Current Genetics
Current Genetics 3, 251-253 (1981)
© Springer-Verlag 1981
Short Communication A Uniparental Mutant of Chlamydomonas reinhardtii Resistant to Chloramphenieol P. Bennoun, P. Delepelaire and M: Delosme Institut de Bio!ogiePhysico-Claimique,13, rue Pierre et Marie Curie, 75005 Paris, France
Summary. A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed. Key words: Chlamydomonas - Uniparental mutation Chloramphenicol resistance
Introduction
Chlamydomonas mutants resistant to various antibiotics
affecting 70S translation and displaying uniparental maternal inheritance have been extensively studied (for a review, see Gillham 1978). Chloramphenicol-resistant mutants in this organism, however, have not been reported. We describe here one such mutant isolated following treatment of wild-type cells with 5-fluorodeoxyuridine. This drug was shown previously to increase the yield of uniparentally inherited mutations conferring resistance to spectinomyein and streptomycin (Wurtz et al. 1979), or resulting in impaired photosynthesis (Shepherd et al. 1979, Bennoun et al. 1978).
Offprint requests to: P. Bennoun
Results and Discussion Wild-type cells of C. reinhardtii strain 137C mt÷ were inoculated into Tris-acetate-phosphate medium (TAP; Gorman and Levine 1965) at a concentration of 10 4 cells/ml in the presence of 1 mM 5-flourodeoxyuridine (FUD). Following growth to stationary phase, cells were diluted 50 times in fresh TAP medium supplemented with 100 7/ ml chloramphenicol and exposed to an illumination of 1,500 lux. Chloramphenicol-resistant, surviving cells were obtained in one of the four flaks inoculated with FUD-treated cells. Following cloning on agar plates, one clone designated CAP1 was analyzed further, as described below. The growth of the chloramphenicol-resistant mutant CAP1 was estimated by drop4est on agar plates (see Fig. 1). Under heterotrophic conditions, i.e. in the presence of acetate and in the dark, CAP1 grows as fast as the wild-type (Fig. 1, lane 1). Under heterotrophic conditions, but in the presence of 200 7/ml chloramphenicol, the growth of CAP1 is unaffected whereas that of the wild-type is strongly reduced (Fig. 1, lane 2). Under phototrophic conditions, i.e. with atmospheric CO2 as the sole carbon Source and under an illumination of 3,000 lux, the growth of CAP1 is temperature sensitive: at the usual growth temperature (25°c) CAP1 and wildtype grow at the same rate whereas at 20 °C or 34 °C, the growth of CAP1 is reduced as compared to that of the wild-type (Fig. 1, lane 3). Under the same phototrophlc conditions, the addition of 200 7/ml chloramphenicol prevents the growth of both wild-type and CAP1 (not shown in the figure). The CAP1 mutant displays a pleiotropic phenotype: in addition to its resistance to chloramphenicol under heterotrophic conditions, this mutant exhibits temperature sensitive growth in the absence of antibiotics under phototrophic conditions. This led us to question whether 0172-8083/81/0003/0251/$01.00
252
P. Bennoun et al.:
Fig. 1. Drop growth tests. Each petri plate carries 1 drop of a wild-type cell suspension containing % 104 cells surrounded by four drops of a CAP1 cell suspension of equivalent initial cell density. The photograph was taken following incubation for one week under the following conditions: Lane 1 : TAP medium, dark grown. Lane 2: TAP medium suppemented with 200 7/ml chloramphenicol, dark grown. Lane 3: Minimal medium, light grown (3,000 lux). Temperature is as indicated on the figure
Fig. 2. Electrophoretogram of thylakoid membrane polypeptides of Chlamydomonas wild-type and mutant CAP1 grown heterotrophically at 25 ° and pulse-labelled in 14C acetate for one hour with and without 100 q,/ml ehloramphenicol. A constant concentration (12%) of aerylamide was used. Left: Slot 1 : CAP1 + Chloramphenicol; Slot 2: WT + Chloramphenieol; Slot 3: CAP1; Slot 4: WT. Right: Autoradiograph of the gel shown on the left
ChlamydomonasMutant Resistant to Chloramphenicol
