Mol Gen Genet (1981) 181:292-295 © Springer-Verlag 1981

Is It Possible to Isolate Methionine Auxotrophs in

Chlamydomonas reinhardtii?

Consideration of Photodynamic Action of the Amino Acid Kazuo Nakamura, Sandra L. Lepard, and Susan J. MacDonald Deparmtent of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.

Summary. In order to address the problem of amino acid auxotroph scarcities in photosynthetic organisms, an attempt was made to recover methionine auxotrophs in a unicellular green alga, Chlamydomonas reinhardtii. Evidence of methionine permeation into the algal cells was provided by the successful competition of the amino acid with its antimetabolite, methionine sulfoximine. No methionine auxotrophs were isolated despite the frequent recovery of both nicotinamide and arginine auxotrophs, selected as controls, and of methionine sensitive mutants. The use of either N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light as mutagens appeared not to alter the mutation spectrum. Medium containing methionine was shown to be inhibitory to the growth of cells in culture under fluorescent light and this toxicity was further stimulated in the presence of riboflavin. In view of our results and the observations of other studies, the non-recoverability of methionine auxotrophs is discussed as a function of the photodynamic action of methionine.

Introduction Attention has been drawn to the striking difference in mutation spectra between non-photosynthetic and photosynthetic organisms. While amino acid auxotrophs are frequent in bacteria and fungi, they are very scarce in green alga Chlamydomonas (c.f. Li et al. 1967). No amino acid auxotrophs have been obtained in C. eugametos and C. moewusii, and only arginine-requiring mutants have been described in C. reinhardtii (Ebersold et al. 1962). In Arabidopsis thaliana no amino acid auxotrophs, in the true sense, have been isolated (Li et al. 1967). Analysis has indicated that neither the mutagens employed nor differing conditions in the same or different laboratories seriously alters the mutation spectrum within a species (Li et al. 1967). Several hypotheses to explain the specificity of forward mutation in Chlamydomonas have been discussed (Ebersold 1962; Li et al. 1967 ; Gowans 1976). One of the more credible hypotheses is that certain auxotrophs may not be recoverable due to a lack of uptake or an inability of the exogenously supplied amino acids to enter the normal metabolic pathways. However, despite the observation that C. eugarnetos can incorporate 14Chistidine or 14C-phenylalanine into soluble proteins (Wang, 1972), efforts to recover histidine auxotrophs (Wang 1972) and phenylalanine auxotrophs (McBride and Gowans 1970) have failed.

It should be noted that, despite its importance, data directly concerning the limited mutation spectrum has remained in the hands of researchers, and has scarcely been published except as dissertations (e.g. Wang 1972) or as sole statements in indirectly involved subjects (e.g. McBride and Gowans 1970). This obviously results from the difficulty of showing positive proof of a negative phenomenon. Our preliminary observations that methionine is transported into the cells of C. reinhardtii, coupled with our recent isolation of tryptophan sensitive mutants in C. eugametos and in C. reinhardtii, which are also sensitive to methionine and cystine (Nakamura et al. 1979), has led us to offer an alternate hypothesis: During the fluorescent irradiation of Chlarnydomonas cells in culture, the putative mutants requiring photolabile amino acids may bring about their own demise by transporting toxic photoproduct(s), the result of photolysis of the exogenously supplied amino acids, into the cell. Any attempt to explain the limited mutation spectrum along this line of consideration has not been reported to date. In this paper, results on the recoverability rates of methionine sensitive mutants and of auxotrophs selected for methionine, nicotinamide, and arginine are presented. Circumstantial evidence is discussed which strongly supports the aforementioned hypothesis of the photodynamic effect of the amino acid upon the mutation spectrum. Materials and Methods

Strains. Wild type stock of Chlamydomonas reinhardtii, 137e mating type +, was obtained from Dr. N.W. Gillham of Duke University, North Carolina. Medium. Minimal medium was used as described by Gowans (1960). Various concentrations of L-methionine, L-methionine-DL-sulfoximine, and riboflavin were added to the minimal medium where indicated. For mutagenesis, the enriched medium (abbreviated NAM) consisted of minimal medium plus nicotinamide (1 ~g/ml), L-arginine hydrochloride (10 gg/ml), and L-methionine (40 gg/ml). For solid media 1.5% Difco bacto agar was added. All media were adjusted to pH 7 + 0.2.

Growth. Pre-experimental cultures were grown in liquid minimal medium at 25° C under 24 h fluorescent light (General Electric, Daylight) with an intensity of 4.8 × 103 lux. Growth in liquid media was measured as a function of turbidity at 750 nm. Cells were counted using a hematocytometer.

