Wild-type Chlamydomonas Reinhardtii Research Articles

Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii.

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5′ element and the coding region. Using 5′ elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5′ UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5′ rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control.

Acclimation of Chlamydomonas reinhardtii to extremely strong light

Most photosynthetic organisms are sensitive to very high light, although acclimation mechanisms enable them to deal with exposure to strong light up to a point. Here we show that cultures of wild-type Chlamydomonas reinhardtii strain cc124, when exposed to photosynthetic photon flux density 3000 μmol m−2 s−1 for a couple of days, are able to suddenly attain the ability to grow and thrive. We compared the phenotypes of control cells and cells acclimated to this extreme light (EL). The results suggest that genetic or epigenetic variation, developing during maintenance of the population in moderate light, contributes to the acclimation capability. EL acclimation was associated with a high carotenoid-to-chlorophyll ratio and slowed down PSII charge recombination reactions, probably by affecting the pre-exponential Arrhenius factor of the rate constant. In agreement with these findings, EL acclimated cells showed only one tenth of the 1O2 level of control cells. In spite of low 1O2 levels, the rate of the damaging reaction of PSII photoinhibition was similar in EL acclimated and control cells. Furthermore, EL acclimation was associated with slow PSII electron transfer to artificial quinone acceptors. The data show that ability to grow and thrive in extremely strong light is not restricted to photoinhibition-resistant organisms such as Chlorella ohadii or to high-light tolerant mutants, but a wild-type strain of a common model microalga has this ability as well.

Growth and hydrogen production by three Chlamydomonas strains cultivated in a commercial fertilizer

In the present investigation, we report the growth and hydrogen production of two wild type Chlamydomonas reinhardtii strains isolated from a tropical oxidation pond in Costa Rica. The performance of these two new isolates was compared to that of Chlamydomonas reinhardtii CC124. All the strains were grown both in conventional Tris-Acetate-Phosphate medium (TAP) and in a commercial fertilizer medium (NPK 20-20-20). The growth of the new two isolates in medium formulated with fertilizer was higher than that attained with the reference strain (CC124). However, the hydrogen production performance of the strain CC124 in TAP-S and fertilizer were comparable, while the two new strains performed better in fertilizer, although the total hydrogen production was lower than that achieved with CC124. By using fertilizer it is possible to reduce the cost of chemical reagents by about 63% compared to TAP. Another advantage of the fertilizer is that it does not contain sulfur, therefore it can be directly used for hydrogen production using the Melis protocol.

Optimization of the droplet electroporation method for wild type Chlamydomonas reinhardtii transformation

We performed the transformation of a wild type Chlamydomonas reinhardtii by optimizing previously developed droplet EP method. For more effective and faster optimization, we used DNA dying fluorescent molecule (Yo-Pro-1) for finding optimal EP conditions instead of using protein expression based evaluation method. By examining wider range of electrical parameter space together with the analysis of total current flow of EP process, we found optimal EP conditions. The obtained optimal EP conditions were verified by CFP transgene expression experiments. By applying the optimal EP conditions to the transformation of C. reinhardtii, we obtained transformants and analyzed them using PCR. Finally, implications and future work are discussed.

Survey of the anaerobic metabolism of various laboratory wild-type Chlamydomonas reinhardtii strains

Given the wide diversity of wild-type Chlamydomonas reinhardtii genotypes due to the presence of genes belonging to either of different gene haplotypes (Gallaher, S.D., Fitz-Gibon, S.R., Glaesener, A.G., Pellegrini, M. and Merchant, S.S. 2015, The Plant Cell, doi 10.1105/tpc.15.00508), we performed a phenotypic characterization of representative strains whose anaerobic metabolism has been studied either by our laboratory or by others. Our results reveal major differences in the levels of H2 production and in the pattern of fermentative metabolite generation, highlighting the activation of different fermentative pathways in each organism. Some of the differences could be attributed to differences in the haplotypes of the various genes involved in these pathways. Moreover, the nature of the metabolites produced under dark anaerobiosis versus sulfur-deprivation-induced anaerobiosis are different in the analyzed strains, emphasizing the differential activation of metabolic pathways promoted under the two conditions and highlighting the importance of utilizing appropriate C. reinhardtii strains for addressing specific research questions.