both characters are co-transmitted in crosses. In the cross, CAP1 mt + x CAP1+ rot-, nineteen tetrads were analyzed: in seventeen of these tetrads both characters of the mt + parent were transmitted exclusively to the four meiotic progeny; in the remaining two tetrads the characters of the mt- parent were transmitted. In the reciprocal cross, CAP1 mt- x CAP1 + mt +, only the characters of the mt + parent were transmitted to the four meiotic progeny of all 12 tetrads analyzed. These data show a clear uniparental maternal co-transmission of both the resistance to chloramphenicol and the thermosensitivity. Similarly in those cases of exceptional transmission by the m t - parent, both rot- associated characters were transmitted to the tetrad products. Heterotrophically-grown (25 °C) wild type and CAP1 cells were pulselabelled with ~4C acetate in the presence or absence of chloramphenicol (Chua and Gillham 1977) to determine whether chloroplast protein synthesis in CAP1 was also resistant to the antibiotic. Thylakoid membrane polypeptides were separated by polyacrylamide gel electrophoresis (Chua et al. 1975) and an autoradiograph of the stained gel was obtained (Fig. 2). Whereas the synthesis of the chloroplast made polypeptides 2, 4, 5 and 6 is completely inhibited by 100 7/ml chloramphenicol in wild-type cells, the synthesis of these polypeptides is only slightly reduced by 100 7/ml chloramphenicol in CAP1 cells (slots 2 and 4 compared to 1 and 3). Thus although the chloroplast is not directly involved in heterotrophic growth, chloroplast protein synthesis in CAP1 is resistant to chloramphenicol under these conditions. Furthermore, when CAP1 cells are grown heterotrophically for six generations in the presence of chloramphenicol, they retain 75% of their photosynthetic ability whereas wild-type cells retain less than 5% (measured by oxygen flash yield according to procedure of Joliot and Joliot 1968). The thermosensitivity of CAP1 was investigated further by analysing thylakoid membrane following phototrophic growth at 34 °C. Under these conditions, a specific set of polypeptides is deficient in CAP1 as compared to wild-type (Fig. 3, slots 2 and 3). These are the very same ones which are deficient following growth of the wild-type in the presence of an inhibitor of 70s translation (Chua and Gillham 1977), particularly CP1, polypeptides 4, 5 and 6 which are known to be synthesized inside the chloroplast. This experiment indicates that chloroplast translation or transcription is defective in CAP1 grown phototrophically at 34°C, in the absence of chloramphenicol. It is worth mentioning that the thermosensitivity of CAP1 occurs only under phototrophic conditions. Under heterotrophic or mixotrophic conditions at 35 ~C, CAP1 grows as well as the wild-type does and shows no alteration in its thylakoid membrane polypeptides pattern. Under mixotropic conditions, i.e. in the presence of acetate and with an illumination of
L Bennoun et al.: ChlamydomonasMutant Resistant to Chloramphenieol
Fig. 3. Electrophoretogramof thylakoid membranepolypeptides of Chlamydomonaswild-type and mutant CAP1, using a 7,5/15% linear acrylamide gradient gel. Slot 1:CAP1 Grown heterotrophieally at 34 °C Slot 2:CAP1 grown phototrophically at 34°C Slot 3: WT grown phototrophically at 34 °C
3,000 lux, the growth of CAP1 at 25 °C is prevented by 200 7/ml Ichloramphenicol on agar plates. We also observed that CAP1 shows the same sensitivity as the wildtype to streptomycin, spectinomycin and erythromycin when grown heterotrophically. The complex phenotype of the CAP1 mutant raises two kinds of problems: 1) How a maternal mutation, thought to be located in chloroplast DNA, leads to chloramphenicol-resistant growth and chloramphenicol-resistant chloroplast protein synthesis in heterotrophically grown cells. Similar problems have been encountered with other antibiotic resistant mutants of Chlamydomonas displaying maternal inheritance (see Gillham 1978). 2) Why does the chloroplast of CAP1 show different properties when cells are grown heterotrophically or phototrophically: chloroplast protein synthesis resistant to chloramphenicol in the former case, thermosensitivity of chloroplast protein synthesis in the latter. Several alternative hypotheses might be proposed to explain our observations: a) The mutation present in CAP1 alters the permeability of the cells toward chloramphenicol.