Offprint requests to: Dr. Kazuo Nakamnra, Department of Biological

Induction of Mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Pre-experimental cultures of wild type ceils, grown to a

Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4

concentration of 2 to 5 x 1 0 6 viable cells/ml, were centrifuged and resuspended in a sterile solution of MNNG in phosphate buffer

0026-8925/81/0181/0292/$01.00

293 (0.02 M, p H - 7 ) to give a final concentration of 40 ~tg MNNG/ml of cells. The suspension was mechanically agitated at room temperature for either 15 or 30 min after which the cells were washed twice by centrifugation with phosphate buffer, diluted with sterile H 2 0 , and plated on solid NAM medium in Petri dishes. The plates were then placed under continuous light for 10 14 days.

Induction of Mutations with Ultraviolet (UV) Light. Appropriate numbers of wild type cells from a pre-experimental culture were spread over solid NAM medium and then exposed to UV light (General Electric germicidal lamp G8 T5) with an intensity of 180 gW/cm 2 for time periods ranging from 2 to 4 rain. After irradiation the plates were placed in the dark for 12 h and then in continuous light for 10 14 days.

40

Mutant Isolation and Identification. Following 10 14 days of growth, colonies were replica plated onto 5 types of solid media: minimal, minimal plus methionine (40 ~tg/ml), minimal plus methionine (300 gg/ ml), minimal plus nicotinamide (1 gg/ml), and minimal plus arginine (10 gg/ml). Plates were placed under continuous light for 7-10 days and mutant colonies then isolated. Mutants were further confirmed by growth tests on the appropriate media.

Results

Methionine versus Methionine Sulfoximine Antagonism The most straightforward explanation for the scarcity of amino acid requiring m u t a n t s would be the impermeability of the cell m e m b r a n e to amino acids other t h a n arginine. If the m e m b r a n e was impermeable, any mutational defect in a synthetic pathway would be fatal to the cell even in the presence of methionine. Figure 1 shows that the inhibitory effect of methionine sulfoximine, an antimetabolite of methionine, is reversed by the addition of increasing a m o u n t s of methionine. This strongly indicates that the cells are permeable to methionine. Additional evidence in support of cell permeability to methionine may be found in the observation of a methionine sulfoximine resistant m u t a n t in the same strain (Hastings et al. 1965).

Plating Effi'ciency Due to the photolabile nature of methionine and the possible production of toxic photoproduct(s) ( N a k a m u r a et al. 1979) it was necessary to determine the o p t i m u m range of methionine concentrations which would allow for the selection of possible methionine auxotrophs. To achieve this ceils were properly diluted, plated on minimal medium supplemented with varying concentrations of L-methionine and incubated under the light. The results (Table 1) showed that although colony counts decreased as the concentration of methionine increased, no appreciable effect was evident up to methionine concentrations of 50 gg/ml. We therefore chose a methionine concentration of 40 ~tg/ml in our mutagenesis experiments for the isolation of methionine auxotrophs. This concentration is also within the range of that used in the isolation of bacterial and fungus mutants. (see for example: Boone et al. 1956; Perkins 1949; T a t u m 1945; Burkholder 1947). It should be noted that whereas no detectable inhibitory effect by methionine (500 gg/ml) was detected on the wild type cells placed in liquid culture (Fig. 1), a significant inhibitory effect was exerted on plated cells by a concentration of 400 ~tg/ml (Table 1). This can be explained by more oxygen being present in the plated cultures t h a n in the n o n a e r a t e d liquid cultures, toxicity of methionine presumably resulting from an oxidation process.

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5

10

20

40

80

METHIONINE SULFOXIMINE (pg/ml)

Fig. 1. Methionine versus methionine sulfoximine antagonism. Cells were grown in 15 x I50 mm test tubes containing 5 ml medium, each having methionine concentrations of 0 ilg/ml (o o), 100 ~g/ml (A-A), or 500 ~tg/ml ( z x). Relative growth as % of value obtained in the absence of methionine sulfoximine is shown. The absolute optical density values in the absence of methionine sulfoximine were 0.126, 0.125, 0.134 respectively for methionine concentrations of 0, 100 and 500 gg/ml. Each point represents the mean growth conducted concurrently in triplicate Table 1. Effect of methionine on wild type ceils Concentration of methionine (~g/ml) added to minimal medium

Relative plating efficiency (% of value obtained from minimal medium)

0 10 25 50 100 200 300 400

100 107 99 100 94 67 4I 4

About 140 cells were plated on petri dish containing 25 ml solid medium and cultured under continuous light for 10 days. Values were based on average colony counts of 3 or 4 plates.

Mutagenesis During our attempts to recover methionine auxotrophs from a wild type, mutagenic selections were made for nicotinamide and arginine-auxotrophs (Ebersold et al. 1962) and methionine sensitive m u t a n t s ( N a k a m u r a et al. 1979) for the purpose of controls. Table 2 shows the effectiveness of U V and M N N G treatments in inducing forward mutation. A comparison of the types of auxotrophs a n d methionine sensitive m u t a n t s induced by the two mutagens does not reveal any m a r k e d difference in the m u t a t i o n spectra (Table 3). It should be pointed out that no methionine auxotrophs were recovered, despite the frequent recovery of the other three types of mutants.

