A Galactoglycerolipid Lipase Is Required for Triacylglycerol Accumulation and Survival Following Nitrogen Deprivation in Chlamydomonas reinhardtii

Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo–synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.     

Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe₂S₂] ferredoxins, products of PETF, FDX2–FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP⁺ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E_m = −321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (∼11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (−350 to −450 mV) that function as electron donors at reductive steps in various metabolic pathways (1–3). In photosynthetic organisms, the well studied ferredoxin (Fd⁴; the product of the PETF gene) is the most abundant iron-containing protein in the chloroplast and is central to the distribution of photosynthetically derived reductive power (4).The most well known Fd-dependent reaction is the transfer of electrons from photosystem I (PSI) to NADPH, catalyzed by Fd:NADP⁺ oxidoreductase (FNR). The NADPH produced by this reaction donates electrons to the only reductant-requiring step in the Calvin cycle and other steps in anabolic pathways that require NADPH as reductant. In addition, reduced Fd directly donates electrons to other metabolic pathways by interacting with various enzymes in the chloroplast. This includes Fd:thioredoxin reductase (FTR), which converts a light-driven electron signal into a thiol signal that is transmitted to thioredoxins (TRXs) present in the plastid as different types (or different isoforms). Once reduced, TRXs interact with specific disulfide bonds on target enzymes, modulating their activities (5). Other Fd targets include hydrogenase, which is responsible for hydrogen production in anaerobic conditions in green algae; glutamine-oxoglutarate amidotransferase in amino acid synthesis; nitrite and sulfite reductases in nitrate and sulfate assimilation, respectively; stearoyl-ACP Δ9-desaturase in fatty acid desaturation; and phycocyanobilin:Fd oxidoreductase in synthesis of phytochromobilin (6). Fd also functions in non-photosynthetic cells. Here, FNR catalyzes the reduction of Fd by NADPH produced in the oxidative pentose phosphate pathway, enabling Fd-dependent metabolism to occur in the dark (7, 8).The single-celled green alga, Chlamydomonas reinhardtii is an excellent reference organism for studying both metabolic adaptation to nutrient stress and photosynthesis (9–13). The Chlamydomonas genome encodes six highly related plant type ferredoxin genes (9). Until recently, only the major photosynthetic ferredoxin, Fd (encoded by PETF), which mediates electron transfer between PSI and FNR, had been characterized in detail (14).Many land plants are known to have multiple ferredoxins. Typically, they are differently localized on the basis of their function. Photosynthetic ferredoxins reduce NADP⁺ at a faster rate and are localized to the leaves, whereas non-photosynthetic ferredoxins are more efficiently reduced by NADPH and are localized to the roots. Arabidopsis thaliana has a total of six ferredoxin isoforms (15). Of these, two are photosynthetic and localized in the leaves. The most abundant, AtFd2, is involved in linear electron flow, and the less abundant (5% of the ferredoxin pool), AtFd1, has been implicated in cyclic electron flow (16). There is one non-photosynthetic ferredoxin located in the roots, AtFd3, which is nitrate-inducible. This protein has higher electron transfer activity with sulfite reductase in in vitro assays compared with other Arabidopsis ferredoxin isoforms, suggesting in vivo function of AtFd3 in nitrate and sulfate assimilation (15, 17). In addition, there is one evolutionarily distant ferredoxin, AtFd4, of unknown function with a more positive redox potential present in the leaves and two other proteins which are “ferredoxin-like” and uncharacterized (15). Zea mays has four ferredoxin isoforms, two photosynthetic and two non-photosynthetic (18). One of the non-photosynthetic isoforms is specifically induced by nitrite, suggestive of a role in nitrate metabolism (19). A cyanobacterium, Anabaena 7120, has two ferredoxins, vegetative and heterocyst type (by analogy to leaf and root types, respectively). The heterocyst type is present only in cells that have differentiated into nitrogen-fixing cells, indicating that this form may serve to transfer electrons to nitrogenase (20).We hypothesize that the presence of as many as six ferredoxin isoforms in a single-celled organism like C. reinhardtii allows for the differential regulation of each isoform and therefore the prioritization of reducing power toward certain metabolic pathways under changing environmental conditions. To test this hypothesis, expression of the genes (PETF and FDX2–FDX6) encoding the six ferredoxin isoforms in Chlamydomonas reinhardtii was monitored under various conditions in which well characterized ferredoxin-dependent enzymes are known to be expressed. In addition, we also analyzed the interaction of Fd and Fdx2 with several ferredoxin-interacting proteins, such as NiR, FNR, and FTR, and determined the kinetic parameters of the corresponding reactions.We found that each of the FDX genes is indeed differently regulated in response to changes in nutrient supply. In the case of FDX2 whose product is most similar to classical Fd, we suggest that it has specificity for nitrite reductase based on its pattern of expression and activity with nitrite reductase.     