253
b) The phenotype o f CAP1 results from multiple mutations displaying uniparental maternal inheritance. c) The complex CAP1 phenotype results from modifications at the organelle ribosomal level. To take into account the phenotype of heterotrophic cells of CAP1, i.e. growth and chloroplast protein synthesis resistant to chloramphenicol, one has toassume that both mitochondrial and chloroplast ribosomes are resistant. The possibility that a chloroplast gene product might be required for both mitochondrial and chloroplast ribosomes has been suggested by other (for a review, see Gillham 1978). Until direct examination of mitochondrial ribosomes is achieved, this possibility remains open. One must take into account the observation that chloroplast protein synthesis is resistant to chloramphenicol in heterotrophlc CAP1 cells but sensitive in phototrophic cells. This hypothesis thus requires that chloroplast ribosomes or ribosomal assembly be different in heterotrophic and phototrophlc cells. The modification of a component of chloroplast ribosomes might lead to their resistance to chloramphenicol under conditions promoting proper assembly into functional 70s ribosomes, but thermosensitive growth and chloramphenicol sensitivity under conditions inhibiting normal ribosomal assembly or function. Direct examination of chloroplast ribosomes from phototrophically and heterotrophically gown CAP1 and wild-type cells will be necessary to evaluate such an hypothesis.
References Bennoun P, Masson A, Pieeioni R, Chua NH (1978) Chloroplast Development. Elsevier North-Holland, Biomedical Press, pp 721-726 Bennoun P, Diner BA, Wollman FA, Sehmidt G, ChuaNH (1980) Proceedings of the Fifth International Congress on Photosynthesis. In press Chua NH, Marlin K, Bennoun P (1975) J Cell Biol 67:361-377 Chua NH, Gillham NW (1977) J Cell Biol 74:441-452 GiUham NW (1978) OrganeUeheredity. Raven Press, New York Gorman DS, Levine RP (1965) Proc Natl Aead Sei USA 54: 1665-1669 Joliot P, Joliot A (1968) Biochim Biophys Aeat 153:625-634 Shepherd HS, Boyton JE, Giilham NW (1979) Proe Natl Aead Sci USA 76:1353-1357 Wurtz EA, Sears BB, Rabert DK, Shepherd HS, Gillham NW, Boynton JE (1979) Mot Gen Genet 170:235- 242
Communicated by K. P. VanWinkle-Swifl Received January 17/March 10, 1981
Current Genetics 3, 251-253 (1981)
© Springer-Verlag 1981
Short Communication A Uniparental Mutant of Chlamydomonas reinhardtii Resistant to Chloramphenieol P. Bennoun, P. Delepelaire and M: Delosme Institut de Bio!ogiePhysico-Claimique,13, rue Pierre et Marie Curie, 75005 Paris, France
Summary. A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed. Key words: Chlamydomonas - Uniparental mutation Chloramphenicol resistance
Introduction
Chlamydomonas mutants resistant to various antibiotics
affecting 70S translation and displaying uniparental maternal inheritance have been extensively studied (for a review, see Gillham 1978). Chloramphenicol-resistant mutants in this organism, however, have not been reported. We describe here one such mutant isolated following treatment of wild-type cells with 5-fluorodeoxyuridine. This drug was shown previously to increase the yield of uniparentally inherited mutations conferring resistance to spectinomyein and streptomycin (Wurtz et al. 1979), or resulting in impaired photosynthesis (Shepherd et al. 1979, Bennoun et al. 1978).