EJ]ect of Photosensitized Methionine To seek an explanation for the non-recoverability of methionine auxotrophs in the preceding experiments, the suspected presence

294 Table 2. Forward mutations induced by UV and MNNG

Mutagenic treatment

UV (2 min)

No. of colonies 1907 replicated Survivors (%) 7 No. of mutants 5 Mutants (%) 0.26

UV (3 min)

UV (4 rain)

MNNG (15 rain)

MNNG (30 rain)

3174

1740

1641

2783

0.8 20 0.63

0.08 16 0.91

20 10 0.61

9 25 0.90

Table 3. Classification of biochemical mutants induced by UV and

MNNG a Type of mutants

Arginine requirement

Nicotinamide methionine requirement requirement

methionine sensitive

UV MNNG

2 4

7 6

32 25

0 0

Data derived from colonies isolated in Table 2. Note: Additional attempts to isolate only methioniue auxotrophs yielded the following results: Mutagenic Treatment

UV b (3 rain)

UV b (4 rain)

MNNG (90 min)

No. of colonies replicated Survivors (%) No. of Mutants

2270 3 0

500 0.3 0

2120 0.2 0

b UV intensity of 140 gw/cm 2

Table 4. Effect of riboflavin and methionine present in medium on

wild type cells Minimal

Relative plating 100 efficiency (% of value obtained from minimal medium)

Minimal + Minimal + Minimal + methionine riboflavin methionine (40 p,g/ml) (1 gg/ml) (40 gg/ml) + riboflavin (1 ~g/ml) 103

108

0

Approx 160 cells were plated on petri dishes containing 25 ml solid medium and cultured under continuous light for 10 days. Values based on average colony counts of 5 plates of a photodynamically produced toxicity by methionine was examined. The involvement of a photochemical process was suggested in our other line of studies (unpublished), where it was conclusively shown that the degree of inhibition on cell growth was directly related to pre-illumination of the media with different types of fluorescent light varying in range of wavelength and peak emittance. In an experiment to simulate the production of toxic photoproduct(s) either in the medium or within the cell during the period of fluorescent illumination, riboflavin was arbitrarily chosen as a photosensitizer. The results (Table 4) shows that growth of wild type cells, from single cells to forma-

tion of colony, is, in fact, seriously affected by the dual presence of methionine and riboflavin in the medium, each of which is not inhibitory when individually present. Discussion

The results (Table 3) indicate that there is a specificity of mutation spectrum leading to the non-recovery of methionine auxotrophs. Explanations regarding the possibility that methionine auxotrophs do not exist due to the impermeability of the cell membrane are not borne out by the results in Fig. 1. The suggestion that the genes responsible for the methionine biosynthetic pathway do not mutate is very unlikely since methionine auxotrophs are abundant in bacteria and fungi (c.f. Li et al. 1967). Also any explanation which involves the specificity of the mutagens themselves is rather remote considering the similar results obtained by two different mutagens (Table 3). It therefore seems logical to conclude that while there is an incidence of methionine auxotrophs, these putative mutants are in fact non-recoverable due to their nonviability. There is no full explanation as to why the putative methionineauxotrophs should not be viable at a concentration of methionine (40 gg/ml) which is not inhibitory to the growth of wild type strains. However, it should be noted that methionine in the presence of riboflavin in the media inhibited the growth of wild type cells into colony (Table 4), and that methionine sensitive strains in both C. reinhardtii and C. eugametos which are also sensitive to tryptophan and cystine (Nakamura et al. 1979) show an increased sensitivity to these amino acids in the presence of a photosensitizer, riboflavin or hematoporphyrin (unpublished). Furthermore in aqueous solutions a photosensitizing effect of chlorophyll has been reported (Jorie et al. 1969). Therefore, it is quite possible that the chlorophyll present in the Chlamydomonas cells acts as a photosensitizer during the mutagenic experiments and subsequent illumination in culture. The putative methionine auxotrophic cells, by having an excess uptake of methionine, would bring about their own demise, since photodynamic action would load their intercellular systems with toxic photoproduct(s). The photodynamic action hypothesis for the limited mutation spectrum is compatible with the results of the mutagenic experiments by other investigators on Chlamydomonas. The failure to recover phenylalanine auxotrophs (McBride and Gowans 1970) and histidine auxotrophs (Wang 1972) in C. eugametos, despite the permeability of the cell, would be explained on the basis of the generally known photoreactive nature of these two amino acids (e.g., Smith and Hanawalt 1969). Arginine, presumably not photoreactive under culture conditions, would allow the recovery of arginine auxotrophs of C. reinhardtii. It is also intersting to point out that in Li et al's tabulation (1967) no riboflavin auxotrophs have been isolated in three species of Chlamydomonas nor in Arabidopisis, although they are frequent among bacteria and fungi. The hypothesis that the limited mutation spectrum could be attributable to some kind of genetic redundancy (Wetherell and Krauss 1957) has been tested fruitlessly by Wang (1972) in his effort to obtain histidine mutants by repeated sequential ultraviolet irradiation of the same cells. Since Wang's thesis did not describe the details of his procedures and results it is difficult to rule out the genetic redundancy hypothesis based solely on his results. It was commented by Gowans (1976) that Wang's failure was not convincing enough to warrant abandonment of this hypothesis. However, even if the redundancy hypothesis remains valid, any potential methionine requiring mu-

