Carotenoids synthesized by UV-induced mutants of a non-acid-fast strain ofMycobacterium phlei

The composition of the cell pigment of three mutants (PN₁, PN₂, PN₃) of a non-acid-fast strain ofMycobacterium phlei (PN) induced with UV-radiation was investigated. It was found that the mutants contain carotenes typical for the original parent strain, but that the quantity of the colourless precursor, phytoene, is substantially lower, and, on the contrary, the amount of lycopene is increased (especially in mutants PN₂ and PN₃). In addition, all mutants synthesized xanthins in considerable quantities: the PN₁ mutant above all with the β-carotene carbon skeleton, whereas for the PN₂ and PN₃ mutants xanthins with the lycopene structure were typical. The hit of the genome of the original strain caused by the mutagen is thus manifested even in the synthesis of secondary metabolites.     

Dinitrogen fixation (Acetylene reduction) and nitrogen accumulation in peas cultivated under the standard growth conditions

InPisum sativum cultivated under standard growth conditions the extent of N₂ fixation with time estimated by the acetylene reduction assay (PN₂F) and rates of the actual nitrogen accumulation of plant biomass (ANA) were calculated from six independent growth experiments. In the plants inoculated with indigenous soilRhizobium populations and cultivated on 0.63 mmol/L nitrate level the percentage PN₂F:ANA ratios ranged from 25.7 to 61.5%. In peas inoculated with the inoculant strain the PN₂F:ANA ratios were markedly higher, ranging from 59.8 to 65.1%. The plants cultivated on N-free nutrient solutions showed both PN₂F:ANA and C₂H₄N₂ ratios to be somewhat higher compared with the 0.63 mmol/L nitrate cultivated plants.     

Synthesis and characterization of iron and ruthenium complexes bearing P-N ligands based on 8-hydroxyquinoline

The synthesis of bidentate aminophosphine ligands (PN^quin) based on 8-hydroxyquinoline is described. These ligands react with cis-Fe(CO)₄Br₂ to give selectively octahedral complexes of the type cis,cis-Fe(PN^quin)(CO)₂Br₂. There is only one isomer formed where the two CO and the two bromide ligands adopt a cis configuration. The reaction of [RuCp(CH₃CN)₃]PF₆ with PN^quin ligands affords the halfsandwich complexes [RuCp(PN^quin)(CH₃CN)]PF₆ in high isolated yields. Likewise, treatment of [Ru(η⁶-p-cymene)(μ-Cl)Cl]₂ with PN^quin in the presence of AgCF₃SO₃ affords halfsandwich complexes of the type [Ru(η⁶-p-cymene)(PN^quin)Cl]CF₃SO₃. All ligands and complexes are characterized by NMR and IR spectroscopy. The X-ray structure of representative compounds is reported. In addition, the relative stability of isomeric structures and conformers of Fe(PN^quin-Ph)(CO)₂Br₂ is studied by means of DFT calculations.     

Kinetics of proflavin binding to bacteriophages T2L and T4D

We have measured the kinetics of proflavin binding to T-even bacteriophages—the 700 S and 1000 S forms of T2L, T4D, and T4D os41—by difference spectroscopy at 430 nm. Measurements were carried out from 22° to 37°C. Binding is very slow to encapsulated DNA compared to free DNA, requiring hours to reach equilibrium. The kinetic data are compatible with the two-step mechanism where P is proflavin, N is nucleotide, and PN₁ and PN₂ are complexes. Computer integration of the rate equations allows evaluation of the rate constants; previous equilibrium measurements gave thermodynamic parameters. For all phage studied, the bimolecular step is endothermic with high positive entropy; the second, unimolecular step is highly exothermic with small negative entropy change. Both forms of T2L bind proflavin with essentially the same rate, as do T4D and the osmotic shock resistant mutant T4D os41. This suggests that the encapsulated DNA is equally accessible to proflavin in both forms of each phage. However, T4D binds dye appreciably faster than T2L, indicating that capsid permeability or DNA environment (glucosylation or packing) is different in the two species.     