Offprint requests to: P. Bennoun
Results and Discussion Wild-type cells of C. reinhardtii strain 137C mt÷ were inoculated into Tris-acetate-phosphate medium (TAP; Gorman and Levine 1965) at a concentration of 10 4 cells/ml in the presence of 1 mM 5-flourodeoxyuridine (FUD). Following growth to stationary phase, cells were diluted 50 times in fresh TAP medium supplemented with 100 7/ ml chloramphenicol and exposed to an illumination of 1,500 lux. Chloramphenicol-resistant, surviving cells were obtained in one of the four flaks inoculated with FUD-treated cells. Following cloning on agar plates, one clone designated CAP1 was analyzed further, as described below. The growth of the chloramphenicol-resistant mutant CAP1 was estimated by drop4est on agar plates (see Fig. 1). Under heterotrophic conditions, i.e. in the presence of acetate and in the dark, CAP1 grows as fast as the wild-type (Fig. 1, lane 1). Under heterotrophic conditions, but in the presence of 200 7/ml chloramphenicol, the growth of CAP1 is unaffected whereas that of the wild-type is strongly reduced (Fig. 1, lane 2). Under phototrophic conditions, i.e. with atmospheric CO2 as the sole carbon Source and under an illumination of 3,000 lux, the growth of CAP1 is temperature sensitive: at the usual growth temperature (25°c) CAP1 and wildtype grow at the same rate whereas at 20 °C or 34 °C, the growth of CAP1 is reduced as compared to that of the wild-type (Fig. 1, lane 3). Under the same phototrophlc conditions, the addition of 200 7/ml chloramphenicol prevents the growth of both wild-type and CAP1 (not shown in the figure). The CAP1 mutant displays a pleiotropic phenotype: in addition to its resistance to chloramphenicol under heterotrophic conditions, this mutant exhibits temperature sensitive growth in the absence of antibiotics under phototrophic conditions. This led us to question whether 0172-8083/81/0003/0251/$01.00
252
P. Bennoun et al.:
Fig. 1. Drop growth tests. Each petri plate carries 1 drop of a wild-type cell suspension containing % 104 cells surrounded by four drops of a CAP1 cell suspension of equivalent initial cell density. The photograph was taken following incubation for one week under the following conditions: Lane 1 : TAP medium, dark grown. Lane 2: TAP medium suppemented with 200 7/ml chloramphenicol, dark grown. Lane 3: Minimal medium, light grown (3,000 lux). Temperature is as indicated on the figure
Fig. 2. Electrophoretogram of thylakoid membrane polypeptides of Chlamydomonas wild-type and mutant CAP1 grown heterotrophically at 25 ° and pulse-labelled in 14C acetate for one hour with and without 100 q,/ml ehloramphenicol. A constant concentration (12%) of aerylamide was used. Left: Slot 1 : CAP1 + Chloramphenicol; Slot 2: WT + Chloramphenieol; Slot 3: CAP1; Slot 4: WT. Right: Autoradiograph of the gel shown on the left
ChlamydomonasMutant Resistant to Chloramphenicol
both characters are co-transmitted in crosses. In the cross, CAP1 mt + x CAP1+ rot-, nineteen tetrads were analyzed: in seventeen of these tetrads both characters of the mt + parent were transmitted exclusively to the four meiotic progeny; in the remaining two tetrads the characters of the mt- parent were transmitted. In the reciprocal cross, CAP1 mt- x CAP1 + mt +, only the characters of the mt + parent were transmitted to the four meiotic progeny of all 12 tetrads analyzed. These data show a clear uniparental maternal co-transmission of both the resistance to chloramphenicol and the thermosensitivity. Similarly in those cases of exceptional transmission by the m t - parent, both rot- associated characters were transmitted to the tetrad products. Heterotrophically-grown (25 °C) wild type and CAP1 cells were pulselabelled with ~4C acetate in the presence or absence of chloramphenicol (Chua and Gillham 1977) to determine whether chloroplast protein synthesis in CAP1 was also resistant to the antibiotic. Thylakoid membrane polypeptides were separated by polyacrylamide gel electrophoresis (Chua et al. 1975) and an autoradiograph of the stained gel was obtained (Fig. 2). Whereas the synthesis of the chloroplast made polypeptides 2, 4, 5 and 6 is completely inhibited by 100 7/ml chloramphenicol in wild-type cells, the synthesis of these polypeptides is only slightly reduced by 100 7/ml chloramphenicol in CAP1 cells (slots 2 and 4 compared to 1 and 3). Thus although the chloroplast is not directly involved in heterotrophic growth, chloroplast protein synthesis in CAP1 is resistant to chloramphenicol under these conditions. Furthermore, when CAP1 cells are grown heterotrophically for six generations in the presence of chloramphenicol, they retain 75% of their photosynthetic ability whereas wild-type cells retain less than 5% (measured by oxygen flash yield according to procedure