Hairy root induction of<Emphasis Type="Italic"> Papaver somniferum</Emphasis> var.<Emphasis Type="Italic"> album</Emphasis>, a difficult-to-transform plant,by<Emphasis Type="Italic"> A. rhizogenes</Emphasis> LBA 9402

Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain [GV 3101 (PMP90RK, p35SGUS-2)] and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L. hypocotyls. Five weeks after the infection with A. rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium. Six hairy-root cultures were established. Transformation was confirmed by polymerase chain reaction analysis. One clone was analysed for its alkaloid production. The total alkaloid content was higher in the transformed roots (0.46±0.06% DW) than in the untransformed roots (0.32±0.05% DW). The transformed roots accumulated three times more codeine (0.18±0.02% DW) than intact roots (0.05±0% DW). Moreover, morphine (0.255±0.03% DW) and sanguinarine (0.014±0% DW) were found in the liquid culture medium.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - LS Linsmaier and Skoog     

Production of β-glucosidase using immobilised<Emphasis Type="Italic"> Piromyces</Emphasis> sp. KSX1 and<Emphasis Type="Italic"> Orpinomyces</Emphasis> sp. 478P1 in repeat-batch culture

Two anaerobic fungi, one a monocentric strain (Piromyces sp. KSX1) and the other a polycentric strain (Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. β-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, β-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more β-glucosidase than strain 478P1, the maximum specific β-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor. Electronic Publication     

Production of γ-aminobutyric acid by <Emphasis Type="Italic">Streptococcus salivarius</Emphasis> subsp. <Emphasis Type="Italic">thermophilus</Emphasis> Y2 under submerged fermentation

Summary. γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 °C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 °C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5′-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 ± 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions. Authors’ address: Zhao-Xin Lu, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

Polyphasic characterization of “<Emphasis Type="Italic">Aspergillus nidulans</Emphasis> var. <Emphasis Type="Italic">roseus</Emphasis>” ATCC 58397

Polyphasic characterization of the echinocandin B producer Aspergillus nidulans var. roseus ATCC 58397 strain was carried out to elucidate its taxonomical status. According to its carbon source utilization and secondary metabolite spectrum as well as the partial β-tubulin, calmodulin, and γ-actin gene sequences, A. nidulans var. roseus belongs to the Emericella rugulosa species. Auxotroph mutants of A. nidulans var. roseus ATCC 58397 and E. rugulosa CBS 171.71 and CBS 133.60 formed stable heterokaryons on minimal medium with several A. nidulans strains, and in the case of A. nidulans var. roseus, even cleistothecia were developed.     

Production of the <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> endo-1,4-β-mannanase in <Emphasis Type="Italic">A. niger</Emphasis>

The β-mannanase gene (man1) from Aspergillus aculeatus MRC11624 (Izuka) was patented for application in the coffee industry. For production of the enzyme, the gene was originally cloned and expressed in Saccharomyces cerevisiae. However the level of production was found to be economically unfeasible. Here we report a 13-fold increase in enzyme production through the successful expression of β-mannanase of Aspergillus aculeatus MRC11624 in Aspergillus niger under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpd _P) and the A. awamori glucoamylase terminator (glaA_T). The effect of medium composition on mannanase production was evaluated, and it was found that the glucose concentration and the organic nitrogen source had an effect on both the volumetric enzyme activity and the specific enzyme activity. The highest mannanase activity levels of 16,596 nkat ml⁻¹ and 574 nkat mg⁻¹ dcw were obtained for A. niger D15[man1] when cultivated in a process-viable medium containing corn steep liquor as the organic nitrogen source and high glucose concentrations.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

<Emphasis Type="Italic">Sphingomonas rosea</Emphasis> sp. nov. and <Emphasis Type="Italic">Sphingomonas swuensis</Emphasis> sp. nov., rosy colored <Emphasis Type="Italic">β</Emphasis>-glucosidase-producing bacteria isolated from soil

Two strains PB196^T and PB62^T of Gram-negative, non-motile, and non-spore-forming bacteria, were isolated from soil in South Korea and characterized to determine their taxonomic positions. 16S rRNA gene sequence analysis showed that the two strains belonged to the genus Sphingomonas. The highest degree of sequence similarity of strain PB196^T was found with PB62^T (98.9%), Sphingomonas humi PB323^T (98.9%), Sphingomonas kaistensis PB56^T (98.2%), and Sphingomonas astaxanthinifaciens TDMA-17^T (98.0%). The highest degree of sequence similarity of strain PB62^T was found with Sphingomonas humi PB323^T (98.8%), Sphingomonas astaxanthinifaciens TDMA-17^T (98.2%), and Sphingomonas kaistensis PB56^T (98.1%). Chemotaxonomic data revealed that they possessed ubiquinone-10 (Q-10) as common in the genus Sphingomonas, that the predominant fatty acids were summed feature 7 (C_18:1 ω7c/ω9t/ω12t), summed feature 4 (C_16:1 ω7c/C_15:0 iso 2OH), C_16:0, and C_17:1 ω6c, and that they contained sphingoglycolipid, phosphatidylglycerol (PG), and phosphatidyle-thanolamine (PE) in common but they showed difference for diphosphatidylglycerol (DPG). Based on these data, PB196^T (=KCTC 12339^T =JCM 16604^T) and PB62^T (=KCTC 12336^T =JCM 16605^T =KEMB 9004-005^T) should be classified as type strains of two novel species, for which the names Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov. are proposed, respectively.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

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.     

