Proc. NatI. Acad. Sci. USA Vol. 74, No. 2, pp. 610-614, February 1977 Cell Biology

A rapid procedure for selective enrichment of photosynthetic electron transport mutants (metronidazole/ferredoxin/Chlamydomonas/photosystem I reactions/mutant selection)

GREGORY W. SCHMIDT, KARL S. MATLIN, AND NAM-HAI CHUA The Rockefeller University, New York, N.Y. 10021

Communicated by Philip Siekevltz, November 9,1976

ABSTRACT Metronidazole (2-methyl-5-nitroimidazole1-ethanol) is shown to be effective for the selective enrichment of mutants of Chlamydomonas reinhardtii that possess impaired -type photosynthetic electron transport. More than 99.91% of cells are killed when incubated in the presence of 6-10 mM metronidazole for 24 hr under illumination of 7500 lux. Survival of wild-type cells in darkness and of mutants that are blocked at different steps in photosynthetic electron transport is nearly 100% when incubated in the presence of the drug under identical conditions. The toxicity of metronidazole is demonstrated to depend upon its reduction by photosynthetic electron transport. Light-dependent oxygen utake mediated by metronidazole is shown to require active photosystem I in vitro and in vivo. Ferredoxin is necessary for metronidazole reduction by thylakoid membrane fractions enriched in photosystem I activity. We propose that the toxicity of metronidazole is due to the formation of lethal derivatives of the drug or to the accumulation of hydrogen peroxide, which could occur upon autooxidation of metronidazole reduced by one electron. The results indicate that mutants of C. reinhardtii, and probably other photosynthetic organisms, with any lesion in photosynthetic electron transport from the oxidizing side of photosystem II to ferredoxin can be isolated by metronidazoke treatment of mutagenized cultures.

presence of metronidazole (2-methyl-5-nitroimidazole-1-ethanol). The toxicity of the drug is shown to be dependent upon its reduction by ferredoxin in reactions that require active photosystem I (PS I).

MATERIALS AND METHODS Conditions of Cell Culture. Wild-type (137c, mt+) and mutant strains of Chlamydomonas reinhardtii were grown in Tris-acetate-phosphate medium (9). Except where indicated, cultures were incubated at 260 on gyratory shakers. Preparation of Thylakoid Membrane Fractions. Thylakoid membranes were purified as previously described (S). For assays of photosynthetic electron transport activities, the thylakoids were suspended in 25 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid (Hepes)-KOH (pH 7.5). Preparation of PS I Particles. Preparations of thylakoid membrane particles enriched in PS I activities were obtained by digitonin treatment of crude membranes (10). Wild-type cells (8 X 108) were harvested by centrifugation at 2500 X g and washed with 10 mM potassium phosphate (pH 7.0)/20 mM KCI/2.5 mM MgCl2. Washed cells were resuspended in 40 ml of the same buffer and were disrupted at 00 by sonication for 1 min with a Heat-System Ultrasonics sonifier. Pellets obtained by centrifugation at 48,000 X gma. for 15 min were resuspended in 15 ml of the buffer and homogenized with a motor-driven Teflon pestle. The thylakoids (300 ,g of chlorophyll per ml) were treated with 0.5% (wt/vol) digitonin by stirring for 30 min at 00. The mixture was centrifuged for 30 min at 50,000 X gmax. The supernatant fraction was subsequently centrifuged at 144,000 X gmax for 60 min. The pelleted fraction was resuspended in the same buffer and used for assays of photosynthetic electron transport activities. Viable Cell Counts. Survival of cells of wild-type and mutant strains of C. reinhardtii was monitored by spreading aliquots of serially diluted cultures on 1.5% agar plates containing Tris-acetate-phosphate medium. Plating efficiency was approximately 85%. Chemicals and Solutions. All chemicals used were analytical grade. Digitonin was recrystallized twice after decolorization with activated charcoal of solutions of 85% ethanol (vol/vol) brought to near saturation at 700. Metronidazole (Flagyl) was obtained through the courtesy of Ms. E. Roman, Searle & Co., Puerto Rico. Stock solutions of metronidazole (60 mM) were prepared by autoclaving in Tris-acetate-phosphate medium.