295 tants would not be recovered under ordinary fluorescent illuminated culture conditions, due to the toxic photoproducts produced by methionine. Given that our hypothesis is correct, it should be possible to obtain methionine auxotrophs by testing the treated cells for growth in the dark on acetate supplemented medium. However, the feasibility of such experiments is severely limited by either practical considerations or by the experimental procedure itself especially where the mutagenic agent involves irradiation of a methionine supplemented medium. Before undertaking this project one must examine the light wavelengths and exposure periods which will trigger photoactivation. We are currently undertaking these experiments. In conclusion, the photodynamic action of methionine seems to be responsible for the lack of methinone auxotrophs in ChIamydomonas reinhardtii. Furthermore it is likely that a similar explanation could be extended to several other photolabile amino acids and other metabolites of a photodynamic nature to account, in part, for the phenomenon of the limited mutation spectrum in photosynthetic organisms. It is hoped that the future study of the photodynamic action of the amino acids in conjunction with chlorophyll will shed more light on the mechanisms behind the differences in mutation spectra between photosynthetic and non-photosynthetic forms. Acknowledgements. We thank Lora Booth and Larry Skiba for their technical assistance in preliminary part of this work. This work was supported by a National Research Council of Canada Grant (A2966) and the University of Lethbridge Research Funds both to K. Nakamura.

Ebersold, WT (1962) Biochemical genetics. In: Lewin RA (ed) Physiology and biochemistry of algae. Academic Press, New York, pp 731739 Ebersold WT, Levine RP, Levine EE, Olmsted MA (1962) Linkage maps in Chlamydomonas reinhardi. Genetics 47:531-543 Gowans CS (1976) Genetics of Chlamydomonas moewusii and Chlarnydomonas eugarnetos. In: Lewin RA (ed) The genetics of algae. University of California Press, Berkeley, pp 145-173 Gowans CS (1960) Some genetic investigations on Chlamydomonas eugametos. Z Vererbungsl 91:63-73 Hastings PJ, Levine EE, Cosbey E, Hudock MO, Gillham NW, Surzycki SJ, Loppes NR, Levine RP (1965) The linkage groups of Chlamydomonas reinhardi. Microb Genet Bull 23:17 19 Jori G, Galiazzo G, Scoffone E (1969) Photodynamic action of porphyrins on amino acids and proteins. I. Selective photooxidation of methionine in aqueous solution. Biochemistry 8 : 2868-2875 Li SL, Redei GP, Gowans CS (1967) A phylogenetic comparison of mutation spectra. Mol Gen Genet 100:77-83 McBride AC, Gowans CS (1970) Comparative sensitivity of three species of Chlamydomonas to analogs of metabolites. J Phycol 6:54-56 Nakamura K, Kozloff RA, Gowans CS (1979) Isolation of tryptophan sensitive strains of the green alga Chlamydomonas. Env Exp Botany 19:311 314 Perkins DD (1949) Biochemical mutants in the smut fungus Ustilago maydis, Genetics 34 : 607 626 Smith KC, Hanawalt PC (1969) Molecular photobiology. Academic Press, New York, p 229 Tatum EL (1945) X-ray induced mutant strains of Escherichia coli. Proc Natl Acad Sci USA 31:215 219 Wang W (1972) Mutational and physiological studies of Chlamydomonas eugametos. Ph D dissertation, University of Missouri-Columbia Wetherell DF, Krauss RW (1957) Y-ray induced mutations in Chlamydomonas eugametos. Am J Bot 44:609 619

References

Boone DM, Stauffer JF, Stahmann MA, Keitt GW (1956) Venturia inaequalis (CKE.) Wint. VII. Induction of mutants for studies on genetics, nutrition, and pathogenicity. Amer J Bot 43:199-204 Burkholder PR, Giles NH Jr (1947) Induced biochemical mutations in Bacillus subtilis. Amer J Bot 34:345-348

Communicated by Ch. Auerbach

Received August 27 / November 10, 1980