Deep genomic analysis of the Chlorella sorokiniana SAG 211-8k chloroplast

The chloroplast genome contains information that is applicable in many scientific fields, such as plant systematics, phylogenetic reconstruction and biotechnology, because its features are highly conserved among species. To date, several complete green algal chloroplast genomes have been sequenced and assembled. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Chlorella sorokiniana SAG 211-8k is reported and compared for the first time to the chloroplast genomes of 10 Chlorellaceae. The recently updated Chlorella sorokiniana cpDNA sequence, assembled as a circular map of 109?811 bp, encodes 113 genes. Similar to other Chlorella strains, this chloroplast genome does not show a quadripartite structure and lacks the large rRNA operon-encoding Inverted Repeat (IR). The Chlorella sorokiniana plastid encodes the tRNA(Ile)-lysidine synthetase (tilS), which is responsible for modifying the CAU anticodon of a unique tRNA. Gene ordering and clustering highlight the close relationships among Chlorella clade members and the preservation of crucial gene clusters in photosynthetic strains. The features of Chlorella sorokiniana presented here reinforce the monophyletic character of Chlorellaceae and provide important information that sheds light on chloroplast genome evolution among species of Chlorella.     

Cloning,functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k)

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.     

Glucose-6P dehydrogenase in Chlorella sorokiniana (211/8k): an enzyme with unusual characteristics

In Chlorella sorokiniana (211/8k), glucose-6 phosphate dehydrogenase (G6PDH—EC 1.1.1.49) activity is similar in both N-starved cells and nitrate-grown algae when expressed on a PCV basis. A single G6PDH isoform was purified from Chlorella cells grown under different nutrient conditions; the presence of a single G6PDH was confirmed by native gels stained for enzyme activity and by Western blots. The algal G6PDH is recognised only by antibodies raised against higher plants plastidic protein, but not by chloroplastic and cytosolic isoform-specific antisera. Purified G6PDH showed kinetic parameters similar to plastidic isoforms of higher plants, suggesting a different biochemical structure which would confer peculiar regulative properties to the algal G6PDH with respect to higher plants enzymes. The most remarkable property of algal G6PDH is represented by the response to NADPH inhibition. The algal enzyme is less sensitive to NADPH effects compared to higher plants G6PDH: Ki_NADPH is 103 μM for G6PDH from nitrogen-starved C. sorokiniana, similarly to root plastidic P2-G6PDH. In nitrate-grown C. sorokiniana the Ki_NADPH decreased to 48 μM, whereas other kinetic parameters remained unchanged. These results will allow further investigations in order to rule out possible modifications of the enzyme, and/or the expression of a different G6PDH isoform during nitrate assimilation.     

Isolation and partial characterization of a lead-accumulating Brassica juncea mutant

A new screening method for non-destructive, high-sensitivity, high-throughput isolation of plant mutants capable of accumulating large amounts of heavy metals has been developed. This method is based on incubating seedlings in a solution containing radioisotopes of the metals of interest and visualizing the tissue accumulation of these metals with a phosphorimager. We used this technique to isolate mutants of Brassica juncea (L.) Czern with increased accumulation of Cd and Pb for use in phytoremediation, an emerging technology using plants to remediate polluted soil and water. Approximately 50,000 M2 seedlings were screened and 21 mutants were recovered that retained increased accumulation through the third generation. Mutant 7/15–1 is characterized by enhanced Pb accumulation per unit of root fresh weight, stunted root growth, and decreased root cell size. Data indicate that roots of 7/15–1 contain more cell-wall material on a fresh-weight basis than roots of the wild-type, which may at least partially explain its ability to accumulate more Pb. Received: 22 September 1998 / Accepted:19 December 1998     

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Ferredoxin-NADP⁺ reductase (FNR, EC I.18.1.2) from the green algae Chlorella fusca Shihira et Kraus 211–15, was purified to homogeneity. The molecular mass was 36.8 kDa as determined by SDS-polyacrylamide gel electrophoresis. The enzyme exhibits the typical spectrum of a flavoprotein with an absorption maximum at 459 nm and an A_273/459 ratio of 7.2. It contains one mol of FAD per mol of protein and the calculated extinction coefficient is 9.8 m M cm⁻¹. Four different forms of the purified enzyme were detected by isoelectric focusing (pI between 5.4 and 5.9), even when protease inhibitors were used during the first steps of the purification. Kinetic parameters were determined for several FNR-catalyzed reactions. NADP⁺ photoreduction gave comparable rates when either ferredoxin or flavodoxin was used.     