Photosynthetic mutants of higher plants and algae have been useful tools for defining the functions of chloroplast components. The order in which electron transport reactions occur, polypeptides that are essential for the structural and functional integrity of chloroplast membranes, and some of the steps involved in chloroplast ribosome assembly have been elucidated with mutants of Chlamydomonas reinhardtl (1-4). Photosynthetic mutants are potentially useful for identification of structural genes for chloroplast polypeptides and of regulatory genes that affect chloroplast biogenesis. Previously developed methods for isolation of photosynthetic mutants suffer from major drawbacks. Screening for mutants with enhanced chlorophyll fluorescence (5) does not permit enrichment of photosynthetic mutants over the wild type. Photosynthetic mutants of Euglena and Chlamydomonas can be recovered selectively because they can survive the presence of arsenate (6, 7). The shortcomings of the arsenate procedure include an unknown mode of action and the prevalence of arsenate permeability mutants among the recovered strains (8). Because mutants that are blocked in photosynthetic CO2 fixation are also isolated (7, 8), arsenate resistance is not a property restricted to those with impaired photosynthetic electron transport. In this paper, we dembnstrate that only mutants that have lesions in photosynthetic electron transport are viable after incubation of cells of Chlamydomonas reinhardtfi in the

RESULTS

Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) is widely used clinically for the treatment of infections of anaerobic bacteria and protozoa (11). It is a nitroheterocyclic compound possessing a low redox potential (-325 mV at pH 6.9) (12). The toxicity of metronidazole is thought to depend upon reduction

Abbreviations: DCMU, 3,4-dichlorophenyl dimethylurea; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PS I, photosystem I; PS II, photosystem II. 610

Proc. Natl. Acad. Sci. USA 74 (1977)

Cell Biology: Schmidt et al. Table 1. PS I-dependent oxygen uptake with thylakoid membranes and digitonin particles prepared from C. reinhardtii Thylakoid membranes Wildtype No additions + 0.2 mM Methyl viologen + 4 mM Metronidazole + 8 mM Metronidazole F1 + 0.2 mM Methyl viologen + 8 mM Metronidazole Digitonin particles No additions + 6 mM Metronidazole + 0.2 mM Methyl viologen + 0.6 MM Ferredoxin + 0.6 ,AM Ferredoxin and 6 mM metronidazole

Table 2. Photosynthetic electron transport-dependent oxygen exchange by wild-type and PS I mutant cells of C. reinhardtii

Rate of 02 uptake (,mol/mg of chlorophyll hr) 130 266 166 254 9 7 118 89 706 295 443

PS I-dependent oxygen uptake with methyl viologen and metronidazole as electron acceptors was measured with a Clark-type oxygen electrode (16). Thylakoid membranes and digitonin particles enriched in PS I were prepared as described in Materials and Methods. Reaction mixtures with thylakoid membranes contained: 30 jig/ml of chlorophyll; 40 mM Hepes-KOH (pH 7.0); 20 mM KCl; 2.5 mM MgCl2; 2 mM NH4Cl; 1 mM NaN3; 0.1 mM 2,6-dichlorophenolindophenol; 3 mM sodium ascorbate; and 10 ,M 3,4-dichlorophenyl dimethylurea (DCMU). The reaction mixtures for the digitonin particles were identical except the chlorophyll concentration was 2.5 Ag/ml and saturating amounts of plastocyanin purified from C. reinhardtii by the method of Gorman and Levine (17) were used. Ferredoxin was prepared from C. reinhardtii (18). Chlorophyll was measured in acetone extracts according to Arnon (19).

of its nitro group by ferredoxin in the pyruvate decarboxylation pathway of certain anaerobes (13, 14). We reasoned that selective killing of photosynthetic organisms with intact photosynthetic electron transport chains also might occur through light-dependent metronidazole reduction by ferredoxin. Edwards et al. (15) previously have shown that metronidazole can mediate PS I-dependent oxygen uptake and inhibit ferredoxin-dependent NADP reduction by spinach chloroplasts. In Table 1, the effectiveness of metronidazole as an oxidant for PS I in thylakoid membrane preparations from C. reinhardtii is compared with methyl viologen. In the absence of any added electron acceptor, 02 uptake with thylakoid membranes from wild-type cells proceeds at a considerable rate. This is presumably due to the presence of endogenous ferredoxin which is autooxidizable upon its photoreduction by PS I (20). Addition of low concentrations of methyl viologen stimulates PS I-dependent 02 uptake 2-fold, but 40 times higher concentrations of metronidazole are required to achieve maximal rates. These results suggest that metronidazole has a low affinity for its electron donor, the redox reaction involving metronidazole is noncatalytic, or both. To demonstrate further that 02 uptake mediated by metronidazole requires active PS I, we used thylakoid membranes purified from a PS I-deficient mutant, Fl. This mutant has been shown to lack the 700 nm absorption change and the chlorophyll-protein complex associated with PS I reaction centers (21). Table 1 shows that in this mutant the rate of photooxidation of reduced 2,6-dichlorophenolindophenol is less than 5% that of wild type. Although it is clear that metronidazole can effect PS I-dependent 02 uptake, it cannot be determined whether the drug

