Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

87 FEMS Microbiology Letters 120 (1994) 87-92 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier FEMSLE 06034 Viktor Kriv~ansloj a, Margita Obernauerovfi and Jfilius Sublk .,a v° • a, Jitka Ulrichov~ b, Vil6m Slmanek b Department of Microbiology and Virology, Faculty of Sciences, Comenius University, Mlynskd dolina B-2, 842 15 Bratislava, Slovak Republic, and b Institute of Medical Chemistry, Medical Faculty, Palack~ University, 775 15 Olomouc, Czech Republic a (Received 18 March 1994; revision received 5 April 1994; accepted 25 April 1994) Abstract: Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome. Key words: Alkaloid; Chelerythrine; Sanguinarine; Saccharomyces cerevisiae; Petite mutant; Yeast Introduction The quaternary benzo/c/phenanthridine alkaloids chelerythrine and sanguinarine (Fig. 1) are known for their antibacterial [1], antimycotic [2] and anti-inflammatory [3] effects. Several reviews have been devoted to their chemical properties, biological activities and pharmacology [46]. In spite of the large effort the exact mode of their action has not yet been elucidated. Nevertheless, some biological activities of chelerythrine and sanguinarine are thought to be due to their * Corresponding author. Tel.: (07) 724 689; Fax: (07) 729 064; e-mail: subik.devin.fns.uniba.sk. SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 1 8 0 - Y interaction with specific cellular proteins [7,8] and their capacity to bind to DNA [9]. In this study we have tested the effect of both alkaloids on yeast cells and revealed the mitochondrial mutagenicity of chelerythrine. O ° , O Fig. 1. Structure of benzo/c/phenanthridine alkaloids: Chelerythrine R 1 = R 2 = CH3; Sanguinarine R I + R 2 = CH 2. In an aqueous solution of the appropriate pH, the iminium ion covalently adds hydroxide ion to form a pseudobase or alkanolamine adduct. Downloaded from https://academic.oup.com/femsle/article/120/1-2/87/506257 by guest on 21 December 2022 Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine 88 Materials and Methods Media and growth conditions From an initial concentration of 5 × 105 cells ml-1 the strains were grown aerobically at 28°C in liquid YNB medium ( W / O amino acids) with 2% glucose, or in semisynthetic medium containing 0.5% peptone, 0.5% yeast extract, 0.5% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO 4 • 7H20, 0.01% NaCI, 0.01% CaCI 2, 0.003% FeCI 3 • 6H20 with 0.5-2.0% glucose or 2% glycerol as carbon source. Media were buffered to the indicated pH by 0.1 M citrate-phosphate, sodium phosphate or Tris. HCI buffers. Alkaloids isolated from above-ground parts of Macleaya cordata [10] were added from sterile stock solutions to autoclaved media before inoculation. Cells were counted in a haemocytometer. Cell viability, detection of mutants and genetic methods Cell viability was estimated after plating ceils onto solid semisynthetic medium containing 2% glucose and 2% agar. Respiration-deficient mutants were detected by colony size upon plating cells on solid semisynthetic medium containing 0.1% glucose and 2% glycerol or by staining colonies with 2,3,5-triphenyltetrazolium chloride [11]. Suppressiveness of the petite clones and the loss of mitochondrial genetic markers were estimated by employing standard genetic techniques and solid glycerol media containing 4 m g m1-1 chloramphenicol or 0.5 /zg m1-1 mucidin [12]. Results and Discussion Chelerythrine and sanguinarine prevented the growth of yeast cells cultured in semisynthetic medium with 2% glucose. The minimum inhibitory concentrations (MIC) of both alkaloids were strain-dependent and identical (Table 1). At concentrations lower than MIC values for both alkaloids, chelerythrine and sanguinarine decreased the growth rate and the final growth yield of the wild-type strain DTXII cultured in semisynthetic medium with 0.5% glucose. At concentrations of 50 and 100 ~g ml -t the final growth yield was decreased to the same level as observed with the corresponding respiration-deficient strain DTXIIA (Fig. 2), indicating that alkaloids interfere with aerobic growth of yeast on ethanol. In accordance with this observation, both alkaloids (50 and 100/~g ml-1) already prevented the growth of yeast in semisynthetic medium with 2% glycerol. The growth inhibitory action of chelerythrine and sanguinarine was found to be pHdependent. The lowest MIC values for the alkaloids were observed at pH 6.0 (insert of Fig. 2). Cultures of the wild-type strain DTXII grown in the presence of chelerythrine in semisynthetic medium with glucose (0.5-2%) contained a considerable portion of respiration-deficient mutants. Their percentage in the culture increased Table 1 Minimum inhibitoryconcentrations (MIC) for chelerythrine and sanguinarinein semisyntheticmediumwith 2% glucose, pH 4.5 Yeast strain MIC (~g ml- 1) Chelery- Sanguithrine narine 200 Saccharomyces cerevisiae TXII 200 100 Saccharomyces cerevisiae 7 100 Saccharomyces cerevisiae Myslenice 50 50 Saccharomyces cerevisiaeJS3-7 150 150 Saccharomycescerevisiae RE-1A 200 200 B2uyverornyceslactis IFO 1267 50 50 Downloaded from https://academic.oup.com/femsle/article/120/1-2/87/506257 by guest on 21 December 2022 Strains The following strains of Saccharomyces cerevisiae were used in this study: DTXII, a wild-type prototrophic diploid; DTXIIA, its cytoplasmic respiration-deficient mutant; RE-1A ( M A T a lys2 rho ÷ capri-321 eryrl-514 mucr2-101); 26-4 (MATa leul thr2-1 rho°), IL166-6C (MATa ural rho+); D7 ( M A T a / M A T a ade2-119/ade2-40 trp5a / trp5b cyh2 / + ile1-92 /ilel-92); JS3-7D ( M A T a adel lys2 ura3 pdr3-1); $6-1 ( M A T a K - R - ) and Myslenice (K1R1), an industrial wine yeast strain with killer phenotype. In addition, the killer strain of Kluyveromyces lactis IFO 1267 (MATa kl k2) was used. Unless indicated otherwise, the presented results are the means of three to five independent experiments. 