ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

Phytoene desaturase (PDS) is a rate‐limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin‐producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ‐carotene. The PDS gene in Chlorella was shown to be up‐regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS‐L516R was still active and exhibited ～36‐fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.     

Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.     

Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

Development of a Virus-Induced Gene-Silencing System for Functional Analysis of the RPS2-Dependent Resistance Signalling Pathways in Arabidopsis

Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at An erratum to this article is available at .     

Flagellin (FliC) protein sequence diversity among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> does not correlate with H serotype diversity

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure and function characterization of the phytoene desaturase related to the lutein biosynthesis in Chlorella protothecoides CS-41

Phytoene desaturase is the key enzyme involved in the biosynthesis pathway of lutein. The unicellular microalga, Chlorella protothecoides CS-41, had been selected for the heterotrophic production of high concentrations of lutein. In this study, a cDNA copy of the pds gene from C. protothecoides was obtained using the rapid amplification of cDNA ends (RACE) technique. Phylogenetic analysis of the deduced amino acid sequence revealed that the phytoene desaturases derived from the algal family. Expression of the pds gene in Escherichia coli produced a single protein of 61 kDa. The PDS activity of the expressed protein was confirmed by the production of ζ-carotene as the result from the action of the enzyme’s desaturation activity, which was identified by high-performance liquid chromatography and heterologous complementation analysis. Using random and site-directed mutagenesis, a single amino acid mutation (N144D) was identified and confirmed. This mutant encodes an inactive enzyme, which implies that amino acid 144 is crutial to the activity of the PDS enzyme. Therefore, by gene cloning and expression in prokaryotic cells, the gene for ζ-carotene production or as part of the biosynthetic pathway of lutein had been characterized from Chlorella protothecoides CS-41.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

A novel carotenoid biosynthesis gene coding for ζ-carotene desaturase: functional expression,sequence and phylogenetic origin

A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting -carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56 740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of -carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between -carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the -carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.     

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

栀子八氢番茄红素合成酶基因的分离及表达分析

为探讨栀子(Gardenia jasminoides)果实中藏花素的合成机理,克隆了栀子类胡萝卜素生物合成的关键酶八氢番茄红素合成酶(GjPSY)基因的全长cDNA。结果表明,推导的GjPSY氨基酸序列与双子叶植物来源的GjPSY亲缘关系较近。采用HPLC检测栀子果实中的藏花素-1含量为(3.96±1.48) mg g-1,在叶片中未检出。通过RT-PCR分析表明,GjPSY在栀子叶片和果实中均有表达,且表达水平一致。因此推测,GjPSY的转录水平与果实中藏花素-1的合成无关。     

Nuclear—organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is ‘plastid autonomous’. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C₄₀ carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear—organelle interactions.     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Cloning and knockout of phytoene desaturase gene in <Emphasis Type="Italic">Sphingomonas elodea</Emphasis> ATCC 31461 for economic recovery of gellan gum

A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.