A highly efficient cell penetrating peptide pVEC-mediated protein delivery system into microalgae

Interest in microalgae has significantly increased due to its potential as a promising bioresource in various fields. However, a technique for intracellular delivery of proteins into microalgae has not established yet, although it is potentially valuable for advanced algal research. Here, we propose a cell penetrating peptide pVEC-mediated protein delivery tool for microalgae. We discovered that the peptide pVEC from vascular endothelial cadherin was more effective in transporting exogenous proteins into algal cells than other peptides including R9, Transportan, TAT and Penetratin. pVEC-mediated tool was shown to deliver proteins of various sizes from 6kDa to 150kDa into wild-type Chlamydomonas reinhardtii and to also work in other algal species such as Nannochloropsis salina and Chlorella vulgaris when delivering a 66kDa protein. In addition, we show that our proposed pVEC-mediated protein delivery system is based on both the endocytic and non-endocytic pathways simultaneously through a mechanism study using inhibitors such as low temperature, N-ethylmaleimide, 5-(N-ethyl-N-isopropyl) amiloride, chlorpromazine, sodium azide, and methyl-beta-cyclodextrin. The results show that the proposed simple and efficient protein delivery tool could contribute to advanced algal research for a wide array of applications for microalgae in various industries.

Does the singlet minus triplet spectrum with major photobleaching band near 680-682 nm represent an intact reaction center of Photosystem II?

We use both frequency- and time-domain low-temperature (5-20 K) spectroscopies to further elucidate the shape and spectral position of singlet minus triplet (triplet-bottleneck) spectra in the reaction centers (RCs) of Photosystem II (PSII) isolated from wild-type Chlamydomonas reinhardtii and spinach. It is shown that the shape of the nonresonant transient hole-burned spectrum in destabilized RCs from C. reinhardtii is very similar to that typically observed for spinach. This suggests that the previously observed difference in transient spectra between RCs from C. reinhardtii and spinach is not due to the sample origin but most likely due to a partial destabilization of the D1 and D2 polypeptides. This supports our previous assignments that destabilized RCs (referred to as RC680) (Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870), with a major photobleaching band near 680-682 nm and the absence of a photobleaching band near 673 nm, do not represent the intact RC residing within the PSII core complex. Time-resolved absorption difference spectra obtained for partially destabilized RCs of C. reinhardtii and for typical spinach RCs support the above conclusions. The absence of clear photobleaching bands near 673 and 684 nm (where the PD1 chlorophyll and the active pheophytin (PheoD1) contribute, respectively) in picosecond transient absorption spectra in both RCs studied in this work indicates that the cation can move from the primary electron donor (ChlD1) to PD1 (i.e., PD1ChlD1(+)PheoD1(-) → PD1(+)ChlD1PheoD1(-)). Therefore, we suggest that ChlD1 is the major electron donor in usually studied destabilized RCs (with a major photobleaching near 680-682 nm), although the PD1 path (where PD1 serves as the primary electron donor) is likely present in intact RCs, as discussed in Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870.

Induction of triacylglycerol production in Chlamydomonas reinhardtii: Comparative analysis of different element regimes

In this study, impacts of different element absence (nitrogen, sulfur, phosphorus and magnesium) and supplementation (nitrogen and zinc) on element uptake and triacylglycerol production was followed in wild type Chlamydomonas reinhardtii CC-124 strain. Macro- and microelement composition of C. reinhardtii greatly differed under element regimes studied. In particular, heavy metal quotas of the microalgae increased strikingly under zinc supplementation. Growth was suppressed, cell biovolume, carbohydrate, total neutral lipid and triacylglycerol levels increased when microalgae were incubated under these element regimes. Most of the intracellular space was occupied by lipid bodies under all nutrient starvations, as observed by confocal microscopy and transmission electron micrographs. Results suggest that sulfur, magnesium and phosphorus deprivations are superior to nitrogen deprivation for the induction triacylglycerol production in C. reinhardtii. On the other hand, FAME profiles of the nitrogen, sulfur and phosphorus deprived cells were found to meet the requirements of international standards for biodiesel.