of Joliot and Joliot 1968). The thermosensitivity of CAP1 was investigated further by analysing thylakoid membrane following phototrophic growth at 34 °C. Under these conditions, a specific set of polypeptides is deficient in CAP1 as compared to wild-type (Fig. 3, slots 2 and 3). These are the very same ones which are deficient following growth of the wild-type in the presence of an inhibitor of 70s translation (Chua and Gillham 1977), particularly CP1, polypeptides 4, 5 and 6 which are known to be synthesized inside the chloroplast. This experiment indicates that chloroplast translation or transcription is defective in CAP1 grown phototrophically at 34°C, in the absence of chloramphenicol. It is worth mentioning that the thermosensitivity of CAP1 occurs only under phototrophic conditions. Under heterotrophic or mixotrophic conditions at 35 ~C, CAP1 grows as well as the wild-type does and shows no alteration in its thylakoid membrane polypeptides pattern. Under mixotropic conditions, i.e. in the presence of acetate and with an illumination of
L Bennoun et al.: ChlamydomonasMutant Resistant to Chloramphenieol
Fig. 3. Electrophoretogramof thylakoid membranepolypeptides of Chlamydomonaswild-type and mutant CAP1, using a 7,5/15% linear acrylamide gradient gel. Slot 1:CAP1 Grown heterotrophieally at 34 °C Slot 2:CAP1 grown phototrophically at 34°C Slot 3: WT grown phototrophically at 34 °C
3,000 lux, the growth of CAP1 at 25 °C is prevented by 200 7/ml Ichloramphenicol on agar plates. We also observed that CAP1 shows the same sensitivity as the wildtype to streptomycin, spectinomycin and erythromycin when grown heterotrophically. The complex phenotype of the CAP1 mutant raises two kinds of problems: 1) How a maternal mutation, thought to be located in chloroplast DNA, leads to chloramphenicol-resistant growth and chloramphenicol-resistant chloroplast protein synthesis in heterotrophically grown cells. Similar problems have been encountered with other antibiotic resistant mutants of Chlamydomonas displaying maternal inheritance (see Gillham 1978). 2) Why does the chloroplast of CAP1 show different properties when cells are grown heterotrophically or phototrophically: chloroplast protein synthesis resistant to chloramphenicol in the former case, thermosensitivity of chloroplast protein synthesis in the latter. Several alternative hypotheses might be proposed to explain our observations: a) The mutation present in CAP1 alters the permeability of the cells toward chloramphenicol.
253
b) The phenotype o f CAP1 results from multiple mutations displaying uniparental maternal inheritance. c) The complex CAP1 phenotype results from modifications at the organelle ribosomal level. To take into account the phenotype of heterotrophic cells of CAP1, i.e. growth and chloroplast protein synthesis resistant to chloramphenicol, one has toassume that both mitochondrial and chloroplast ribosomes are resistant. The possibility that a chloroplast gene product might be required for both mitochondrial and chloroplast ribosomes has been suggested by other (for a review, see Gillham 1978). Until direct examination of mitochondrial ribosomes is achieved, this possibility remains open. One must take into account the observation that chloroplast protein synthesis is resistant to chloramphenicol in heterotrophlc CAP1 cells but sensitive in phototrophic cells. This hypothesis thus requires that chloroplast ribosomes or ribosomal assembly be different in heterotrophic and phototrophlc cells. The modification of a component of chloroplast ribosomes might lead to their resistance to chloramphenicol under conditions promoting proper assembly into functional 70s ribosomes, but thermosensitive growth and chloramphenicol sensitivity under conditions inhibiting normal ribosomal assembly or function. Direct examination of chloroplast ribosomes from phototrophically and heterotrophically gown CAP1 and wild-type cells will be necessary to evaluate such an hypothesis.
References Bennoun P, Masson A, Pieeioni R, Chua NH (1978) Chloroplast Development. Elsevier North-Holland, Biomedical Press, pp 721-726 Bennoun P, Diner BA, Wollman FA, Sehmidt G, ChuaNH (1980) Proceedings of the Fifth International Congress on Photosynthesis. In press Chua NH, Marlin K, Bennoun P (1975) J Cell Biol 67:361-377 Chua NH, Gillham NW (1977) J Cell Biol 74:441-452 GiUham NW (1978) OrganeUeheredity. Raven Press, New York Gorman DS, Levine RP (1965) Proc Natl Aead Sei USA 54: 1665-1669 Joliot P, Joliot A (1968) Biochim Biophys Aeat 153:625-634 Shepherd HS, Boyton JE, Giilham NW (1979) Proe Natl Aead Sci USA 76:1353-1357 Wurtz EA, Sears BB, Rabert DK, Shepherd HS, Gillham NW, Boynton JE (1979) Mot Gen Genet 170:235- 242
Communicated by K. P. VanWinkle-Swifl Received January 17/March 10, 1981
