35°C in the presence of lipid hydroperoxide. The mutants failed to induce the GSHPx at 35°C. However, the enzyme induced at 28°C and prepared from both mutants was stable after incubation at 37°C for I h.

Radiation (1), halocarbons (2, 3), some drugs (4--6), and herbicides (7, 8) are known to be causative of oxidative stresses, being able to peroxidize the biological membrane in vivo. Lipid hyroperoxide is one of the sources of reactive oxygen species, and it is degraded to yield some radicals such as hydroxyl radical (HO.), alkoxy radical (LO°), and peroxyl radical (LOOo) in the presence of transition metal ions. Both prokaryotic and eukaryotic microorganisms have defensive mechanisms to protect themselves against oxidative stresses. For example, the oxyR-controlled regulon of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium (9, 10), and the superoxide dismutase-catalase system as well as cytochrome c peroxidase in Saccharomyces cerevisiae (11), are well known. However, the defensive mechanism for the oxidative stress caused by lipid hydroperoxide has not been studied in detail in microorganisms. As the first step in this study, we screened several yeast strains for resistance against lipid hydroperoxide, and f o u n d t h a t Hansenula mrakii IFO 0895 could grow in a medium containing 4 mM linoleic acid hydroperoxide in which none of the other yeast strains tested could grow (12). The resistance was supposedly caused by a glutathione peroxidase (GSHPx) which was induced when the yeast was cultured in the presence of lipid hydroperoxide. Recently, we found that GSHPx was also induced by hydroxyl radical (HOo) and superoxide anion (O2-) when the protoplasts of H. mrakii were exposed to such reactive oxygen species (Tran et al., manuscripts submitted). In order to investigate the role of GSHPx against oxidative stress in the yeast, we isolated lipid hydroperoxide-sensitive mutants from H. mrakii, and their phenotypic characteristics were examined. Cells of H. mrakii IFO 0895 obtained from the Institute for Fermentation, Osaka, Japan, were cultured in a nutrient medium (1% glucose, 0.5% peptone, 0.2% yeast extract, 0.03% K2HPO4, 0.03% KHEPO4, 0.01% MgC12; pH 5.5) at 30°C for 16 h. An appropriate amount of the culture was transferred into a test tube (2.2 x 20 cm) contalning 10 ml of the same fresh medium, and the cells were * Corresponding author.

cultured at 30°C until the optical density of the culture at 610 nm (OD610) reached approximately 1.0. Cells were collected by centrifugation (5,000 rpm, 10 min, 4°C), washed once with sterilized 0.85% NaC1 solution, and resuspended in 10 ml of the same solution. The cell suspension was transferred into a petri dish and the cells were exposed to UV-irradiation at 20-cm distance from a UV-lamp with gentle stirring on a clean bench. After 60-s exposure, the cells were diluted appropriately with sterilized 0.85% NaCI solution and spread onto nutrient agar plates. The cells were grown at 30°C for 24 h, and each plate was replica plated onto an SD minimal agar (2.0% glucose, 0.67% yeast nitrogen base, 2.0% agar; p H 5.5) plate containing 0 m M or 2 m M tert-butyl hydroperoxide (tert-BHP), respectively. Cells were cultured at 30°C. Colonies that disappeared and/or were small in size on the agar plate containing 2 mM tert-BHP were picked up from the corresponding master plate, and replica plated again as described above. Approximately 3 x 105 cells were screened for sensitivity to tert-BHP at 30°C, and two candidates (M1 and M9) were obtained. They showed slow growth and made small colonies on the SD agar plate containing 2 mM tert-BHP at 30°C, whereas wild type cells of H. mrakii could make large colonies under the same conditions. To confirm the phenotypes of M1 and M9, each cell was cultured in a liquid medium containing tert-BHP, linoleic acid hydroperoxide (LOOH) and linolenic acid hydroperoxide (LnOOH), respectively, with various concentrations, at 28°C and 35°C. Lipid hydroperoxides were prepared by the photosensitized oxidation method using methylene blue, and purified according to the method of Terao et al. (13, 14). As shown in Fig. 1, wild type cells as well as M1 and M9 cells showed almost same growth rates at 28°C, and they could grow in a 2 mM tert-BHP-, LOOH-, and LnOOH-containing medium. At 35°C, wild type cells showed the same growth rate at 28°C in the medium contalning each lipid hydroperoxide. M1 could not grow in the medium containing 1 mM tert-BHP, 2 m M LOOH, and 1 mM L n O O H at 35°C. On the other hand, M9 could grow in the medium containing I mM lipid hydroperoxide after a 1- to 2-d lag time, although the growth of M9 was 229

230

INOUE ET AL.

