Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Spectroscopic evidence for an all-ferrous [4Fe–4S]<Superscript>0</Superscript> cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from <Emphasis Type="Italic">Acidaminococcus </Emphasis><Emphasis Type="Italic">fermentans</Emphasis>

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]⁺ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]⁰ cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔE _Q = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S₄ sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin S _t = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = ?0.66 cm^?1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]⁰ cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]⁰ cluster found in biology.     

Rhizosphere processes induce changes in dissimilatory iron reduction in a tidal marsh soil: a rhizobox study

Background and aims

Although the role of microbial iron respiration in tidal marshes has been recognized for decades, the effect of rhizosphere processes on dissimilatory ferric iron reduction (FeR) is poorly known. Herein, we examined the FeR surrounding the root zone of three tidal marsh plants.

Methods

Using in situ rhizoboxes, we accurately separated rhizobox soil as one rhizosphere zone, and three bulk soil zones. Dissimilatory and sulfidic-mediated FeR were quantified by accumulation of non-sulfidic Fe(II) and Fe sulfides over time, respectively.

Results

The rates of dissimilatory FeR attained 42.5 μmol Fe g^?1 d^?1 in the rhizosphere, and logarithmically declined by up to 19.1 μmol Fe g^?1 d^?1 in the outer bulk soil. The rates of sulfidic-mediated FeR were less than 2 μmol Fe g^?1 d^?1 among all zones. Poorly crystalline Fe(III), DOC and DON, porewater Fe²⁺, and SO₄^2? were all enriched in the rhizosphere, whereas non-sulfidic Fe(II) and Fe sulfides gradually accumulated away from the roots. Iron reducers (Geobacter, Bacillus, Shewanella, and Clostridium) had higher populations in the rhizosphere than in the bulk soil. Higher rates of dissimilatory FeR were observed in the Phragmites australis and Spartina alterniflora rhizoboxes than in the Cyperus malaccensis rhizoboxes.

Conclusions

The radial change pattern of dissimilatory FeR rates were determined by allocation of poorly crystalline Fe(III) and dissolved organic carbon. The interspecies difference of rhizosphere dissimilatory FeR was associated with the root porosity and aerenchyma of the tidal marsh plants.

     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

Characterization and Proteomic Analysis of the <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 <Emphasis Type="Italic">xenB</Emphasis> Knockout Mutant Under RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Stress

Pseudomonas sp. HK-6 is able to utilize RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) as its sole nitrogen source. The role of the xenB gene, encoding xenobiotic reductase B, was investigated using HK-6 xenB knockout mutants. The xenB mutant degraded RDX to a level that was 10-fold less than that obtained with the wild-type HK-6 strain. After 60 days of culture with 25 or 50 μM RDX, no residual RDX was detected in the supernatants of the wild-type aerobically grown cultures, whereas approximately 90 % of the RDX remained in the xenB mutant cultures. The xenB mutant bacteria exhibited a 10²–10⁴-fold decrease in survival rate compared to the wild-type. The expression of DnaK and GroEL proteins, two typical stress shock proteins (SSPs), in the xenB mutant increased after immediate exposure to RDX, yet dramatically decreased after 4 h of exposure. In addition, DnaK and GroEL were more highly expressed in the cultures with 25 μM RDX in the medium but showed low expression in the cultures with 50 or 75 μM RDX. The expression levels of the dnaK and groEL genes measured by RT-qPCR were also much lower in the xenB genetic background. Analyses of the proteomes of the HK-6 and xenB mutant cells grown under conditions of RDX stress showed increased induction of several proteins, such as Alg8, alginate biosynthesis sensor histidine kinase, and OprH in the xenB mutants when compared to wild-type. However, many proteins, including two SSPs (DnaK and GroEL) and proteins involved in metabolism, exhibited lower expression levels in the xenB mutant than in the wild-type HK-6 strain. The xenB knockout mutation leads to reduced RDX degradation ability, which renders the mutant more sensitive to RDX stress and results in a lower survival rate and an altered proteomic profile under RDX stress.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Cloning and characterization of two distinct water-forming NADH oxidases from <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> for the regeneration of NAD

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K _M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k _cat/K _M of 1.0 and 0.2 μM^?1 s^?1, respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD⁺ was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD⁺ regeneration in dehydrogenase-catalyzed oxidations.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

Effects of zinc on the production of alcohol by <Emphasis Type="Italic">Clostridium carboxidivorans</Emphasis> P7 using model syngas

Renewable energy, including biofuels such as ethanol and butanol from syngas bioconversed by Clostridium carboxidivorans P7, has been drawing extensive attention due to the fossil energy depletion and global eco-environmental issues. Effects of zinc on the growth and metabolites of C. carboxidivorans P7 were investigated with model syngas as the carbon source. The cell concentration was doubled, the ethanol content increased 3.02-fold and the butanol content increased 7.60-fold, the hexanol content increased 44.00-fold in the medium with 280 μM Zn²⁺, when comparing with those in the control medium [Zn²⁺, (7 μM)]. Studies of the genes expression involved in the carbon fixation as well as acid and alcohol production in the medium with 280 μM Zn²⁺ indicated that fdhII was up-regulated on the second day, acs A, fdhII, bdh35 and bdh50 were up-regulated on the third day and bdh35, acsB, fdhI, fdhIII, fdhIV, buk, bdh10, bdh35, bdh40 and bdh50 were up-regulated on the fourth day. The results indicated that the increased Zn²⁺ content increased the alcohol production through increase in the gene expression of the carbon fixation and alcohol dehydrogenase.     

Characterization of alcohol dehydrogenase from <Emphasis Type="Italic">Kangiella koreensis</Emphasis> and its application to production of all-<Emphasis Type="Italic">trans</Emphasis>-retinol

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min^?1 mg^?1, a K _m of 1.8 μM, and a k _cat of 1.7 min^?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD ⁺ as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l^?1, and 2,200 mg all-trans-retinal l^?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l^?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l^?1 h^?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

Biomass and terpenoids produced by mutant strains of <Emphasis Type="Italic">Arthrospira</Emphasis> under low temperature and light conditions

The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14?°C, 43.75 μmol photons m^?2 s^?1 and performed breed conservation at 2.5?°C, 8.75 μmol photons m^?2 s^?1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16?°C and 43.75 μmol photons m^?2 s^?1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057?±?80 mg l^?1, 68.7?±?2.5 mg l^?1 day^?1, 1839?±?44 mg l^?1, 60.6?±?1.8 mg l^?1 day^?1, and 2113?±?64 mg l^?1, 77.7?±?2.5 mg l^?1 day^?1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10?°C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28?°C, achieving 1140 mg l^?1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-d-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.     

Characterization of a novel <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> xanthine dehydrogenase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications.

Results

The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40–45 °C, was stable under alkaline conditions (pH 7–11) and the half-inactivation temperature was 60 °C. The K _m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s^?1 and 2.7 μM^?1 s^?1, respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized.

Conclusion

The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.