Detection of algal lipid accumulation due to nitrogen limitation via dielectric spectroscopy of Chlamydomonas reinhardtii suspensions in a coaxial transmission line sample cell

In this study, dielectric characterization of algae cell suspensions was used to detect lipid accumulation due to nitrogen starvation. Wild-type Chlamydomonas reinhardtii (CC-125) was cultivated in replete and nitrogen-limited conditions in order to achieve a range of lipid contents, as confirmed by Nile Red fluorescence measurements. A vector network analyzer was used to measure the dielectric scattering parameters of a coaxial region of concentrated cell suspension. The critical frequency fc of the normalized transmission coefficient |S21(*)| decreased with increasing lipid content but did not change with cell concentration. These observations were consistent with a decrease in cytoplasmic conductivity due to lipid accumulation in the preliminary transmission line model. This dielectric sensitivity to lipid content will facilitate the development of a rapid, noninvasive method for algal lipid measurement that could be implemented in industrial settings without the need for specialized staff and analytical facilities.

Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas reinhardtii

It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin a (Pheo a) within the D1 protein (Pheo(D1)), while Pheo(D2) (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q(y)-states of Pheo(D1) and Pheo(D2) bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986 - 998; Cox et al. J. Phys. Chem. B 2009, 113, 12364 - 12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo(D1) is near 672 nm, whereas Pheo(D2) (~677.5 nm) and Chl(D1) (~680 nm) have the lowest energies (i.e., the Pheo(D2)-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q(y) absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472 - 11482; Germano et al. Biophys. J. 2004, 86, 1664 - 1672]. To provide more insight into the site energies of both Pheo(D1) and Pheo(D2) (including the corresponding Q(x) transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo(D1) is genetically replaced with chlorophyll a (Chl a). We show that the Q(x)-/Q(y)-region site energies of Pheo(D1) and Pheo(D2) are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803 - 8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare directly the effects of electroporation and sonication in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it nonspecifically disrupts cell-surface barriers. (E-mail: prausnitz@gatech.edu)

Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii.

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPase-deficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite- and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.

Comparative profiling of lipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas reinhardtii

Excess light can impose severe oxidative stress on photosynthetic organisms. We have characterized high-light responses in wild-type Chlamydomonas reinhardtii and in the npq1 lor1 double mutant. The npq1 lor1 strain lacks two photoprotective carotenoids, lutein and zeaxanthin, and experiences acute photo-oxidative stress upon exposure to excess light. To examine the ability of npq1 lor1 cells to respond to photo-oxidative stress, we measured changes in lipid-soluble antioxidants following a shift from low light to high light in the wild type and the double mutant. The size of the xanthophyll cycle pool increased in both the wild type and mutant during the first 6 h of exposure to high light levels, but then decreased in the mutant during photo-oxidative bleaching. The level of alpha-tocopherol (vitamin E) was constant in the wild type and mutant during the first 6 h; then it increased by three-fold in the wild type but declined in npq1 lor1 cells. We also used cDNA microarrays and RNA gel-blot analysis to monitor differences in gene expression. Both strains showed an initial light-stress response in the form of a transient increase in expression of (1) GPXH, a glutathione peroxidase gene that has been shown to respond specifically to singlet oxygen and lipid peroxidation; (2) SMT1, a gene for a putative sterol C-methyltransferase; and (3) LI818r, a stress-responsive member of the light-harvesting complex superfamily. These transient changes in gene expression in high light were followed by a second series of changes in npq1 lor1, coincident with declines in lipid-soluble antioxidants but preceding detectable photo-oxidative damage to proteins and lipids. Thus, the response of npq1 lor1 to high light is unexpectedly complex, with initial changes in lipid-soluble antioxidants and RNA levels that are associated with acclimation in the wild type and a second wave of changes that accompanies photo-oxidative bleaching.

Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella.

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke–central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair–radial spoke interactions involves passive central pair reorientation to changes in bend plane.

Evidence from time resolved studies of the P700[rad]+/A1[rad]− radical pair for photosynthetic electron transfer on both the PsaA and PsaB branches of the photosystem I reaction centre

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild-type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron–sulphur centres, Fe–SX/A/B, can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.

Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research

We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chl a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves.

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.