J. FERMENT.BIOI~NO.,

2~°c [Wadtype

"~

TABLE 2. Effectof temperature on stability of GSHPx from lipid hydroperoxide-sensitive mutants

M9

MI

Strain

11

Wild type MI M9

O.1H ~ Cone. (mM) 0 1 2 Additive

12

12

0

t-BliP LOOH LnOOH

12 12

12

t-BHP LOOH LnOOH

0 12

12

12

t-BliP LOOH LnOOH

3S°C 1 [wadt~l~

i0.1 Cone. (taM) Additive

M1

' 0 12

12

12

0

t-BHP LOOH LnOOH

L_

1 2 12

12

t-BIEPLOOH LnOOH

0 12 12 12

t-BHP IX)OH LnOOH

also completely inhibited by 2 m M lipid hydroperoxide at 35°C. We have already reported that the resistance against lipid hydroperoxide of the wild type cells of H. mrakii was owing to the G S H P x which was induced when the cells were cultured in the presence o f lipid hydroperoxide (12). Hence, the activities of G S H P x in M1 and M9 were examTABLE 1. GSHPx activities in lipid hydroperoxide-sensitive mutants

Wild type M1 M9

GSHPx activity (mU/mg-protein) tert-BHP added into medium 0mM lmM 2mM 28°C 35°C 28°C 35°C 280C 35°C NDa ND ND

ND ND ND

31 25 28

26 NDb 26

180 (100) 130 (100) ll0 (100)

176 (98) 134 (103) l l l (101)

183 (102) 130 (100) 113 (103)

Cells (wild type, MI and M9) were cultured in a 2-l Sakaguchi flask containing 1 1SD medium at 28°C. When the OD6~0reached approximately 1.0, 2 mM tert-BHP was added to each culture and cultivation was continued until OD~10=8.0. GSHPx from each strain was incubated in a mixture containing 10 mM potassium phosphate buffer (pH7.0) and 0.2raM phenylmethylsulfonyl fluoride at various temperatures for 1 h. After incubation, the enzyme activity was determined. Parentheses show the relative activities to the activity at 28°C in each strain.

lllL

FIG. I. Effectof temperature on growth of lipid hydroperoxidesensitive mutants. Ceils (wild type, MI and M9) were cultured in SD medium at 28°C until the stationary phase. A portion of each culture was transferred into test tubes (l.0x 16.5 cm) containing 5 ml SD medium with lipid hydroperoxides ( ~ , 0raM; ~ , 1 raM; ~ , 2 raM), and cells were cultured at 28°C (upper panel) and 35°C (lower panel), respectively. Cell growth was monitored by measuring the OD6t0. Each bar indicates the OD6~0of the 3-d culture, t-BHP, tertbutyl hydroperoxide; LOOH, linoleic acid hydroperoxide; LnOOH, linolenic acid hydroperoxide.

Strain

GSHPx activity (mU/mg-protein) 28°C 35°C 37°C

180 130 110

140 NDb NDb

Cells (wild type, MI and M9) were cultured in a 2-1Sakaguchi flask containing I l SD medium at 28°C and 35°C, respectively. When the OD610 reached approximately 1.0, tert-BHP was added and cultivation was carded out until OD610:8.0. One unit of GSHPx was defined as the amount of enzyme oxidizing 1 pmol of glutathione per minute at 25°C. Protein was determined by the method of Lowry et al. 05). a Not detected. b Cells did not grow after the addition of tert-BHP, so the ceils were collected at the same time that the wild type cells were harvested.