Syntopic frogs reveal different patterns of interaction with the landscape: A comparative landscape genetic study of Pelophylax nigromaculatus and Fejervarya limnocharis from central China

Amphibians are often considered excellent environmental indicator species. Natural and man‐made landscape features are known to form effective genetic barriers to amphibian populations; however, amphibians with different characteristics may have different species–landscape interaction patterns. We conducted a comparative landscape genetic analysis of two closely related syntopic frog species from central China, Pelophylax nigromaculatus (PN) and Fejervarya limnocharis (FL). These two species differ in several key life history traits; PN has a larger body size and larger clutch size, and reaches sexual maturity later than FL. Microsatellite DNA data were collected and analyzed using conventional (F_ST, isolation by distance (IBD), AMOVA) and recently developed (Bayesian assignment test, isolation by resistance) landscape genetic methods. As predicted, a higher level of population structure in FL (F_ST′ = 0.401) than in PN (F_ST′ = 0.354) was detected, in addition to FL displaying strong IBD patterns (r = .861) unlike PN (r = .073). A general north–south break in FL populations was detected, consistent with the IBD pattern, while PN exhibited clustering of northern‐ and southern‐most populations, suggestive of altered dispersal patterns. Species‐specific resistant landscape features were also identified, with roads and land cover the main cause of resistance to FL, and elevation the main influence on PN. These different species–landscape interactions can be explained mostly by their life history traits, revealing that closely related and ecologically similar species have different responses to the same landscape features. Comparative landscape genetic studies are important in detecting such differences and refining generalizations about amphibians in monitoring environmental changes.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Partial physical and functional map of aMycobacterium aurum carotenogenesis operon

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A₁₁), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC²-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.     

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i _Bl and i _Ec , respectively) were expressed in lycopene producing E. coli strains by pTlyci_Bl and pTlyci_Ec plasmids, under the control of tac promoter. The results showed that the overexpression of i _Ec improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci_Ec. In contrast, the expression of i _Bl increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci_Bl. The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i _Ec in E. coli Tlyci_Ec-mev and 181 ± 9 mg/gDCW with i _Bl in E. coli Tlyci_Bl-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci_Bl-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.     

Preparation and characterization of a new rhenium(V) complex containing the

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids

The carotenogenic (crt) gene cluster from Brevibacterium linens, a member of the commercially important group of coryneform bacteria, was cloned and identified. An expression library of B. linens genes was constructed and a fragment of the crt cluster was obtained by functional complementation of a colourless B. flavum mutant, screening transformed cells for production of a yellow pigment. Subsequent screening of a cosmid library resulted in the cloning of the wholecrt cluster from B. linens. All genes necessary for the synthesis of the aromatic carotenoid isorenieratene were identified on the basis of sequence homologies. In addition a novel type of lycopene cyclase was identified by complementation of a lycopene-accumulating B. flavum mutant. Two genes, named crtYc and crtYd, which code for polypeptides of 125 and 107 amino acids, respectively, are necessary to convert lycopene to β-carotene. The amino acid sequences of these polypeptides show no similarity to any of the known lycopene cyclases. This is the first example of a carotenoid biosynthetic conversion in which two different gene products are involved, probably forming a heterodimer. Received: 17 July 1999 / Accepted: 7 December 1999     

Phototrophic cultivation of NaCl‐tolerant mutant of Spirulina platensis for enhanced C‐phycocyanin production under optimized culture conditions and its dynamic modeling

Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C‐phycocyanin (C‐PC ) and other valuable chemicals like carotenoids and γ‐linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C‐PC production in NaCl‐tolerant mutant of Spirulina platensis . In this study, highest C‐PC (147 mg · L^?1) and biomass (2.83 g · L^?1) production was achieved when a NaCl‐tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl‐tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C‐PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (μ_max) for NaCl‐tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half‐saturation constant (K_s), indicating that this strain could grow well at high concentration of NaNO₃. C‐PC production rate (C_max) in mutant cells increased by 12.2% at almost half the value of K_s as compared to its wildtype counterpart. Moreover, the inhibition constant (K_i) value was 207.85% higher in NaCl‐tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C‐PC even at high concentrations of NaNO₃.     