Katanin Localization Requires Triplet Microtubules in Chlamydomonas reinhardtii

Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19) and p80 (pf15) subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin) alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.     

Efficient Phagocytosis Requires Triacylglycerol Hydrolysis by Adipose Triglyceride Lipase

Macrophage phagocytosis is an essential biological process in host defense and requires large amounts of energy. To date, glucose is believed to represent the prime substrate for ATP production in macrophages. To investigate the relative contribution of free fatty acids (FFAs) in this process, we determined the phagocytosis rates in normal mouse macrophages and macrophages of adipose triglyceride lipase (ATGL)-deficient mice. ATGL was shown to be the rate-limiting enzyme for the hydrolysis of lipid droplet-associated triacylglycerol (TG) in many tissues. Here, we demonstrate that Atgl^−/− macrophages fail to efficiently hydrolyze cellular TG stores leading to decreased cellular FFA concentrations and concomitant accumulation of lipid droplets, even in the absence of exogenous lipid loading. The reduced availability of FFAs results in decreased cellular ATP concentrations and impaired phagocytosis suggesting that fatty acids must first go through a cycle of esterification and re-hydrolysis before they are available as energy substrate. Exogenously added glucose cannot fully compensate for the phagocytotic defect in Atgl^−/− macrophages. Hence, phagocytosis was also decreased in vivo when Atgl^−/− mice were challenged with bacterial particles. These findings imply that phagocytosis in macrophages depends on the availability of FFAs and that ATGL is required for their hydrolytic release from cellular TG stores. This novel mechanism links ATGL-mediated lipolysis to macrophage function in host defense and opens the way to explore possible roles of ATGL in immune response, inflammation, and atherosclerosis.     

Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.     

缺氮介导的莱茵衣藻油脂合成过程的内参及标志物蛋白质的筛选鉴定

微藻被认为是最有潜力的生物能源原料之一,了解油脂合成机理、提升油脂合成的效率是重要的生物学问题.莱茵衣藻缺氮胁迫是油脂合成机理研究的模式系统,组学研究已经积累了大量的数据,但针对莱茵衣藻缺氮介导的油脂合成过程的内参及标志物蛋白质还鲜有报道.本研究对莱茵衣藻进行了对照和缺氮胁迫培养,比较了多个时间点(0、1、2、4和6d)在2种处理条件下的培养物表型、细胞密度、油脂含量以及总蛋白质含量的变化等特征.结果显示:莱茵衣藻细胞在受到缺氮胁迫后表现为培养物颜色由绿变黄;A750和细胞计数结果显示细胞生长趋于停滞;尼罗红染色定量实验鉴定到油脂含量的显著升高;考马斯亮蓝染色实验检测到总蛋白质含量降低.以20个莱茵衣藻蛋白质为候选,利用蛋白质印迹技术(Western blot,WB)检测了其在不同处理和不同时间点的表达特征变化,通过计算总蛋白质和候选蛋白质含量的皮尔森相关系数(Pearson’s correlation coefficient, PCC)筛选了莱茵衣藻缺氮胁迫的内参蛋白质,发现Histone H3、 RBCL(ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit)和BCR1(biotin carboxylase,ACCase complex 1)在对照和缺氮胁迫条件下均与总蛋白质含量变化呈现极显著或显著正相关,所以被选作内参蛋白质.进而通过比较候选蛋白质的平均相对倍率变化(average relative fold change, ARF),鉴定了莱茵衣藻缺氮胁迫的标志物蛋白质,发现ATPs-β(ATP synthase CF1beta subunit)、 GAP2(glyceraldehyde 3-phosphate dehydratase 2)和RMT1(rubisco large subunit N-methyltransferase 1)蛋白质的ARF值分别为180.59、52.90和12.48,明显高出其他蛋白质,由此把它们选做缺氮胁迫的标志物蛋白质.接下来,对缺氮胁迫早期(0、2、4、8、12、18、24和48 h)的样品进行蛋白质印迹法分析,发现可检测的ATPs-β、GAP2和RMT1的缺氮诱导条带出现的时间分别是8、18和12 h.综上可以认为,在所有候选蛋白质中,ATPs-β是出现最早且变化幅度最大的缺氮处理标志物蛋白质.本研究鉴定的内参和标志物蛋白质对了解缺氮应答及油脂合成机理会有所帮助,所积累的蛋白质表达信息可供研究同行参考.     

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu²⁺ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.     

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.     