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants

A novel plant lectin was isolated from salt-stressed rice (Oryzasativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants. Received: 27 July 1999 / Accepted: 11 November 1999     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Na+:HCO(3-) cotransporters (NBC): cloning and characterization

The sodium bicarbonate cotransporter (NBC1) is essential for bicarbonate transport across plasma membranes in epithelial and nonepithelial cells. The direction of the NaHCO₃ movement in secretory epithelia is opposite to that in reabsorptive epithelia. In secretory epithelia (such as pancreatic duct cells) NBC is responsible for the transport of bicarbonate from blood to the cell for eventual secretion at the apical membrane. In reabsorptive epithelia (such as kidney proximal tubule cells) NBC is responsible for the reabsorption of bicarbonate from cell to the blood. In nonepithelial cells this transporter is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). All but AE4 are presumed to mediate the cotransport of Na⁺ and HCO₃ ⁻ under normal conditions and may be functionally altered in certain pathologic states. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. In addition to the kidney, NBC1 is expressed in pancreatic duct cells, is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO₃ ⁻ secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The characterization of NBC4 is incomplete. The NBC-related protein called NCBE mediates Na-dependent, Cl⁻/Bicarbonate Exchange. The purpose of this review is to summarize recent advances on the cloning of NBC isoforms and related proteins and their role and regulation in physiologic and pathologic states. Received: 26 February 2001/Revised: 14 May 2001     

Purification, characterization and cDNA cloning of soluble carbonic anhydrase from Chlorella sorokiniana grown under ordinary air

Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO₂ was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)⁻¹. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A. These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About 60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized. The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH₂-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an α-type CA with salt-sensitive, periplasm-locating and acidic properties and very different from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively. Received: 25 May 1998 / Accepted: 9 July 1998     

Ferredoxin isoforms from Chlorella vulgaris (Chlorococcales, Chlorophyceae): Molecular characterization and participation in ferredoxin-dependent enzymes

Using hydrophobic chromatography, Chlorella ferredoxin was separated into three components (Fd I, Fd II and Fd III) in ratios of approximately 3:13:1. The three components differed in isoelectric point, peptide mapping, amino acid composition and N-terminal sequence. Fd II and Fd III were found to support fairly high rates of cytochrome c reduction by spinach FNR, while Fd I could not support this reaction at all. The highest value of the specificity constant (k_cat/K_m for NiR was demonstrated for Fd II-dependent activity; however, the lowest value of k_cat/K_m for NiR was obtained using Fd II.     

布鲁氏菌M5-90疫苗株virB2基因缺失株的构建及鉴定

【目的】构建布鲁氏菌M5-90疫苗株virB2基因缺失株。【方法】利用常规分子生物学技术构建自杀载体pGEM-7zf-ΔvirB2-sacB,通过同源重组的方法,将电转化后的布鲁氏菌分别经100 mg/L氨苄抗性筛选和5%蔗糖敏感性筛选,获得基因缺失株。对获得的基因缺失株进行PCR鉴定和稳定性检测。【结果】成功构建M5-90ΔvirB2基因缺失株,并且该缺失株在10代以内未发生回复突变。【结论】为研发新型布鲁氏菌弱毒基因缺失活苗奠定基础。     

Molecular cloning and characterization of a cDNA encoding nitrate reductase from Chlorella ellipsoidea (Chlorophyta)

Nitrate reductase (NR) genes have beencloned from higher plants, fungi and algae.Based on seven of the amino acid residuesmost strongly conserved between Chlorella vulgaries and Chlamydomonasreinhardtii NR gene, a degenerate primerwas designed. This degenerate primer wasused to amplify the corresponding homologyin Chlorella ellipsoidea. A 3304 bpfull-length cDNA was cloned by rapidamplification of cDNA ends (RACE). Thededuced amino acid sequence of this cDNAhas a high degree of similarity withpreviously identified members of the NRgene. This suggests that the amplified cDNAencodes a functional NR. Northern blotexpression analysis suggests that this geneis strongly induced by nitrate, but isrepressed by ammonium. The nucleotidesequence data reported in this paper willappear in the DDBJ/EMBL/GeneBank databasesunder accession number AY275834.     

