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Applied and Environmental Microbiology, 1991, vol. 57, (11), pp. 3183-3186

Role of hydrosulfide ions  (HS -) in Methylmercury Resistance  in Saccharomyces cerevisiae

Ono, B., Ishii, N., Fujino, S. and Aoyama, I.
 

ABSTRACT

Methylmercury-resistant mutants were obtained from Saccharomyces cerevisiae. They were divided into two complementation groups, met2 (homoserine O-acetyltransferase deficiency) and met15 (enzyme deficiency unknown), as reported previously. It was found that met15 was allelic to met17 (O-acetylserine and O-acetylhomoserine sulfhydrylase deficiency). Methylmercury toxicity was counteracted by exogenously added HS-, and both met2 and met17 (met15) mutants overproduced H2S. On the basis of these results, we conclude that met2 and met17 (met15) cause accumulation of hydrosulfide ions in the cell and that the increased level of hydrosulfide is responsible for detoxification of methylmercury.

INTRODUCTION

Mercury is one of the environmental pollutants of most concern because it is highly toxic to living organisms and widely used in industry (4, 6, 10). In studying mercury toxicity, knowledge of the effects of mercury at cellular and subcellular levels is indispensable. For such studies use of model organisms is of importance, and Saccharomyces cerevisiae is appropriate in this respect because it is a unicellular eucaryotic organism, readily manipulated genetically, and easily analyzed biochemically.

Ono et al. (13) showed that Hg²⁺ resistance mutations were dominant and were divided into two groups by their linkage relationships. It was also shown that the cell wall acted as an adsorption filter for Hg²⁺ and that the Hg2+- resistant mutants had the cell wall with increased Hg²⁺- binding capacity. On the other hand, Singh and Sherman (20, 21) isolated methylmercury-resistant mutants and found that the mutants were methionine dependent. The responsible mutations were recessive and were divided into two complementation groups. Although one was identified as met2, which confers deficiency of homoserine O-acetyltransferase (EC 2.3.1.31) (3), the other did not correspond to any of the met mutations known at that time and was named metiS. Whether the met2 and metiS mutations confer methylmercury resistance by the same mechanism remains unknown. To elucidate the biochemical nature of metiS and the mechanism of methylmercury resistance, we isolated methylmercury- resistant mutants and characterized them genetically and biochemically. Here, we present the results of our work.

MATERIALS AND METHODS

Yeast strains and growth conditions.

The yeast strains used in this study are listed in Table 1. Standard yeast growth media were used (11, 18). YPD medium contained 2% glucose, 2% peptone, and 1% yeast extract. The synthetic minimal (SD) medium was described by Wickerham (22). For solid media, 2% agar was added. The growth temperature was 30°C.

Genetic procedures.

Methylmercury-resistant mutants were isolated as described by Singh and Sherman (20, 21) with minor modifications. Cells were spread on SD agar plates, and a filter paper disc (5 mm in diameter) was placed at the center of each plate. Methylmercuric chloride (5 to 10 µl of a 12 mM solution) was applied onto the discs, and then the plates were incubated for 5 to 7 days. Complementation tests were achieved by masS mating. Strains to be tested were mixed with tester strains on YPD agar medium. The plates were incubated overnight, and the cells were transferred to selective plates (SD medium). The plates were incubated for 4 days, and then the growth was scored.

Growth kinetics.

An aliquot of the overnight culture was added to fresh YPD liquid medium; the initial cell density was adjusted to approximately 10⁴ cells ml-1. Methylmercury, NaSH, and/or homoserine was added to the growth medium at the desired concentration. Growth was monitored by measuring the increase of the optical density at 600 nm with an automatic cell growth analyzer (Bioscreen C; Labsystems Japan Co., Ltd., Tokyo, Japan).

Measurement of H2S production.

Cells were inoculated on LA agar medium (0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% ammonium sulfate, 0.1% lead acetate, and 2% agar) and were incubated for a week at 30°C. 'qn this plate, H2S-producing colonies develop dark-brown color due to formation of PbS.

A more quantitative analysis was made as follows. Cells were grown overnight in YPD liquid mediumn, harvested, washed twice with water, and then suspended in' Sp liquid medium (360 ml) containing 30 ,uM methionine. N2 gas (5 ml min- 1) was passed through the culture vessel and then through a trap containing 3.75 ml of 1% zipc acetate and 0.125 ml of 12% sodium hydroxide (5). The cqltlire was placed in a rotary shaker (30 rpm). After cutIvation, trapped H2S was assayed by the method of Siegel (1 ). H2S production (micromoles per cell per hour) was deduced by dividing the amount of trapped H2S by the cumulative cell number and the duration of cultivation.

RESULTS

Mutant isolation. Methylmercury-resistant mutants were isolated as described in Materials and Methods. 4 clear zone of growth inhibition was formed around the filter paper disc containing methylmercury, but there were a few colonies within this region. They were picked, subcloned, and tested for methylmercury resistance by the filter disc method. We obtained five independent methylmercury-resistant mutants (MMR1 through MMR5) in this way. Resistance levels of the mutants were quantitated by growth in YPD liquid medium containing various concentrations of methylmercury. A typical result is shown in Fig. 1.

