LOCALIZATION OF OXYTETRACYCLINE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Oxytetracycline (OTC) is an important antimicrobial used in aquaculture. However, residues of OTC have been isolated from nontarget aquatic organisms, sediments, and water located near aquaculture facilities. Identifying OTC in plant material is particularly difficult due to interference from pigments and polyphenol substances but is important especially for algae since they are a primary food source for fish in early life stages. In this study, we describe the effect of OTC (0.1, 1, 10, 25, 50, 100 μg · mL^?1) on cell growth, and the localization of OTC (0, 1, 25, 100 μg · mL^?1) in vacuoles of Chlamydomonas reinhardtii P. A. Dang. (wildtype, ATCC 18798). We also present a method for semiquantifying OTC in living cells using fluorescent microscopy and Adobe Photoshop. We exposed algal cells to OTC and sampled after 2 or 7 d exposure. On day 7, OTC significantly inhibited algal growth at 1, 10, 25, 50, and 100 μg · mL^?1. When viewed with fluorescent microscopy, cells exposed to the 25 and 100 μg · mL^?1 contained yellow fluorescent areas, ≤1 μm in diameter that were easily discernable against the red fluorescence of the intracellular chl. The fluorescent areas corresponded to small spherical vacuoles (i.e., polyphosphate bodies that contain calcium and magnesium complexed with polyphosphate) seen in the cells by LM. Since OTC has a high affinity for divalent cations, we suggest that OTC is localized in these vacuoles.     

ISOLATION AND PRELIMINARY CHARACTERIZATION OF THREE CHLAMYDOMONAS STRAINS INTERFERTILE WITH CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Three new strains of the unicellular green alga Chlamydomonas reinhardtii Dangeard were isolated from soil. The isolates differed from one another and from standard laboratory strains of C. reinhardtii in a number of traits, including heavy metal resistance, protein composition, and mitochondrial DNA length. The new isolates also exhibited distinctive restriction fragment length polymorphisms in their nuclear, chloroplast, and mitochondrial genomes. The new isolates were interfertile with the standard laboratory strains and appeared to transfer chloroplast and mitochondrial genomes in a similar manner, that is, predominantly from the material (mt⁺) and paternal (mt^?) parents, respectively.     

DEEP-ETCH ANALYSIS OF ADHESION COMPLEXES BETWEEN GAMETIC FLAGELLAR MEMBRANES OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The ultrastructure of adhesion complexes between gametic flagellar membranes of Chlamydomonas reinhardtii Dangeard was analyzed using the quick-freeze deep-etch technique. The sexual agglutinin fibrils interact by forming hybrid fibers that frequently branch, forming extensively cross-bridged meshworks. This pattern of interaction mimics a prominent mode of cell wall formation in Chlamydomonas, supporting the notion that the agglutinins evolved from cell wall proteins and that sexual adhesion and cell wall assembly are homologous events.     

COPPER SORPTION AND RELEASE BY CYCLOTELLA MENEGHINIANA (BACILLARIOPHYCEAE) AND CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Initial Cu⁺⁺ sorption by Cyclotella meneghiniana Kütz. (Cu⁺⁺-sensitive) and Chlamydomonas reinhardtii Dangeard (Cu⁺⁺-resistant) was rapid in the first 5 min of Cu⁺⁺ incubation with little sorption after 2 h. On a cell to cell basis, Cyclotella sorbed ca. five times more Cu⁺⁺ from the medium than Chlamydomonas. In MBL medium with EDTA Cyclotella and Chlamydomonas cells sorbed 21.0 and 4.41 nM Cu⁺⁺/10⁶ cells respectively in 6 h with 0.3 mg Cu⁺⁺/l in the medium. Proportionally similar quantities of Cu⁺⁺ were sorbed when the cells were Cu⁺⁺ incubated in MBL + citrate or filtered lake water. Cleaned cell walls of Cyclotella sorbed little Cu⁺⁺ (1.7 nM/10⁶ cells) as compared to living cells (17.5 nM Cu⁺⁺/10⁶ cells) in 3 h. Therefore, in living Cyclotella most of the Cu⁺⁺ taken up must be absorbed by the protoplasm or perhaps by the organic layer surrounding the silica wall. Cleaned cell walls of Chlamydomonas sorbed 3.5 nM Cu⁺⁺/10⁶ cells and living Chlamydomonas cells sorbed 2.6 nM Cu⁺⁺/10⁶ cells. This indicates that most of the Cu⁺⁺ sorbed by Chlamydomonas cells remained bound to the cell wall and probably did not readily enter into the protoplasm: When placed in Cu⁺⁺ free medium after Cu⁺⁺ incubation, Cyclotella and Chlamydomonas cells released 46 and 59% respectively of the Cu⁺⁺ sorbed.     