611

Rate of 02 exchange

(yrmolr/mg of chlorophyll-hr) Wild-type No additions + 12 mM Metronidazole + 12 mM Methyl viologen + 12 mM Metronidazole + 10

PLMDCMU F1 No additions + 18 mM Methyl viologen + 18 mM Metronidazole

+ 65 (evolution)

- 96 (uptake) - 78 (uptake) 0 0 0 0

The cells were suspended in 25 mM Hepes-KOH (pH 7.0) to a final chlorophyll concentration of 60 ,g/ml and preincubated for 15 min in darkness in the presence of the electron acceptors for PS I. The rate of oxygen exchange dependent upon photosynthetic electron transport was calculated by correcting for the respiration rate of cells kept in darkness. For other details, see legend of Table 1.

is reduced directly by the primary electron acceptor of PS I or indirectly via ferredoxin. Therefore, fractions enriched in PS I were prepared by digitonin treatment of thylakoid membranes (10). The endogenous rates of PS I-dependent 02 uptake is low relative to that in the presence of methyl viologen in digitonin particles (Table 1). Metronidazole could not substitute for methyl viologen in the Mehler reaction mediated by these particles, indicating it cannot be reduced directly by the primary electron acceptor for PS I. Addition of ferredoxin to the digitonin particles stimulates light-dependent 02 uptake about 3-fold. However, in reaction mixtures containing ferredoxin plus metronidazole the reaction is stimulated even further. From these results, we conclude that the photoreduction of metronidazole by PS I is dependent upon the presence of ferredoxin. Table 2 shows that metronidazole, like methyl viologen (22), can mediate the Mehler reaction in intact cells. Oxygen uptake in the presence of metronidazole and methyl viologen-is completely inhibited by DCMU, indicating that activities of intact photosynthetic electron transport are required. No oxygen exchange in the presence of methyl viologen or metronidazole occurs in mutant Fl. Therefore, metronidazole cannot accept electrons directly from photosystem II (PS II), which is intact in F1 (21). The studies in vitro and in vivo demonstrate that metronidazole can be reduced by photosynthetic electron transport in C. reinhardtii. Furthermore, in C. reinhardtii and anaerobes alike, ferredoxin is required for reduction of the drug (14). Since it is toxic to anaerobes in its reduced form, we determined whether metronidazole kills cells of C. reinhardtii with active photosynthetic electron transport. The toxicity of metronidazole in wild-type cells of C. reinhardtii is shown in Fig. 1. Approximately 99.99% of the wildtype cells are killed during incubation in the presence of 10 mM metronidazole for 24 hr in the light. In contrast, the recovery of F34, a PS II mutant (3), is nearly 100% after metronidazole treatment under conditions that kill almost the entire wild-type cell population. The specificity of metronidazole as a selection agent was tested further with wild-type and mutants of C. reinhardtii that are blocked at various photosynthetic steps. In Table 3, only 0.03% of the wild-type cells survive 6 mM metronidazole after

Proc. Natl. Acad. Sci. USA 74 (1977)

Cell Biology: Schmidt et al.

612

Table 4. Protocol for isolation of photosynthetic electron transport mutants of C. reinhardtii by the metronidazole enrichment procedure

2

(1) Mutagenesis of wild-type cells. (2) Grow aliquots of cells for 3 days to allow expression of mutations. (3) Add metronidazole to a final concentration of 6-12 mM at a cell density of 2 to 5 X 105 cells per ml. (4) Illuminate with 7500 lux at 340 for 24 hr. (5) Plate -5 X 104 cells on Tris-acetate-phosphate medium. (6) Replica plate-colonies to minimal medium. (7) Isolate acetate-requiring and pigment-deficient mutants. (8) Analyze mutant phenotype: (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thylakoid membrane polypeptides. (b) Measure electron transport activities. (c) Determine mode of inheritance of mutations.