Effect of inhibitors on ammonium assimilation in Chlorella sorokiniana in light and darkness

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO₂, and was severely inhibited by 100 μ M atrazine or 10 μ M 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU). In N-limited cells, assimilation of NH₄⁺ took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by l -methionine- dl -sulfoximine (MSX), but not by atrazine or DCMU.
The rate of photosynthetic O₂ evolution was 2.9±0.9 mmol ml⁻¹ packed cell volume (PCV) h⁻¹ in N-sufficient cells, and 0.64±0.12 mmol ml⁻¹ PCV h⁻¹ in N-limited cells. In the latter resupply of ammonium resulted in a rapid activation by 22%;, followed by a time-dependent increase of the photosynthetic O₂ evolution, which after 12 h reached the same rate as in N-sufficient cells.
Respiratory consumption of oxygen in darkness in N-sufficient and N-limited cells was 0.10±0.03 and 0.11±0.02 mmol ml⁻¹ PCV h⁻¹, respectively. Addition of ammonium was without effect on respiration of N-sufficient cells, but resulted in a 4-fold stimulation of respiration of N-limited cells. Such stimulation took place also in cells treated with DCMU, atrazine, or MSX, and it was also promoted by methylammonium. The stimulation of respiration lasted for several hours.     

Mapping of two genes for resistance to clubroot (Plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by means of RFLP and AFLP markers

A genetic map covering 615 cM in 12 linkage groups was assembled based on 92 RFLP and AFLP markers segregating in a population of 107 doubled haploid lines (DH lines) of Brassica oleracea. The DH-line population was obtained through microspore culture from the of two homozygous parents: DH-line Bi derived from the cabbage landrace Bindsachsener, and DH-line Gr from broccoli cv ‘Greenia’. Sixty-five percent of the loci, and in some cases complete linkage groups, displayed distorted segregation ratios, a frequency much higher than that observed in populations of the same species. DH-line Bi was resistant to clubroot, which is caused by a Dutch field isolate of Plasmodiophora brassicae. Resistance in the DH-line population was determined in two ways: by assigning symptom grades to each plant, and by measuring the fresh weights of the healthy and affected parts of the root system of each plant. Using a multiple QTL mapping approach to analyze the fresh weight data, we found two loci for clubroot resistance; these were designated pb-3 and pb-4. The additive effects of these loci were responsible for 68% of the difference between the parents and for 60% of the genetic variance among DH-line means. Also, indications for the presence of two additional, minor QTLs were found. Analysis of symptom grades revealed the two QTLs pb-3 and pb-4, as well as one of the two minor QTLs indicated by analysis of the fresh weight data. Received: 29 April 1996 / Accepted: 10 May 1996     

Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis

Upon herbivore attack, maize (Zea mays L.) emits a mixture of volatile compounds that attracts herbivore enemies to the plant. One of the major components of this mixture is an unusual acyclic C₁₁ homoterpene, (3E )-4,8-dimethyl-1,3,7-nonatriene (DMNT), which is also emitted by many other species following herbivore damage. Biosynthesis of DMNT has been previously shown to proceed via the sesquiterpene alcohol, (E )-nerolidol. Here we demonstrate an enzyme activity that converts farnesyl diphosphate, the universal precursor of sesquiterpenes, to (3S)-(E )-nerolidol in cell-free extracts of maize leaves that had been fed upon by Spodoptera littoralis. The properties of this (E )-nerolidol synthase resemble those of other terpene synthases. Evidence for its participation in DMNT biosynthesis includes the direct incorporation of deuterium-labeled (E )-nerolidol into DMNT and the close correlation between increases in (E )-nerolidol synthase activity and DMNT emission after herbivore damage. Since farnesyl diphosphate has many other metabolic fates, (E )-nerolidol synthase may represent the first committed step of DMNT biosynthesis in maize. However, the formation of this unusual acyclic terpenoid appears to be regulated at both the level of (E )-nerolidol synthase and at later steps in the pathway. Received: 20 August 1999 / Accepted: 27 October 1999     

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants

Summary. Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.