89 200150 ~ "~E o,2~-~G 100 0~ ~ _ so o 0 25 50 7.5 Alkaloids (~.g ml"1) 100 Fig. 2. Inhibition of yeast growth on glucose by chelerythrine ((3, e) and sanguinarine (zx, A). Growth yield of the wildtypestrain DTXII (open symbols) and the respiration-deficient mutant DTXIIA (filled symbols) in semisynthetic medium with 0.5%glucose, pH 4.5 was evaluated after 48 h. The insert shows theeffect of pH on the minimum inhibitory concentration ofchelerythrine and sanguinarine for the strain DTXII grown insemisynthetic medium with 2% glucose. 100 I - I I I - A I I I so I B 75 o 0 . 50 25 0 0 6 Time 12 (h) 18 24 o 25 I Chelerythrine(~g I 75 ml"1) I lOO Fig. 3. Induction of respiration-deficient mutants in S. cerevisiae by chelerythrine as a function of time and alkaloid concentration. (A) Strain DTXII was grown in semisynthetic medium (pH 4.5) with 2% glucose containing 25 g.g ml-1 chelerythrine. (B) Strain RE-1A was grown 24 h in semisynthetic medium (pH 4.5) with 2% glucose containing the indicated concentrations of chelerythrine. The results of a representative experiment are shown. Downloaded from https://academic.oup.com/femsle/article/120/1-2/87/506257 by guest on 21 December 2022 O0 4 5 b 7 pH - "EIo0 ~ with both the time of exposure and the concentration of chelerythrine (Fig. 3). Under similar conditions no induction of respiration-deficient mutants was observed with different concentrations of the structurally related sanguinarine. The differential effect of both alkaloids on the mitochondrial genome can be attributed to their slightly different physico-chemical properties and their accessibility to mitochondrial DNA under physiological conditions [4-6,9]. Chelerythrine was found to induce respiration-deficient mutants only under the conditions of growth. When growth of yeast cells in semisynthetic medium with 2% glucose was prevented by cycloheximide, inhibiting the synthesis of proteins in the cytoplasm, or when yeast cells were treated by chelerythrine in buffer, no formation of respiration-deficient mutants was observed. Under the latter conditions, yeast cells were very sensitive to chelerythrine and their survival was very low (Table 2). The mass formation of respiration-deficient mutants by chelerythrine in diploid yeast strain DTXII indicated the cytoplasmic nature of their mutations. This was proved by the lack of complementation to respiration competence in 9O Table 2 Survival of ceils and induction of respiration-deficient mutants by chelerythrine in S. cerevisiae TXII under non-growing conditions Conditions Viability (%) Petite mutants (%) 25 100 100 0.3 77.0 25 97 100 0.02 25 1.9 0.3 2.0 Initial concentration 1 × 106 cells m l - 1. Media were buffered to pH 5.0. Viability and fraction of petite mutants were evaluated after 24 h of chelerythrine treatment. Table 3 Suppressiveness and mitochondrial genotypes of petite mutants induced by chelerythrine (50 tzg m1-1) in S. cerevisiae RE-1A grown for 24 h in semisynthetic medium with 2% glucose, pH 4.5 Petite mutants 97 Frequency of suppressive petite clones (%) 73.4 Suppressiveness range (%) 1.3-18.6 Genotypes of suppressive petites (% of total) cap r mttc r cap r muc r drug ° 0 36.4 18.2 45.4 Fifteen randomly selected petite clones were analysed. Typical results of one of the two independent experiments are shown. diploids obtained from a cross of randomly picked respiration-deficient mutants of haploid strain RE-1A induced by chelerythrine with the rho ° tester strain 26-4 containing no mitochondrial DNA. The majority of cytoplasmic petite mutants induced in the haploid yeast strain RE-1A by chelerythrine (50/zg ml-1) exhibited suppressiveness (Table 3). Some of them contained either chloramphenicol resistance or mucidin resistance mitochondrial markers originally present in the respiration-competent strain. Most of the analysed suppressive clones did not carry any original mitochondrial markers. None of the suppressive petite clones was found to contain both markers together, indicating severe damage to the mitochondrial genome. The petite mutants exhibiting no suppressiveness had deleted their mitochondrial DNA completely. Chelerythrine-induced disintegration of the mitochondrial genome in S. cerevisiae, demonstrated for the first time in this paper, can be explained by the intercalative binding of the alkaloid to mitochondrial DNA [9]. This mutagenic effect of chelerythrine in yeast is in contrast to a recent study failing to demonstrate its genotoxic and mutagenic activity in Escherichia coli [13] and should be taken into consideration for the longterm medical use of this phytotherapeutic. 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