A Photorespiratory Mutant of Chlamydomonas reinhardtii

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO₂ concentration for photoautrophic growth in spite of the apparent induction of a functional CO₂ concentrating mechanism in air-adapted cells. In 2% O₂ the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O₂, photosynthetic O₂ evolution is severely inhibited in the mutant by preillumination in limiting CO₂, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O₂ evolution measurements. Net CO₂ uptake is also inhibited when the cells are exposed to air (21% O₂, 0.035% CO₂, balance N₂) for longer than a few minutes. [¹⁴C]Phosphoglycolate accumulates within 5 minutes of photosynthetic ¹⁴CO₂ fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO₂ requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.     

A Reduced Activity of Catalase as a Basis for Light Dependent Methionine Sensitivity of a Chlamydomonas reinhardtii Mutant

Pre-illumination of methionine-supplemented medium enhancedthe inactivation of the light dependent methionine sensitivemutant cells of Chlamydomonas reinhardtii and, to a lesser extent,the wild-type cells, confirming that the photodamage is dueto production of toxic produces) in the medium. Exogenouslyadded catalase protected the mutant cells from growth inhibition.Starch gel electrophoresis showed lower catalase activity inthe mutant cells than the wild type. Catalase activity whichwas followed by measuring O₂ evolution after the addition ofH₂O₂ to the cell suspensions was consistently lower in the mutantthan the wild type. The results indicate that light sensitivityin the presence of methionine expressed by this mutant is dueto reduced activity of catalase. (Received August 31, 1989; Accepted October 9, 1989)     

Mutant strains of Chlamydomonas reinhardtii that move backwards only

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak- like projections found within the B-tubules of outer doublets 5 and 6.     

Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii

Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

Transformation of CW-15 Mutant Cells of Chlamydomonas reinhardtii Dang. with pCTVHyg Plasmid

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10⁶ cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ Hyg^R recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg^R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.     

Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a “near dark” condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable.     

Mutant Male Chlamydomonas reinhardtii Cells with Few Chloroplast Nucleoids and Exceptional Chloroplast Gene Transmission

mt⁻ (male) mutant cell of Chlamydomonas reinhardtii was isolated. It was reduced in size and showed few chloroplast (cp) nucleoids. When smaller mutant cells, obtained through a 3 μm pore filter, were crossed with mt ⁺ wild type cells, the frequency of transmission of cp genes was not different from the wild type cross. The cell size and the number of cp nucleoids appear to have no effect on the transmission of cp genes. Received 27 August 1999/ Accepted in revised form 16 February 2000     

Periplasmic Carbonic Anhydrase Structural Gene (Cah1) Mutant in Chlamydomonas reinhardtii

To survive in various conditions of CO₂ availability, Chlamydomonas reinhardtii shows adaptive changes, such as induction of a CO₂-concentrating mechanism, changes in cell organization, and induction of several genes, including a periplasmic carbonic anhydrase (pCA1) encoded by Cah1. Among a collection of insertionally generated mutants, a mutant has been isolated that showed no pCA1 protein and no Cah1 mRNA. This mutant strain, designated cah1-1, has been confirmed to have a disruption in the Cah1 gene caused by a single Arg7 insert. The most interesting feature of cah1-1 is its lack of any significant growth phenotype. There is no major difference in growth or photosynthesis between the wild type and cah1-1 over a pH range from 5.0 to 9.0 even though this mutant apparently lacks Cah1 expression in air. Although the presence of pCA1 apparently gives some minor benefit at very low CO₂ concentrations, the characteristics of this Cah1 null mutant demonstrate that pCA1 is not essential for function of the CO₂-concentrating mechanism or for growth of C. reinhardtii at limiting CO₂ concentrations.     

Chemoresponses of Chlamydomonas reinhardtii

Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.     

