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 YOSHIHARU INOUE,* LINH-THUOC 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 reac- tive oxygen species, and it is degraded to yield some radi- cals 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 oxida- tive stresses. For example, the oxyR-controlled regulon of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium (9, 10), and the superoxide dismu- tase-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 hydro- peroxide, 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 in- duced 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 ex- posed to such reactive oxygen species (Tran et al., manu- scripts 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 Insti- tute 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) con- talning 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 col- lected by centrifugation (5,000 rpm, 10 min, 4°C), washed once with sterilized 0.85% NaC1 solution, and resus- pended 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; pH 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 cor- responding master plate, and replica plated again as de- scribed 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 hydro- peroxide (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 con- talning each lipid hydroperoxide. M1 could not grow in the medium containing 1 mM tert-BHP, 2 m M LOOH, and 1 mM LnOOH 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 [Wad type MI M9

"~ 11

O.1H ~ Cone. 0 12 12 12 0 12 12 12 0 12 12 12 (mM)

Additive t-BliP LOOH LnOOH t-BHP LOOH LnOOH t-BliP LOOH LnOOH

3S°C 1 [ wad t~l~ M1

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0 12 12 12 Cone. 0 12 12 12 0 12 12 12 (taM) Additive t-BHP LOOH LnOOH t-BIEP LOOH LnOOH t-BHP IX)OH LnOOH

FIG. I. Effect of temperature on growth of lipid hydroperoxide- sensitive 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 OD6~0 of the 3-d culture, t-BHP, tert- butyl hydroperoxide; LOOH, linoleic acid hydroperoxide; LnOOH, linolenic acid hydroperoxide.

also completely inhibited by 2 mM 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 GSHPx which was induced when the cells were cultured in the presence of lipid hydroperoxide (12). Hence, the activities of GSHPx in M1 and M9 were exam-

TABLE 1. GSHPx activities in lipid hydroperoxide-sensitive mutants

GSHPx activity (mU/mg-protein) tert-BHP added into medium

Strain 0mM lmM 2mM

28°C 35°C 28°C 35°C 280C 35°C

Wild type ND a ND 31 26 180 140 M1 ND ND 25 ND b 130 ND b M9 ND ND 28 26 110 ND b

Cells (wild type, MI and M9) were cultured in a 2-1 Sakaguchi 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 cultiva- tion 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.

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

GSHPx activity (mU/mg-protein) Strain

28°C 35°C 37°C

Wild type 180 (100) 176 (98) 183 (102) MI 130 (100) 134 (103) 130 (100) M9 ll0 (100) l l l (101) 113 (103)

Cells (wild type, MI and M9) were cultured in a 2-l Sakaguchi flask containing 1 1 SD medium at 28°C. When the OD6~0 reached approx- imately 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 in- cubated 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 deter- mined. Parentheses show the relative activities to the activity at 28°C in each strain.

ined. Cells of the wild type, M1, and M9 were cultured in an SD minimal medium at 28°C and 35°C, respectively. When the OD610 of the medium reached approximately 1.0, tert-BHP was added to each medium, and the cultiva- t ion was continued until the OD6t0 reached 8.0. At 35°C, MI and M9 could not grow after the additions of 1 mM and 2 mM tert-BHP, respectively, so the cells were har- vested at the same time that the wild type cells were col- lected. The activity of GSHPx was assayed in a mixture (1.0 ml) containing 2.5 mM tert ,BHP, 10 mM glutathione, 50raM potassium phosphate buffer (pH7.0) , 0 .3raM NADPH, 3.4 un i t s /ml glutathione reductase and GSHPx. The wild type as well as M1 and M9 induced GSHPx at 28°C and specific activity increased in association with the amount 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 GSHPx. When 2 mM tert-BHP was added, neither M1 nor M9 induced GSHPx.

In order to investigate whether or not the temperature- sensitive phenotypes of M1 and M9 were due to the instability of GSHPx at higher temperature, the GSHPx induced at 28°C and prepared from each strain was in- cubated at 28°C, 35°C, and 37°C, respectively. As shown in Table 2, GSHPx prepared from each strain was stable after at least 1-h incubat ion at 37°C. Several possibil- ities could be speculated; e.g., mutation(s) might occur on the gene(s) encoding some positive regulators involved in the biosynthesis of GSHPx, or mutation(s) might occur to decrease the thermal stability of the mRNA of GSHPx or to decrease the translational efficiency of GSHPx- m R N A at higher temperature. We have started to clone the gene for GSHPx; a comparison of the nucleotide se- quences of the wild type and mutan t GSHPxs would solve this problem.

As described above, we reconfirmed that GSHPx was essential for H. mrakii to survive under the oxidative stress caused by lipid hydroperoxide. Apparent pheno- types of M1 and M9 hereto obtained seemed to be identical. Genetic analyses of these mutants could supply more in- format ion about the oxidative stress-inducible mechan- ism in H. mrakii.

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