ined. Cells of the wild type, M1, and M9 were cultured in an SD m i n i m a l m e d i u m at 28°C a n d 35°C, respectively. W h e n the OD610 of the medium reached approximately 1.0, tert-BHP was added to each medium, and the cultivation was continued until the OD6t0 reached 8.0. At 35°C, M I a n d M9 could not grow after the additions of 1 m M a n d 2 m M tert-BHP, respectively, so the cells were harvested at the same time that the wild type cells were collected. The activity of G S H P x was assayed in a mixture (1.0 ml) containing 2.5 m M tert,BHP, 10 m M glutathione, 5 0 r a M potassium phosphate buffer (pH7.0), 0 . 3 r a M N A D P H , 3.4 u n i t s / m l glutathione reductase and GSHPx. The wild type as well as M1 and M9 induced G S H P x at 28°C and specific activity increased in association with the a m o u n t of tert-BHP added (Table 1). Both wild type and M9 induced the enzyme when 1 raM tert-BHP was added at 35°C, while M1 failed to induce G S H P x . W h e n 2 m M tert-BHP was added, neither M1 nor M9 induced GSHPx. In order to investigate whether or not the temperaturesensitive phenotypes of M1 and M9 were due to the instability of G S H P x at higher temperature, the G S H P x induced at 28°C and prepared from each strain was incubated at 28°C, 35°C, and 37°C, respectively. As shown in Table 2, G S H P x prepared from each strain was stable after at least 1-h i n c u b a t i o n at 37°C. Several possibilities could be speculated; e.g., mutation(s) might occur o n the gene(s) encoding some positive regulators involved in the biosynthesis of G S H P x , or mutation(s) might occur to decrease the thermal stability of the m R N A of G S H P x or to decrease the translational efficiency of GSHPxm R N A at higher temperature. We have started to clone the gene for GSHPx; a comparison of the nucleotide sequences of the wild type and m u t a n t GSHPxs would solve this problem. As described above, we reconfirmed that G S H P x was essential for H. mrakii to survive under the oxidative stress caused by lipid hydroperoxide. A p p a r e n t phenotypes of M1 and M9 hereto obtained seemed to be identical. Genetic analyses of these m u t a n t s could supply more inf o r m a t i o n a b o u t the oxidative stress-inducible mechanism in H. mrakii.

REFERENCES 1. Lai, E.K., Crossley, C., Shridhar, R., Misra, H.P., Janzen, E.G., and McCay, P. B.: In vivo spin trapping of free radicals generated in brain, spleen, and liver during ~"radiation of mice. Arch. Biochem. Biophys., 255, 156-160 (1986). 2. MeCay, P. B., Lal, E. K., Poyer, J. L., DuBose, C. M., and Jan-

VOL 75, 1993 zen, E. G.: Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro. J. Biol. Chem., 259, 2135-2141 (1984).

3 . Poyer, J. L., MeCay, P.B., Weddle, C. C., and Downs, P. E.: 4.

5. 6.

7. 8.

9.

In vivo spin-trapping of radicals formed during halothane metabolism. Biochem. Pharmacol., 30, 1517-1519 (1981). Hiralwa, K., Oka, T., and Yagi, K.: Effect of bleomycin on lipid peroxides, glutathione peroxidase and collagenase in cultured lung fibroblasts. J. Biochem., 93, 1203-1210 (1983). Stuart, M. J., DeAlareon, P. A., and Barvlnchak, g. M.: Inhibition of adriamycin-induced human platel¢~ lipid peroxidation by vitamin E. Am. J. Hematol., 5, 295-303 (1978). Svlngen, B. A., Powis, G., Appel, P. L., and Scott, M.: Protection against adriamycin-induced skin necrosis in the rat by dimethyl sulfoxide and a-tocopherol. Can. Res., 41, 3395-3399 (1981). BindoH, A., Valente, M., and Cavallinu, L.: Inhibition of lipid peroxidation by a-tocopherolquinone and a-tocopherolhydroquinone. Biochem. Internatl., 10, 753-761 (1985). Burk, R.F., Lawrence, R.A., and Lane, J . M . : Liver necrosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. Effect of selenium deficiency. J. Clin. Invest., 65, 1024-1031 (1980). Christman, M. F., Stroz, G., and Ames, B. N.: OxyR, a possible

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regulator of hydrogen peroxide-inducible genes in Escherichia coil and Salmonella typhimurium, is homologus to a family of bacterial regulatory proteins. Proc. Natl. Acad. Sci. USA, 86, 3484-3488 0989). 10. Storz, G., Tartaglia, L.A., and Ames, B.N.: Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation. Science, 248, 189-194 (1990). ll. Yonetani, T.: Cytochrome c peroxidase. Adv. Enzymol., 33, 309-335 (1970).

12. Inoue, Y., Iehiryu, T., Yoshikawa, K., Tran, L. -T., Murata, K., and Kimura, A.: Induction of glutathione peroxidase by linoleic acid hydroperoxide in Hansenula mrakii. Agric. Biol. Chem., 54, 3289-3293 (1990). 13. Terao, J. and Matsnshita, S.: Analysis of photosensitized oxidation products of unsaturated triglyceridc and vegetable oils by gas chromatography-mass spectrometry. Agric. Biol. Chem., 45, 601-608 (1981). 14. Terao, J., Asano, I., and Matsnshlta, S.: Preparation of hydroperoxy and hydroxy derivatives of rat liver phosphatidylcholine and phosphatidylethanolamine. Lipids, 20, 312-317 (1985). 15. Lowry, O.H., Resebrough, N.J., Farr, A.L., and Randall, R . J . : Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951).