Effect of Chlorophyll-Protein Complex I Deficiency on the Physiological Character of a Chlamydomonas reinhardtii Mutant

In the mutant CC-1047 of Chlamydomonas reinhardtii, LDS-PAGE showed that the chlorophyll-protein complex I (CPI) is almost absent. The mutant could not grow in a culture medium without organic carbon source while the wild type (WT) C. reinhardtii grew quickly. When an organic carbon source was added into the culture medium, the mutant grew almost as well as WT. The rate of photosystem 1 (PS1) electron transport (DCPIPMV) and the rate of whole chain electron transport (H₂OMV) of chloroplasts of the CC-1047 mutant were both lower than those of WT. The photophosphorylation activity, photosynthetic O₂ evolution rate, and rate of NADP⁺ photoreduction of CC-1047 were also much lower than the activities of WT. There were some differences in ATPase activity between the mutant and WT. Two different activation ways were used to activate the latent ATPase using methanol and dithiothreitol (DTT) as activation substrate. More methanol and DTT were required for the mutant than WT to obtain the maximum activity. Thus the photosynthetic apparatus could not operate normally when CPI was absent because of the abnormal PS1 electron transport. Meanwhile, the other adjacent complexes of the thylakoid membrane, for example, ATP synthase complex, were slightly affected.     

Blue Light-Induced Lethality of a Cell Wall-Deficient Mutant of the Unicellular Green Alga Chlamydomonas reinhardtii

When synchronized cultures of a cell wall-deficient Chlamydomonasreinhardtii mutant strain were grown under heterotrophic conditionsand subsequently transferred to the light, a considerable decreaseof the cell number was observed during transition to the celldivision phase. Lethality of the wall-deficient cells was inducedby blue light, but not by red or far-red light, and could notbe prevented by addition of the photosystem II inhibitor DCMU.The light-induced lethality was found to be restricted to wall-deficientcells which were agitated by bubbling with filtered air or nitrogenor vigorously shaken during the transition to the cell divisionphase. Therefore, a (blue) light-induced sensitivity to anymechanical stress seems to be the cause for cell death. In heterotrophicallygrowing cultures of the Chlamydomonas wild-type, illuminationwith blue or white light did not cause a decrease of the cellnumber but only a delay of cell divisions. The latter effectwas also observed in case of the wall-deficient mutant. Bothblue light effects are observed during the transition to thecell division phase and can be induced during the same periodof the cell cycle. Furthermore, the (blue) light-induced lethalityof wall-deficient cells was found to be prevented when the transitionto the cell division phase was inhibited by addition of antibiotics.Therefore, we assume that there is a connection between theblue light-induced sensitivity to mechanical stress and theblue light-induced delay of cell divisions. (Received September 3, 1993; Accepted November 12, 1993)     

Cardiolipin of Chlamydomonas reinhardtii 137

The fatty acids of cardiolipin from the phototrophic green alga Chlamydomonas reinhardtii 137⁺ have been quantitatively analysed. Comparison is made at the molecular level between the cardiolipin of Chlamydomonas and that of higher plant tissue.     

Short-Flagella Mutants of Chlamydomonas reinhardtii

Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.     