10

,1

U 10~ 6-

0

6

12

18

as long as port

24

Time of Treatment (Hr)

they

possess

active photosynthetic electron trans-

The protocol for selective enrichment of photosynthetic electron transport mutants from C. reinhardtii is shown in Table 4. Elevated temperatures are used during growth of mutagenized cultures in order that temperature-sensitive as well

FIG. 1. Kinetics of killing of cells of wild type (WT) and F34 in the presence of various concentrations of metronidazole. Cells in Tris-acetate-phosphate medium were treated with metronidazole at 340 undcer illumination of 7500 lux. Viable cell counts were measured as described in Materials and Methods.

as

nontemperature-sensitive mutations

may

be expressed.

Metronidazole is added to dilute cultures to insure high photosynthetic electron transport rates in wild-type cells. After 24 hr of metronidazole treatment the cultures appear chlorotic. Aliquots of about 5 X 104 cells are then plated on Tris-acetate-phosphate medium and incubated at 26°. When colonies appear, replicas are made on acetate-containing and acetatefree medium and acetate-requirers are isolated.

24 hr. In contrast, wild-type cells incubated either.in darkness in the light in the presence of DCMU grow in the presence of metronidazole.,Mutations that result in deficient PS I or PS II also prevent metronidazole toxicity. Cells of mutant ac20crl, which possess impaired chloroplast protein synthesis and cotisequently have deficient Hill reaction activities (25), are also recovered with nearly 10Q% efficiency. Impaired photophosphorylation cant indirectly inhibit photosynthetic electron transport (24), and, as shown with mutant F54, can render affected cells relatively resistant to metronidazole. Chlorophyll-deficient mutants, like ac5, possess intact photosynthetic electron transport (26) but survive metronidazole treatment probably becausq of lower rates of metronidazole reduction on a per cell basis. Mutations that affect photosynthetic carbon fixation do not cause resistance to metronidazole. Photosynthetic carbon fixation does not occur in F60 because it possesses impaired phosphoribulokinase activity (27). Yet, because photosynthetic electron transport is normal in the acetaterequiring mutant F60, cells of the strain are killed effectively by metronidazole. These experiments demonstrate conclusively that metronidazole is extremely lethal to cells of C. reinhardtai

or

DISCUSSION Our studies demonstrate that metronidazole can be used as a highly specific agent for the selective enrichment of mutants of C, reinhardti impaired in photosynthetic electron transport. On the basis of reconstruction experiments, the theoretical enrichment of such mutants over wild-type approaches 4000-fold. The lethal effect of metronidazole requires its reduction by ferredoxin (Table 1). Whether the obligate requirement for ferredoxin is a property of the quaternary structure of metronidazole in addition to its redox potential is not clear. However, other heterocyclic nitro and nitroso compounds, including 2,4-dinitrophenol and parathion, have been shown to be photoreduced only in the presence of ferredoxin (28). The re-

Table 3. Survival of wild-type and mutant strains of C. reinhardtii after treatment with 6 mM metronidazole for 24 hr Ref.

Strain

Wild-type Wild-type Wild-type F34 C1 F54

ac2Ocrl ac5

F60

Lesion

Incubation

Light Dark + 5 gM

DCMU 3 23 24 25 26 27

Light Light Light Light Light Light Light

Conditions of incubation and measurement of viable cell counts wrapped with plastic black electrical tape.

PS II PS I

Photophosphorylation Deficient in 70S ribosomes

Chlorophyll-deficient Phosphoribulokinase

was as described for Fig.

% Survival

0.026 188 142 104 101 26 89 44 0.16

1. Dark cultures were achieved with culture flasks

Proc. Nati. Acad. Sci. USA 74(1977)

Cell Biology: Schmidt et al.

613

DCMU

H20 - -

- --[ s-

-

i--

- -

--PS

---X---Fd--NADP '

Metronidazole CH2CH20H 0

-02N

N

CH3 I

11

* H202

A rapid procedure for selective enrichment of photosynthetic electron transport mutants.

Proc. NatI. Acad. Sci. USA Vol. 74, No. 2, pp. 610-614, February 1977 Cell Biology A rapid procedure for selective enrichment of photosynthetic elect...
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