Altered Flagellar Size-Control in shf-1 Short-Flagella Mutants of Chlamydomonas reinhardtii

Two allelic Mendelian mutations which confer a short flagella phenotype were used to explore flagellar size control in Chlamydomonas reinhardtii. When mutant/wild type quadriflagellate dikaryon cells were constructed, their two short flagella rapidly grew out to near wild type length. The kinetics of elongation suggest that the flagellar assembly process is not intrinsically self-limiting as a number of otherwise attractive models for size control require. Instead, we suggest that there exists a cellular machinery dedicated to flagellar size control and that the short-flagella mutations alter the machinery in some as yet unknown way. One of the mutants shows temperature-sensitive flagellar assembly, and both are flagellaless in acetate media. Genetic analysis indicates that the temperaturesensitive, acetate-sensitive, and short-flagella phenotypes have a common genetic basis. The responsible gene has been named shf-1, and it has been mapped to chromosome VI, approximately 5 map units from the centromere.     

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.     

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.     

Mitochondrial fusion in Chlamydomonas reinhardtii zygotes

Mitochondria are dynamic organelles that were found to fuse and divide in many different cell types. Mitochondrial fusion plays important roles in maintenance of respiratory capacity, dissipation of metabolic energy, and inheritance of mitochondrial DNA. While the molecular machinery of mitochondrial fusion has been characterized in great detail in yeast and mammals, only little is known about mitochondrial fusion in higher plants and algae. We asked whether mitochondrial fusion can be observed in the unicellular green alga Chlamydomonas reinhardtii. Mitochondria were stained with fluorescent dyes in gametes, and mixing of fluorescent markers was detected by fluorescence microscopy in zygotes indicating fusion. Mitochondrial fusion was observed in wild type zygotes, and also in respiratory mutants, albeit with less efficiency. We conclude that mitochondria readily fuse in green algae.     

Receptor-Mediated Calcium Influx in Chlamydomonas reinhardtii

ABSTRACT. Alcian blue acts as a secretagogue and chemorepellent in a variety of unicellular eukaryotes. We report that alcian blue stimulates flagellar excision and induction of RNA encoding flagellar proteins in Chlamydomonas reinhardtii . Flagellar excision by alcian blue is dependent on extracellular Ca²⁺ and is blocked by La³⁺, ruthenium red, and neomycin, and so is similar to flagellar excision by acid shock. However, the adf-l mutant excises its flagella following alcian blue treatment, but not following acid shock, thus genetically distinguishing alcian-blue-induced excision from acid-shock-induced excision. Wild-type, but not adf-1, cells regrow their flagella in the continued presence of alcian blue. Wild-type cells that regrow flagella in the presence of alcian blue fail to excise their flagella in response to either increased concentrations of alcian blue or to acid shock. Alcian blue treatment of cells also induces RNA encoding flagellar components, but in a manner distinct from other means of stimulation. These results suggest that treating Chlamydomonas with the secretagogue alcian blue initiates a Ca²⁺ influx pathway and that prolonged treatment with alcian blue desensitizes the acid-shock-activated Ca²⁺ influx pathway to acid treatment. Alcian blue will thus be a useful excitatory ligand in future studies of receptor-mediated Ca²⁺ signaling in the Chlamydomonas flagellar regeneration system.     

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.     

Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii

Acetamide, a nitrogen and carbon source for Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wild-type through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm⁺ (utilized acetamide as sole N source) and acm^-. All acm⁺ isolates had acetamidase activity and were obligate phototrophs (i.e. dark-diers). Acm^- isolates had either normal urea assimilation (ure⁺) or lacked all urea pathway activities, namely transport, urea carboxylase and allophanate hydrolase (ure^-). Inheritance patterns for both types indicated single nuclear gene mutations. The acm^- ure⁺ type presumably resulted from a defective acetamidase gene, and the acm^- ure^- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wild-type enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence.Abbreviations F-acetamide fluoroacetamide - F-acetate fluoroacetate - TAP tris-acetate-phosphate medium - CDB Chlamydomonas dilution buffer - TCA trichloroacetic acid - AH allophanate hydrolase - UC urea carboxylase - PAR photosynthetically active radiation - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Intracellular Carbon Partitioning in Chlamydomonas reinhardtii

Using enzymic and isotope techniques the intracellular partitioning of newly fixed carbon was studied in synchronized cells of Chlamydomonas reinhardtii. Starch and growth metabolism, i.e. the use of carbon in biosynthesis, were found to be the major sinks for photosynthetically fixed carbon in the alga. Sucrose does not accumulate in significant quantities. The amount of carbon partitioned either into starch or growth varies during the 12 hour light/12 hour dark cell cycle. Starch is accumulated at the beginning and at the end of the light period while a net breakdown is observed in the middle of the light period and in the dark. In contrast, nonsynchronized cells accumulate starch all the time in the light which suggests that carbon partitioning is controlled by the cell cycle. Labeled bicarbonate is incorporated into starch even at times when the total intracellular level of starch is decreasing. This indicates a turnover of the starch pool in the light with synthesis and degradation occurring simultaneously and at different rates.     

Stress-induced protein synthesis in Chlamydomonas reinhardtii

Deoxyribonucleic acid base composition, deoxyribonucleic acid-deoxyribonucleic acid hybridization, and biochemical studies were performed on some enterococci from clinical sources of uncertain taxonomic position. Our results indicate that 6 human strains, a single clinical isolate and a strain from bovine mastitis are genetically distinct from each other and all other previously described Enterococcus species and constitute three new species, for which the names Enterococcus raffinosus, Enterococcus solitarius and Enterococcus pseudoavium are proposed.     

New arginine-requiring mutants in Chlamydomonas reinhardtii

Twelve arginine-requiring mutants of the unicellular green alga Chlamydomonas reinhardtii previously isolated in our laboratory were investigated to find new blocks in the biosynthetic pathway of arginine. In addition to the already described mutants lacking acetylglutamyl phosphate reductase (arg 1), ornithine carbamoyltransferase (arg4) and argininosuccinate lyase (arg7), three new types of mutants were found lacking acetylornithine aminotransferase (arg9-1, arg9-2), acetylornithine glutamate transacetylase (arg10) and argininosuccinate synthetase (arg8-1, arg8-2, arg8-3) respectively. The genetic analysis of these new mutants showed that arg9 and arg8 are unlinked to the other arginine markers and that arg10 probably carries a chromosomal mutation inducing a very high lethality of meiotic products.Abbreviations WT wild-type - mt mating-type - SP spore plating - ZP zygote plating     

Adaptive responses in Chlamydomonas reinhardtii.

The photosynthetic single cellular alga Chlamydomonas reinhardtii has been used as a model organism to examine in detail the physiological, biochemical and molecular processes of photosynthesis, flagella synthesis and movement, mineral stress, interactions between nucleus, chloroplasts and mitochondria and other processes. In this review we summarize part of the current knowledge on adaptive responses in C. reinhardtii when it is exposed to oxidative stress and to changes in light intensity, concentration of minerals, herbicides and metals. The individual responses are linked in order to understand the response of the cell, which is continuously subjected to fluctuations, as a whole.     

Visualization of microbodies in Chlamydomonas reinhardtii

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0?μm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.     

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.     

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.