Location of the recessive gene ym (yellow margin) on chromosome 12 of diploid Solatium tuberosum by means of trisomic analysis

Summary Ten out of twelve primary trisomics of dip-loid S. tuberosum were crossed as females with a recessive mutant for yellow margin (ym ym) obtained from S. phureja. All primary trisomics used proved to be homozygous dominant. Trisomic plants from all ten F₁'s were backcrossed with the mutant and trisomics from eight F₁'s were crossed also with a disomic heterozygous f₁ plant from triple 10 X mutant.In both BC₁ and half sib progeny of each trisomic type the mutant plants were easily identified because of their typical small roundish leaflets with yellow or reddish margins. The observed segregation ratios for normal to mutant were tested against the expected non-critical ratios and against various expected critical ratios.From the results of these tests it is concluded that the gene ym is located on chromosome 12 of the potato. A hypothesis of linkage between ym and a gene l _x for lethality is put forward. It is concluded that l _x is not identical with a previously detected recessive gene l ₂ which is responsible for yellow cotyledons and lethality.     

Generation of lycopene-overproducing strains of the fungus <Emphasis Type="Italic">Mucor circinelloides</Emphasis> reveals important aspects of lycopene formation and accumulation

Objectives

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.

Results

Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.

Conclusions

A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

     

Lycopene intervention reduces inflammation and improves HDL functionality in moderately overweight middle-aged individuals

The management of overweight subjects by interventions aimed at reducing inflammation is highly desirable. To date, observational studies have identified a link between increased dietary antioxidant intake and reduced cardiovascular morbidity. However, direct trial evidence regarding the ability of antioxidants to influence inflammation is lacking. Therefore, this study examined lycopene's ability to lower systemic and high-density lipoprotein (HDL)-associated inflammation in moderately overweight middle-aged subjects. Serum was collected before and after a 12-week intervention from 54 moderately overweight, middle-aged individuals. Subjects were randomised to one of three groups: control diet (< 10 mg lycopene/week), lycopene-rich diet (224–350 mg lycopene/week) and lycopene supplement (70 mg lycopene/week). HDL was subfractionated into HDL_2&3 by rapid ultracentrifugation. Compliance was monitored by assessing lycopene concentration in serum and HDL_2&3. Systemic and HDL-associated inflammation was assessed by measuring serum amyloid A (SAA) levels. HDL functionality was determined by monitoring the activities of paraoxonase-1 (PON-1), cholesteryl ester transfer protein (CETP) and lecithin cholesterol acyltransferase (LCAT). Lycopene increased in serum and HDL_2&3 following both lycopene interventions (P<.001, for all), while SAA decreased in serum following the lycopene supplement and in HDL₃ following both lycopene interventions (P<.05 for all). PON-1 activity increased in serum and HDL_2&3 in both lycopene groups (P<.05, for all). Furthermore, the activity of CETP decreased in serum following the lycopene supplement, while the activity of LCAT increased in serum and HDL₃ following both lycopene interventions (P<.05 for all). These results demonstrate that in moderately overweight, middle-aged subjects, increasing lycopene intake leads to changes to HDL_2&3, which we suggest enhanced their antiatherogenic properties. Overall, these results show the heart-protective properties of increased lycopene intake.     

筛选地中海拟无枝酸菌无痕基因敲除菌株的简便方法

将抗生素抗性基因作为标记筛选无痕基因敲除菌株比较费时,因而建立筛选无痕基因敲除菌株的简便方法。通过敲除茄红素生物合成途径中第一个反应的酶编码基因dxs(1-脱氧-D-木酮糖-5-磷酸合酶基因),获得白色地中海拟无枝酸菌突变菌株,以此菌株为受体菌,对S-丙二酰转移酶基因(mtf)进行无痕敲除。针对菌落本身携带颜色的地中海拟无枝酸菌(橘红色),利用茄红素合成酶基因dxs无痕敲除获得了白色菌株,在此基础上进行mtf的无痕敲除。以茄红素生物合成途径中任意一个反应的酶编码基因作为标记,很容易筛选得到无痕基因敲除的突变菌株。     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.