A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. C hlamydomonas reinhardtii flagella use an asymmetric beat stroke, similar to a breast stroke, to propel cells forward. To generate the asymmetric beat stroke, dynein activity must be regulated both along the length and around the circumference of the flagella. If all dyneins were active at the same time, the flagella would exist in a state of rigor. The dyneins are located in two rows along the length of the doublet microtubules. The inner dynein arms are heterogeneous in composition with at least eight heavy chains and various intermediate and light chains arranged in an elaborate morphology that repeats every 96 nm (Kagami and Kamiya, 1992; Mastronarde et al., 1992). In contrast, the outer dynein arms are biochemically and morphologically homogeneous (Huang et al., 1979; Mitchell and Rosenbaum, 1985; Kamiya, 1988); each outer dynein arm contains three dynein heavy chains and 10 intermediate and light chains. The inner and outer arms appear to have different functions in the formation of the beat stroke; the inner arms generate the waveform of the beat stroke, whereas the outer arms provide additional force to the waveform (Brokaw and Kamiya, 1987).Previous workers had shown that dynein regulation is imposed, in part, by activities of the radial spokes and the central pair complex. Mutant strains that are missing or have altered radial spokes or central pair complexes are paralyzed even if they have a full complement of dyneins (Adams et al., 1981; Piperno et al., 1981). Many extragenic suppressors of this paralysis phenotype do not restore the missing structures, but rather suppress by altering either inner arm or outer arm region structures (Huang et al., 1982a ; Piperno et al., 1992; Porter et al., 1992, 1994). These data suggest that direct or indirect interactions exist between the dynein arms and the radial spokes or central pair complexes.Over 80 proteins in Chlamydomonas flagella are phosphorylated (Piperno et al., 1981), which makes dynein regulation by phosphorylation an attractive model. Hasegawa et al. (1987) showed that a higher percentage of demembranated axonemes reactivate with ATP after treatments that lower cAMP levels or inhibit cAMP-dependent protein kinase (cAPK)¹. In flagella from other organisms, cAMP has an opposite role (for reviews see Tash and Means, 1983; Tash, 1989). An increased frequency of reactivation also occurs after the NP-40–soluble components are extracted from the axonemes, which suggests that the cAPK, target phosphoproteins, and endogenous phosphatases are all integral axonemal components (Hasegawa et al., 1987). In quantitative sliding disintegration assays, the inner dynein arm activity of axonemes that are missing the radial spokes is increased in the presence of pharmacological or specific peptide inhibitors of cAPK (Smith and Sale, 1992; Howard et al., 1994). Reconstitution experiments with axonemes that are missing the radial spokes suggest that radial spokes normally function to activate the inner dynein arms by inhibiting a cAPK (Smith and Sale, 1992; Howard et al., 1994). It is not known if the cAPK directly phosphorylates inner dynein arm components or phosphorylates another axonemal component that then acts on the inner dynein arms (Howard et al., 1994).The f (originally called I1) inner arms are biochemically the best studied inner dynein arm complex. This complex is comprised of two dynein heavy chains and three intermediate chains of 140, 138, and 110 kD; it can be purified by sucrose density centrifugation (Piperno and Luck, 1981; Smith and Sale, 1991; Porter et al., 1992) or ion-exchange chromatography (Kagami and Kamiya, 1992). The purified complex has low ATPase activity and only rarely translocates microtubules in vitro (Smith and Sale, 1991; Kagami and Kamiya, 1992). Deep-etch EM of the purified f inner arm shows a two-headed complex that is connected to a common base by thin stalks (Smith and Sale, 1991). Longitudinal EM image analyses have shown that this complex is located just proximally of the first radial spoke in each 96-nm repeating unit (Piperno et al., 1990; Mastronarde et al., 1992). Mutations at three different loci (PF9/ IDA1, IDA2, and IDA3) result in the complete loss of the f complex (Kamiya et al., 1991; Kagami and Kamiya, 1992; Porter et al., 1992). The PF9/IDA1 locus encodes a dynein heavy chain that is believed to be one of the two heavy chains that are components of the f complex (Porter, 1996).We undertook a new approach to identify axonemal components involved in dynein regulation; we isolated and characterized mutant strains that were unable to perform phototaxis. In Chlamydomonas, phototaxis is a behavior by which cells orient to the direction of incident light. Light direction is detected by the eyespot, an asymmetrically located organelle, and a signal is transmitted to the flagella using voltage-gated ion channels (Harz and Hegemann, 1991). For cells to perform phototaxis, the waveforms of the two flagella are altered coordinately. The trans flagellum, which is located farther from the eyespot, beats with a larger front amplitude than the cis flagellum to turn the cell toward the light (Rüffer and Nultsch, 1991). It seemed likely that the alterations in the beat amplitudes needed for correct phototactic behavior could be caused by differential dynein regulation in the cis and trans flagella. Therefore, we hypothesized that there should be a class of phototactic mutant strains that is not able to perform phototaxis because of defects in the regulation of dyneins. Three of the eight phototactic mutant strains that we characterized had biochemical defects in the f class of inner dynein arms. One of these strains, pf9-4, was missing the entire f complex, and the other two strains, mia1-1 and mia2-1, exhibited a novel f class inner dynein arm biochemical phenotype. These observations suggest that the f inner dynein arm is a target for regulation during phototaxis.     

An Indexed,Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.