Unicellular green algaChlamydomonas reinhardtii as an activation system for 2-aminofluorene

Despite the promutagenic/procarcinogenic potential, polycyclic aromatic amines are widely spread in the environment. Biotransformation of the polycyclic aromatic amine 2-aminofluorene (2-AF) was proved in mammals and higher plants. The algal cell/microbe coincubation assay is an additional system that complemented those proved in mammals and higher plants, useful for detection and conversion of environmental promutagens, mainly in aquatic environments. The unicellular green algae may be a good activating system in coincubation assays in that the algal cells exist as a natural system. To increase the effectiveness of this metabolizing system, different modifications of the standard experimental procedure were conducted. Algae can accumulate and metabolize promutagenic pollutants, some of which may differ from those activated by the animal microsome metabolizing system (S9 mix) and by the plant cell/microbe coincubation assay. 2-AF was activated in the algal cell/ microbe coincubation assay in which wild-type Chlamydomonas reinhardtii cells were used as an activating system and the bacteria Salmonella typhimurium TA98, YG1024, and yeast Saccharomyces cerevisiae D7 as the genetic indicator organisms. It was converted to the mutagenic product(s) for the strain YG1024, but the strain TA98 did not exhibit any increase in the mutant yield of His+ revertants. Consequently, metabolites from 2-AF are substrates for O-acetyltransferase. A direct comparison of algal 2-AF activation with mammalian activation system (S9 mix) proved the higher activity of mammalian microsome system (S9 mix). After the combination of both activation systems, a slight synergetic effect was found. Although the genetic endpoints induced by 2-AF using both modifications of the algal cell/S. cerevisiae coincubation assay and those obtained in intact yeast cells were similar at the equitoxic concentrations, 2-AF activation by the algal supernatant slightly increased the genetic endpoints studied.

Chapter 77 Strategies for Isolation of Flagellar Motility and Assembly Mutants in Chlamydomonas

Publisher Summary Chlarnydornonas is uniquely suited for genetic studies because its flagellated vegetative cells are haploid and its mutants can be genetically analyzed by sexual crosses between different strains. More than 40 mutants are known that are deficient in internal structures of the flagella such as inner and outer dynein arms, central pair microtubules, or radial spoke. This chapter demonstrates a simple method for the isolation of various motility mutants. It is based on enrichment for cells that grow on the bottom of test tube cultures after mutagenesis with UV light. The enrichment methods can similarly be used for the selection of paralyzed or slow-swimming mutants generated by insertional mutagenesis. Using TAP as the medium the mutagenesis initiated the process to culture wild-type Chlamydomonas reinhardtii (plus or minus mating type) to a density of 1–2 x 10 6 /ml in liquid TAP medium under continuous illumination. While enriching of Motility-Deficient Cells, during the second light phase, it is reqired to carefully transfer cells on the bottom of each test tube to another test tube containing 4 ml of fresh medium. The volume of transferred suspension is 50–200 μ I. It is necessary to use a new, sterilized Pasteur pipette for each transfer. When single colonies are apparent, use sterile toothpicks to transfer the colonies to 96-well culture plates, each well containing 200 μ I of liquid medium. When mutants of interest are obtained, mate them with wild type to obtain daughter cells of opposite mating types. This step is necessary because the first-generation mutants often carry a second, undesirable mutation.

Photosynthesis modulates the sign of phototaxis of wild-type Chlamydomonas reinhardtii: Effects of red background illumination and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea

We have found that the sign of phototaxis, i.e. the direction of phototactic migration either towards or away from the light source, of wild-type Chlamydomonas reinhardtii depends on its photosynthetic activity. This accounts for the frequently reported transient positive phototaxis that precedes a negative phototaxis of wild-type cells, as well as the earlier observations that preillumination affects the sign of phototaxis. The bases for our conclusion are as follows. (1) The transient nature of phototaxis was preferentially observable with blue-green actinic light rather than with green actinic light. (2) Red background light induces negative phototaxis under the actinic-light conditions in which, without background light, Chlamydomonas cells show exclusively positive phototaxis. (3) Both the effect of red background light and the transient change in the sign of phototaxis were inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, a specific inhibitor of photosynthesis. The conclusion modifies the accepted view that photosynthesis does not link with the phototaxis of this microorganism, thus constituting a necessary part in elucidating mechanisms of algal phototaxis.