脂肪酶的底物特异性及其应用潜力

不同来源的Lipase对底物油脂中脂肪酸的链长、不饱和度及不饱和脂肪酸的双键位置表现出不同的脂肪酸特异性;对甘油酯中Sn-1(3)和Sn-2位酯键具有不同的位置特异性;对甘油酯中立体对映结构的1位和3位酯键呈现不同的立体特异性,脂肪酶能够催化酯水解和酯合成(或酯交换)反应,用于制备甘油单酯、多不饱和脂肪酸及其酯和具有光学活性的有机化合物,因此它在油酯加工和有机合成中具有很大的应用潜力.     

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein using S-adenosyl-l-methionine (SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved among crenarchaea, and distantly related homologs also exist in Bacteria and Eukarya. aKMT was active over a wide range of temperatures, from ∼25 to 90°C, with an optimal temperature at ∼60 to 70°C. Amino acid residues Y9 and T12 at the N terminus appear to be the key residues in the putative active site of aKMT, as indicated by sequence conservation and site-directed mutagenesis. Although aKMT was identified based on its methylating activity on Cren7, the crenarchaeal chromatin protein, it exhibited broad substrate specificity and was capable of methylating a number of recombinant Sulfolobus proteins overproduced in Escherichia coli. The finding of aKMT will help elucidate mechanisms underlining extensive protein lysine methylation and the functional significance of posttranslational protein methylation in crenarchaea.     

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii

The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA‐ and siRNA‐based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one‐step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G‐Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon‐counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK3 (calcium‐dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.     

On the Substrate Specificity of the Lipase Produced by Candida paralipolytica

The lipase from Candida paralipolytica required activating factors for the hydrolysis of synthetic triglycerides, methyl esters of fatty acid and so on. Of the saturated monoacid triglycerides tested, tricaprylin was hydrolyzed most quickly. On the other hand, the lipase was hardly able to hydrolyze methyl butyrate, methyl caproate, monoolein, Tween 20 and Span 20.

Human blood plasma did not act as an activator, but act rather as an inhibitor of the lipase. Therefore, it seems that the lipase does not belong to the group of lipoprotein lipase.     

Triacylglycerol Lipase in Developing Rat Sciatic Nerve

Triacylglycerol lipase activity, with a pH optimum of 7.5, was demonstrated in cell-free homogenates of rat sciatic endoneurium. 1,2-Diacylglycerol was the major product of triacylglycerol hydrolysis. A rapid decline in lipase activity was found in rats up to 2 months of age. After this time, the decrease continued, but at a much slower rate. Such developmental changes in triacylglycerol lipase activity may, at least in part, account for the slower metabolic turnover of endoneurial triacylglycerol in adult rat sciatic nerve.     

The DrrAB Efflux System of Streptomyces peucetius Is a Multidrug Transporter of Broad Substrate Specificity

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.     

Gravitaxis in Chlamydomonas reinhardtii studied with novel mutants

Many free-swimming unicellular organisms show negative gravitaxis, i.e. tend to swim upward, although their specific densities are higher than the medium density. To obtain clues to the mechanism of this behavior, we examined how a mutation in motility or behavior affects the gravitaxis in Chlamydomonas. A phototaxis mutant, ptx3, deficient in membrane excitability showed weakened gravitaxis, whereas another phototaxis mutant, ptx1, deficient in regulation of flagellar dominance displayed normal gravitaxis. Two mutants that swim backwards only, mbo1 and mbo2, did not show any clear gravitaxis. We also isolated two novel mutants deficient in gravitaxis, gtx1 and gtx2. These mutants displayed normal motility and physical characteristics of cell body as assessed by the behavior of anesthetized cells. However, these cells were found to have defects in physiological responses involving membrane excitation. These observations are consistent with the idea that the gravitaxis in Chlamydomonas involves a physiological signal transduction system, which is at least partially independent of the system used for phototaxis.     

Portrait of a species: Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, the first alga subject to a genome project, has been the object of numerous morphological, physiological, and genetic studies. The organism has two genetically determined mating types (plus and minus) and all stages of the simple life cycle can be evoked in culture. In the nearly 60 years since the first standard laboratory strains were isolated, numerous crosses and exchanges among laboratories have led to some confusion concerning strain genealogy. Here we use analyses of the nuclear internal transcribed spacer regions and other genetic traits to resolve these issues, correctly identify strains currently available, and analyze phylogenetic relationships with all other available similar chlamydomonad types. The presence of a 10-bp indel in ITS2 in some but not all copies of the nuclear ribosomal cistrons of an individual organism, and the changing ratios of these in crosses, provide a tool to investigate mechanisms of concerted evolution. The standard C. reinhardtii strains, plus C. smithii +, plus the new eastern North American C. reinhardtii isolates, comprise one morphological species, one biological species of high sexual intercompatibility, and essentially identical ITS sequences (except the tip of helix I of ITS2). However, variant RFLP patterns characterize strains from each geographic site.     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Rapid biotransformation of arsenate into oxo-arsenosugars by a freshwater unicellular green alga, Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.