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.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Cryopreservation of hairy root cultures of <Emphasis Type="Italic">Maesa lanceolata</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35–40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.     

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.     

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.

     

Production and physicochemical characterization of β-glucan produced by<Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JB115

This study was conducted to develop a bacterial glucan as an animal feed additive. A novel glucan-producing bacterium.Paenibacillus polymyxa JB115, was isolated from Korean soil. The glucan, JB115-BG, produced byP. polymyxa JB115, was confirmed by TLC to be composed of glucose only. By examining FT-IR,¹H NMR, and¹³C NMR spectra, it was proven that JB115-BG has a β-(1→3)- and β-(1→6)-linked glucan structure. The particle size of JB115-BG was distributed in the range of 4–800 μm, with a mean value of 149.1 μm, and its molecular distribution ranged from 6.9∼3,103.7 kDa. It was also observed that 80% of the purified JB115-BG had a molecular distribution above 100 kDa. The obtained results suggest that the glucan JB115-BG can be used as an animal feed additive for the purpose of enhancing immunity.     

Involvement of Carbohydrates in Response to Preconditioning Flooding in Two Clones of <Emphasis Type="Italic">Populus deltoides</Emphasis> Marsh. × <Emphasis Type="Italic">P.</Emphasis> <Emphasis Type="Italic">nigra</Emphasis> L.

Poplar, a fast-growing species widely used outside its original area of distribution, was evaluated in the present study to verify its tolerance and hardening to waterlogging in anticipation of its implementation in rehabilitation practices for marginal lands subjected to flooding. Three water regimes were applied to the plants, grown in pots, with two clones (I-488 and D-64) with different sensitivities to flooding. These plants included a lot consisting of control plants (C), which were irrigated with good drainage, a non-preconditioned lot (NPr), and a third lot that was preconditioned to flooding (Pr). Furthermore, flooding was imposed on NPr and Pr plants by submerging pots up to 5 cm above the collar for 60 days followed by a 40-day recovery period. At the end of these two periods, shoot dry mass, foliar concentrations of certain ions, soluble sugars, starch and proline, and the relative electrolyte leakage were evaluated. The preconditioning treatment conferred different degrees of hardness on the treated plants compared with NPr plants. Our results revealed that high carbohydrate availability (soluble sugars and starch) is suggested to participate in sustaining membrane integrity which may affect the recuperative potential of Pr plants, notably those of clone I-488, from flooding damage.     

Description of “<Emphasis Type="Italic">Desulfotomaculum nigrificans</Emphasis> subsp. <Emphasis Type="Italic">salinus</Emphasis>” as a New Species, <Emphasis Type="Italic">Desulfotomaculum salinum</Emphasis> sp. nov.

This study focused on the physiological, chemotaxonomic, and genotypic characteristics of two thermophilic spore-forming sulfate-reducing bacterial strains, 435^T and 781, of which the former has previously been assigned to the subspecies “Desulfotomaculum nigrificans subsp. salinus”. Both strains reduced sulfate with the resulting production of H₂S on media supplemented with H₂ + CO₂, formate, lactate, pyruvate, malate, fumarate, succinate, methanol, ethanol, propanol, butanol, butyrate, valerate, or palmitate. Lactate oxidation resulted in acetate accumulation; butyrate was oxidized completely, with acetate as an intermediate product. Growth on acetate was slow and weak. Sulfate, sulfite, thiosulfate, and elemental sulfur, but not nitrate, served as electron acceptors for growth with lactate. The bacteria performed dismutation of thiosulfate to sulfate and hydrogen sulfide. In the absence of sulfate, pyruvate but not lactate was fermented. Cytochromes of b and c types were present. The temperature and pH optima for both strains were 60–6°C and pH 7.0. Bacteria grew at 0 to 4.5–6.0% NaCl in the medium, with the optimum being at 0.5–1.0%. Phylogenetic analysis based on a comparison of incomplete 16S rRNA sequences revealed that both strains belonged to the C cluster of the genus Desulfotomaculum, exhibiting 95.5–98.3% homology with the previously described species. The level of DNA–DNA hybridization of strains 435^T and 781 with each other was 97%, while that with closely related species D. kuznetsovii 17^T was 51–52%. Based on the phenotypic and genotypic properties of strains 435^T and 781, it is suggested that they be assigned to a new species: Desulfotomaculum salinum sp. nov., comb. nov. (type strain 435^T = VKM B 1492^T).