While a mutant (MMR1) and the parental strain (IS66-4C) responded similarly to methylmercury up to 0.5 µM, the latter grew more slowly than the former at higher concentrations (1.5 to 5.0 µM); at 5.0 µM, the parental strain did not grow at all while the mutant grew up, even though the initiation of growth was somewhat delayed.

Complementation of methionine dependence.

The five mutants were subjected to complementation tests using strains containing the defined methionine mutations: met] through met8, metlO, metl3 through metiS, and metl7 (Table 2). Two mutants, MMR1 and MMR2, did not complement strains containing metiS or metl7. This result indicates either that the mutants contain both metiS and metl 7 or that meti5 and metl 7 are alleles of the same gene. Since authentic metiS and metl7 strains did not complement each other (Table 2), we conclude that the latter is the case. We hereafter refer to the mutation as metl7 or metl7 (met15) because the enzymatic deficiency has been defined under the name of metl7; metl 7 causes deficiency of a single protein which acts as O-acetylserine sulfhydrylase (EC 4.2.99.8) and O-acetylhomoserine sulfhydrylase (EC 4.2.99.10) (8, 9, 23). Though mutant MMR3 complemented all strains tested, its complementation with the metl7 strain was distinguishably weak. Since met25, which is shown to be allelic to metl 7 by the gene-cloning studies (2, 17), has been reported to complement metl 7 (8), we assume that MMR3 contains a met25- like mutation; unfortunately, a complementation test using an authentic met25 strain was not conducted because such a strain was not available to us. The two remaining mutants, MMR4 and MMR5, did not complement the met2 strain, suggesting that they contain mutations allelic to met2.

Effect of HS- on methylmercury toxicity. From the S-amino acid biosynthetic pathway of S. cerevisiae (15), we suspected that met2 and metl7 cause accumulation of homoserine and HS- and that either or both of these compounds reduce the toxicity of methylmercury. We therefore examined the effect of these compounds added exogenously. Strain IS66-4C was inoculated in YPD liquid medium containing 5.0 µM methylmercury and various concentrations of homoserine or NaSH, and the growth was monitored (Fig. 2). NaSH dose-dependently counteracted methylmercury toxicity up to 0.1 µM. The antagonistic effect of NaSH saturated or tended to decrease at higher concentrations. We attribute this to the toxicity of NaSH itself, because NaSH markedly inhibited growth (data not shown). In contrast, homoserine (up to 540 µM) did not affect the toxicity of methylmercury (data not shown). The results clearly indicate that HS- but not homoserine counteracts the toxicity of methylmercury.

Production of H2S by methylmercury-resistant mutants. Methylmercury-resistant mutants MMR1 (metl 7) and MMR4 (met2) and their parental strain (IS66-4C) were examined for H2S production; a meti mutant (SF115-lA) defective for sulfate reduction (7) was included as a negative control (Fig. 3). While MMR1 and MMR4 yielded dark-brown colonies on LA agar medium, IS66-4C and SF115-lA did not. It was further found that MMR1 and MMR4 produced substantial amounts of H2S while IA66-4C and SF115-lA did not (Table 3).

DISCUSSION

We have shown in this study that both met2 and meti 7 confer methylmercury resistance via increased accumulation of H2S. As far as we tested, the methylmercury-resistant mutants were only slightly resistant to Hg2+, and the Hg2+- resistant mutants (13) were not resistant to methylmercury at a detectable level (data not shown). Therefore, we conclude that methylmercury and Hg2+ resistances are different. Hg2+ reacts effectively with sulfhydryl (-SH) groups (1), and presumably because of this property it massively binds to the cell wall and the cell membrane (13) and inhibits their functions, such as transport. Tyrosine transport (14) and cysteine transport (12) are severely inhibited by Hg2+. Since Hg2+ hardly penetrates into the cell (13), the cell must have developed in the course of evolution a protective aid on the cell surface (i.e., cell wall). In contrast, methylmercury because of its hydrophobicity appears to penetrate readily into the cell and causes damage in the cell.

Although exogenously added HS- reduced the toxicity of Hg2+, the strains carrying met2 or meti 7 were not markedly resistant to Hg2+ (data not shown). Thus, we conclude that methylmercury is mainly detoxified by an increased level of HS- in the cell. Although we have presented evidence that the cell detoxifies methylmercury by using HS-, we still do not know what chemical reaction contributes most in this phenomenon. Since it is known that H2S aids the volatilization of methylmercury (16), removal of methylmercury from the cell via such a mechanism is possible. However, it is also possible that methylmercury is converted to Hg2+ in the cell and then reacts with HS- to yield highly insoluble and less-toxic HgS. Studies of the chemical transformation of mercury in the cell and the involvement of HS- in it are needed for a better understanding of the molecular basis of methylmercury resistance.

ACKNOWLEDGMENT

We thank S. Shinoda (Okayama University) for his encouragement and valuable suggestions provided during the study.

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