EVIDENCE FOR A LACK OF PHOTOSYSTEM SEGREGATION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The distribution of photosystems I and II (PSI and PSII) in cells of Chlamydomonas reinhardtii Dangeard was studied by immunogold electron microscopy using cultures grown autotrophically at moderate irradiance and harvested in the middle of the light period. Sections of Lowicryl-embedded cells were labeled with monospecific heterologous antisera raised against the reaction center proteins of PSI (CP1-e) or the core antenna proteins of PSII (CP40 and CP47). All three antisera labeled both the appressed and the nonappressed thylakoid membranes at essentially similar densities. Labeling with both PSI and PSII antisera was slightly more concentrated over the outer nonappressed membranes of the thylakoid bands (1.7- to 2.4-fold with anti-CP1- e and 1.5- to 1.8-fold with anti-CP47 and anti-CP40). However, since appressed membranes comprised 73% of the total thylakoid membranes, 50%–62% of the PSI and 58%–65% of the PSII labeling were localized on appressed membranes. We conclude that photosystem distribution in C. reinhardtii is similar to that reported for other algae and different from the lateral heterogeneity observed in higher plants.     

MATURATION OF ALGAL ZYGOTES: ALTERNATIVE EXPERIMENTAL APPROACHES FOR CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Young zygotes from crosses of Chlamydomonas reinhardtii Dang. mutant and wild-type strains were incubated, in the presence or absence of light and/or nitrogen to determine whether continuation of conditions inducing gamete formation permits zygospore formation without loss of viability. Different culture media, continuous illumination vs. dark incubation and various durations of the maturation period were tested, for effect on zygospore germination efficiency, zygospore “burst size” and zoospore viability. Following either the routine maturation procedure of dark incubation on standard minimal medium, or following a new procedure of incubation under continuous illumination on N-free medium, zygospore formation can be ensured and high germination efficiencies obtained within 3 days after mating. Tetrad analysis indicates meiosis occurs normally whether zygotes have been matured in the presence or absence of light or nitrogen. Preliminary data suggest an effect of increased maturation time on the transmission of cytoplasmic genes, if a N-free continuous illumination maturation protocol is followed. Two experimental approaches for the maturation of C. reinhardtii zygotes are suggested and advantages of each are discussed.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

MORPHOLOGICAL CHANGES IN MITOCHONDRIAL AND CHLOROPLAST NUCLEOIDS AND MITOCHONDRIA DURING THE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELL CYCLE1

Morphological changes in the organellar nucleoids and mitochondria of living Chlamydomonas reinhardtii Dang were examined during the cell cycle under conditions of 12:12 light:dark. The nucleoids were stained with SYBR‐Green I, and the mitochondria were stained with 3,3‐dihexyloxacarbocyanine iodide. An mocG33 mutant, which contains one large chloroplast nucleoid throughout the cell cycle, was used to distinguish between the mitochondrial and chloroplast nucleoids. Changes in the total levels of organellar DNA levels were assessed by real‐time PCR. Each of the G₁, S, M, and Smt,cp phases was estimated. At the start of the light period, the new daughter cells were in G₁ and contained about 30 mitochondrial and 10 chloroplast nucleoids, which were dispersed and had diameters of 0.1 and 0.2 μm, respectively. During the G₁ phase of the light period, and at the start of the S phase, both nucleoids formed short thread‐like or bead‐like structures, probably divided, and increased continuously in number, concomitantly with DNA synthesis. The nucleoids probably became smaller due to the decrease in DNA of each particle and were indistinguishable. The cells in the S and M phases contained extremely high numbers of scattered nucleoids. However, in the G₁ phase of the dark period, the nucleoids again formed short thread‐like or bead‐like structures, probably fused, and decreased in number. The mitochondria appeared as tangled sinuous structures that extended throughout the cytoplasm and resembled a single large mitochondrion. During the cell cycle, the numbers of mitochondrial nucleoids and sinuous structures varied relative to one another.     

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min^-1.mg^-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg²⁺. The monovalent cation K⁺ acts as a strong activator. The K_m values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with V_max for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).     

UNIPARENTAL INHERITANCE IN CHLAMYDOMONAS EUGAMETOS (CHLOROPHYCEAE)1,2

The gene sr-2 conferring resistance to 500 μ/ml streplomycin exhibits uniparental inheritance in Chlamydomonas eugametos Moewus. The mutation to neamine dependence (nd) is probably of the same type. All meiotic progeny from crosses involving these mutant genes have the phenotype of the historically designated male parent. Unlike C. reinhardtii Dang., no exceptional zygotes have been observed.     

QUALITATIVE ASSAY OF DISSOLVED AMINO ACIDS AND SUGARS EXCRETED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) AND EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Dissolved amino acids and sugars produced by Chlamydomonas reinhardtii Dangeard and Euglena gracilis Klebs were assayed using a combination of radiochromatography and membrane separated spinner flasks. Both species produced similar complements of sugars. The sugars produced In the algae included galactose, glucose, maltose and xylose. The amino acid complements produced Were different for each species. C. reinhardtii excreted aspartate, leucine, methionine, phenlyalanine, tyrosine and Valine. E. gracilis excreted alanine, glutamate, proline and serine. Separation of cells from growth media via membrane filtration produced an overestimate of net amount of dissolved carbon compounds excreted by the cells. However, the radiochromatographic spectra for both filtrates and cell-free compartments of the diffusion flask experiments were identical. It is hypothesized that the process of filtration max enhance leakage of labeled cellular pools rather than cellular disruption in the species investigated.     

