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.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Molecular polymorphism of α-galactosidase <Emphasis Type="Italic">MEL</Emphasis> genes of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts

Molecular genetic analysis of melibiose-fermenting Saccharomyces strains isolated from fermentative processes and natural sources in different world regions was conducted to deduce the evolutionary diversity of Saccharomyces yeasts and find new α-galactosidase MEL genes. The species S. bayanus, S. mikatae, and S. paradoxus were shown to have a single copy of MEL and not accumulate polymeric genes, unlike some S. cerevisiae populations. The polymeric genes MELp1 and MELp2 were identified in S. paradoxus for the first time. Genes identical by 98.7% are located on the chromosomes X and VI, respectively. Phylogenetic analysis indicates that MEL genes of the Saccharomyces yeasts are species-specific.     

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.     

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 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.     

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.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> inhibition of α-amylases of stored-product mite <Emphasis Type="Italic">Acarus siro</Emphasis>

The stored-product mites are the most abundant and frequent group of pests living on the stored food products in Europe. They endanger public health since they produce allergens and transmit mycotoxin-producing fungi. Novel acaricidal compounds with inhibitory effects on the digestive enzymes of arthropods are a safe alternative to the traditional neurotoxic pesticides used for control of the stored-product pests. In this work, we explored the properties of acarbose, the low molecular weight inhibitor of -amylases (AI), as a novel acaricide candidate for protection of the stored products from infestation by Acarus siro (Acari: Acaridae). In vitro analysis revealed that AI blocked efficiently the enzymatic activity of digestive amylases of A. siro, and decreased the physiological capacity of mites gut in utilizing a starch component of grain flour. In vivo experiments showed that AI suppressed the population growth of A. siro. The mites were kept for three weeks on experimental diet enriched by AI in concentration range of 0.005 to 0.25%. Population growth of A. siro was negatively correlated with the content of AI in the treated diet with a half-population dose of 0.125%. The suppressive effect of AIs on stored-product mites is discussed in the context of their potential application in GMO crops     

How promising is <Emphasis Type="Italic">Lasioseius floridensis</Emphasis> as a control agent of <Emphasis Type="Italic">Polyphagotarsonemus</Emphasis><Emphasis Type="Italic">latus</Emphasis>?

The blattisociid mite Lasioseius floridensis Berlese was found associated with the broad mite, Polyphagotarsonemus latus (Banks), on gerbera leaves in Mogi das Cruzes, State of Sao Paulo, Brazil. Blattisociid mites are not common on aerial plant parts, except under high air humidity levels. Some Lasioseius species have been mentioned as effective control agents of rice pest mites, but nothing is known about the biology of L. floridensis. The objective of this study was to evaluate whether the observed co-occurrence of L. floridensis and P. latus was just occasional or whether the latter could be important as food source for the former, assumed by laboratory evaluation of the ability of the predator to maintain itself, reproduce and develop on that prey. Biological parameters of L. floridensis were compared when exposed to P. latus and to other items as food. The study showed that mating is a pre-requisite for L. floridensis to oviposit and that oviposition rate was much higher on the soil nematode Rhabditella axei (Cobbold) (Rhabditidae) than on P. latus. Ovipositon on the acarid mite Tyrophagus putrescentiae (Schrank) was about the same as on P. latus, but it was nearly zero when the predator was fed the fungi Aspergillus flavus Link or Penicillium sp., or cattail (Typha sp.) pollen. Survivorship was higher in the presence of pollen and lower in the presence of A. flavus or Penicillium sp. than in the absence of those types of food. Life table parameters indicated that the predator performed much better on R. axei than on P. latus. To evaluate the potential effect of L. floridensis as predator of P. latus, complementary studies are warranted to determine the frequency of migration of L. floridensis to aerial plant parts, when predation on P. latus could occur.     

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. and (<Emphasis Type="Italic">n </Emphasis>= 9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica ‘C’ (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23–0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Overexpression of <Emphasis Type="Italic">ARO10</Emphasis> in <Emphasis Type="Italic">pdc5</Emphasis>Δmutant resulted in higher isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

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.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

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.