Nitrogen addition and rhizome severing modify clonal growth and reproductive modes of <Emphasis Type="Italic">Leymus</Emphasis> <Emphasis Type="Italic">chinensis</Emphasis> population

The resurrection flowering plant Ramonda serbica inhabits the shallow organo-mineral soil that develops in crevices on northern-facing carbonate rocks in the gorges in the Balkan Peninsula. This type of soil represents a complex substrate whose physical and chemical properties were found to be well suited to the most important requirements for the growth and development of R. serbica as well as for the plant’s survival in the state of anhydrobiosis in periods of drought stress. Considerable amount of organic matter (39.4%) in the soil resulted in the high field capacity (134 ml/100 g soil) as well as the slow changes in the amount of its available water. The suitable soil hydric status, based on the organic remains, supports the slow dehydration of this poikilohydric plant, which is extremely important in allowing the activation of the plant’s protective mechanisms. The pH of the soil solution was slighty alkaline (7.7) mostly due to carbonates in its crystallographic structure. The large amount of incompletely decomposed organic debris resulted in a marked difference between total and available nutrient concentration in the soil. Still, the adequate content of nutrients in the leaves points to efficient mineral consumption by the plant roots. The sufficient bioavailability of nutrients and water was also improved by vesicular–arbuscular mycorrhiza detected in R. serbica roots.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Carbon isotope fractionation during <Emphasis Type="Italic">cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> isomerization of unsaturated fatty acids in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cis–trans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cis–trans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cis–trans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp² carbon linkage of the cis-double bond to an intermediate sp³ within an enzyme–substrate complex. The sp² linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe³⁺), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp² linkage into sp³.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

In-fusion expression and characterization of <Emphasis Type="Italic">β</Emphasis>-xylanase and <Emphasis Type="Italic">β</Emphasis>-1,3-1,4-glucanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Two chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower K_n higher k_cat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.     

Novel peroxidases of <Emphasis Type="Italic">Marasmius scorodonius</Emphasis> degrade <Emphasis Type="Italic">β</Emphasis>-carotene

Two extracellular enzymes (MsP1 and MsP2) capable of efficient β-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).     

Applications of PCR-RFLPs for differentiating two freshwater sponges: <Emphasis Type="Italic">Ephydatia </Emphasis><Emphasis Type="Italic">fluviatilis</Emphasis> and <Emphasis Type="Italic">Ephydatia mülleri</Emphasis>

The two freshwater sponges Ephydatia fluviatilis and Ephydatia mülleri belong to the widespread Spongillidae family. Their morphological tracts are very similar and can be distinguished mainly on the basis of their gemmuloscleres. However, when gemmules are absent it is essential to have an unambiguous species attribution for a population genetic study based on fresh tissues and historical collections. This article reports a simple Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, applied to DNA extracted from both gemmules and fresh tissues in order to discriminate between the two congeneric E. fluviatilis and E. mülleri. Such a biomolecular method is based on the discriminative enzymes’ digestion of each of the three amplified fragments 5.8S-ITS2-28S, D3 domain of the 28S subunit and COI. Two restriction enzymes were tested for a 620–642 bp fragment of 5.8S-ITS2-28S and for a 342 bp fragment of the D3 domain of the 28S, one restriction enzyme was tested for a 681 bp fragment codifying for COI. Obtained digestion patterns were diagnostic for each of the two species, providing a relatively simple, fast and cheap method for species attribution compared to sequencing. Handling editor: C. Sturmbauer     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

Isolation and functional characterization of <Emphasis Type="Italic">Lycopene β-cyclase</Emphasis> (<Emphasis Type="Italic">CYC-B</Emphasis>) promoter from <Emphasis Type="Italic">Solanum habrochaites</Emphasis>

Background  

Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061.     

Synthesis of disaccharides using β-glucosidases from <Emphasis Type="Italic">Aspergillus niger</Emphasis>, <Emphasis Type="Italic">A. awamori</Emphasis> and <Emphasis Type="Italic">Prunus dulcis</Emphasis>

Objective

Glucose conversion into disaccharides was performed with β-glucosidases from Prunus dulcis (β-Pd), Aspergillus niger (β-An) and A. awamori (β-Aa), in reactions containing initial glucose of 700 and 900 g l^?1.

Results

The reactions’ time courses were followed regarding glucose and product concentrations. In all cases, there was a predominant formation of gentiobiose over cellobiose and also of oligosaccharides with a higher molecular mass. For reactions containing 700 g glucose l^?1, the final substrate conversions were 33, 38, and 23.5% for β-An, β-Aa, and β-Pd, respectively. The use of β-An yielded 103 g gentiobiose l^?1 (15.5% yield), which is the highest reported for a fungal β-glucosidase. The increase in glucose concentration to 900 g l^?1 resulted in a significant increase in disaccharide synthesis by β-Pd, reaching 128 g gentiobiose l^?1 (15% yield), while for β-An and β-Aa, there was a shift toward the synthesis of higher oligosaccharides.

Conclusion

β-Pd and the fungal β-An and β-Aa β-glucosidases present quite dissimilar kinetics and selective properties regarding the synthesis of disaccharides; while β-Pd showed the highest productivity for gentiobiose synthesis, β-An presented the highest specificity.

     

Retiolitid graptolites from the collection of Hermann Jaeger II: <Emphasis Type="Italic">Cometograptus</Emphasis>, <Emphasis Type="Italic">Spinograptus</Emphasis> and <Emphasis Type="Italic">Plectograptus</Emphasis>

Graptolites from the Jaeger collection at the Museum für Naturkunde (Berlin, Germany) provide important information on structural details of Silurian (Wenlock–Ludlow) retiolitids as well as for the biostratigraphic and biogeographic distribution of these magnificent graptolites. Species of the genera Cometograptus, Spinograptus and Plectograptus are described from isolated glacial boulder material, collected in northern Germany and from shale specimens found in the Lower Graptolite Shale of Thuringia. The biostratigraphic placement of material derived from glacial erratic boulders, however, is far from being precise. The fauna associated with the neotype of Plectograptus macilentus in the ‘Unterer Graptolithenschiefer’ of Thuringia is discussed and illustrated. Cometograptus alfeisenacki from the Cyrtograptus lundgreni Biozone is recognized as a new species. The genus is discovered for the first time in North German glacial erratic boulders.     

<Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> and its important metabolites — <Emphasis Type="Italic">review</Emphasis>

C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein. This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such as textile and in cosmetics.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.