CHARACTERISTICS OF PHOSPHORUS LIMITATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE) AND ITS PALMELLOIDS1

Chlamydomonas reinhardtii Dang, was grown in a chemostat culture under phosphate limitation. The steady state concentration of phosphate was below the detection limit (< 1 μg P/L) in all runs. The cellular content of phosphorus (Q_p), polyphosphate (Q_pp) and chlorophyll a increased with increasing dilution rate, and the growth rate of the alga was described by Q_p as well as Q_pp in the Droop model. The ratio Q_pp/Q_p and the activity of alkaline phosphatase were maximal at high and low growth rates, respectively. Palmelloids of Chlamydomonas were found at high dilution rates (D > 0.12 h^?1) and became attached to the wall of the culture vessel. They differed from the vegetative stage in both chemical composition and growth rate. Their contents of phosphorus and chlorophyll a were low, as in the vegetative cells, which grew at a low growth rate, whereas the ration Q_pp/Q_p and the activity of alkaline phosphatase were comparable with those of fast growing vegetative cells. The growth rate of the palmelloids was 0.03 h^?1 whereas maximum growth rate (μ_m) for the vegetative cells was 0.21 h^?1.     

METAL‐INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation‐sensitive reporter dye, 5‐(and‐6)‐carboxy‐2′,7′‐dihydrodifluorofluorescein diacetate (H₂DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10^?6 to 10^?9 M, except in the case of Fe(III), which was effective at 10^?18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.     

LOSS OF HYBRID LETHALITY DURING BACKCROSS PROGRAMS INVOLVING CHLAMYDOMONAS EUGAMETOS AND CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE)1

Wild-type strains of the interfertile species Chlamydomonas eugametos (UTEX 9 and 10) and Chlamydomonas moewusii (UTEX 96 and 97) male readily and reciprocally; however, considerable lethality occurs among F₁ hybrid meiotic products. We prepared two hybrid backcross lineages using C. eugametos and C. moewusii. One lineage began with the cross C. eugametos mating-type-plus (mt⁺) × C. moewusii mating-type-minus (mt^?). An F₁ mt⁺ hybrid from this cross was back-crossed to C. moewusii mt^?, and a B₁ mt⁺ hybrid was recovered. The B₁ hybrid was again backcrossed to C. moewusii mt^?, and this process was repeated through the fifth backcross. The other backcross lineage began with the reciprocal cross C. moewusii mt⁺× C. eugametos mt^? and employed C. eugametos as the recurring mt^? parent. This lineage also was continued through the fifth backcross. Meiotic product survival in the reciprocal interspecific crosses was less than 10%. In successive back-cross generations associated with both lineages, this value increased progressively to a maximum of 85–90%, the level observed for the intraspecific crosses. These results are consistent with the hypothesis that multiple genetic differences exist between C. eugametos and C. moewusii and that these are the major source of meiotic product lethality associated with the interspecific crosses. The inheritance of chloroplast genetic markers for resistance to streptomycin (sr-2) and for resistance to erythromycin (er-nM1) was also scored w the interspecific crosses and in the backcrosses. Most hybrid zygospores transmitted the resistance markers of the mt⁺ parent only, or of both parents, with the former zygospore type being more common. Although the intraspecific C. eugametos and C. moewusii crosses differ conspicuously with respect to the fraction of zygospores which transmit chloroplast genetic markers of both parents, the inheritance of chloroplast genetic markers in the interspecific crosses and backcrosses at' scribed here failed to clarify the genetic basis for this difference.     

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

In our previous study, we generated a strain of 19‐P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780‐bp stem. We utilized this RNAi‐induced strain to uncover RNAi‐related genes. Random insertional mutagenesis was performed to generate tag‐mutants that show a RNAi deficient phenotype. The 92‐12C is one such tag‐mutant, which bears a 14‐kb deletion in chromosome 1. Complementation of 92‐12C revealed that a protein gene, including a Cys‐Cys‐Cys‐His‐type zinc finger motif and an ankyrin repeat motif, is essential for effective RNAi in Chlamydomonas reinhardtii (Dangeard). BLAST analysis revealed that the zinc finger protein is homologous to an mRNA splicing‐related protein of other species. Therefore, one of the probable scenarios is that mRNA coding for RNAi‐related proteins cannot be properly spliced, which causes RNAi deficiency in the 92‐12C tag‐mutant.     

IDENTIFICATION OF A MYOSIN-LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

A myosin-like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime-mold myosin recognized a 180,000 M_r protein in western blots of total protein extracts from three different strains, including cyt-1, a cytokinesis-defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy-chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy-chain gene and a myosin-like protein in vegetative cells of the model organism Chlamydomonas.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.