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.     

Detection and morphological characteristics of “Kunimasu” (<Emphasis Type="Italic">Oncorhynchus kawamurae</Emphasis>)/”Himemasu” (<Emphasis Type="Italic">O. nerka</Emphasis>) hybrids in Lake Motosu,Yamanashi Prefecture,Japan

A recently discovered transplanted population of “Kunimasu” (Oncorhynchus kawamurae) in Lake Saiko, Yamanashi Prefecture, Japan, extinct in its original habitat, has been reported as almost never hybridizing with sympatric “Himemasu” (O. nerka). However, analyses of microsatellite and mitochondrial DNA markers disclosed extensive hybridization between these two species in Lake Motosu, Yamanashi Prefecture, a second lake to which Kunimasu had been transplanted. The rates of purebred Kunimasu, Kunimasu/Himemasu hybrids and purebred Himemasu were 0 %, 64.3 % and 35.7 %, respectively, and hybrid specimens had intermediate numbers of pyloric caeca and gill rakers compared with the parental species.     

Does sequence polymorphism of <Emphasis Type=\"Italic\">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type=\"Italic\">Brassica </Emphasis><Emphasis Type=\"Italic\">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.     

Decline of <Emphasis Type=\"Italic\">Cornus florida</Emphasis> and forest succession in a <Emphasis Type=\"Italic\">Quercus–Carya</Emphasis> forest

Cornus florida is a common understory species in many hardwood forests in eastern North America. It plays an important role in nutrient cycling and is an important food resource for many vertebrate species, especially migratory birds. We used data collected over a 16-year period to examine population dynamics of a tagged population of C. florida in a 6.4 ha area in the context of change in the protected Quercus–Carya forest of the Ross Biological Reserve, Indiana. We examined the hypothesis that forest dynamics result from interactions between long-term ecological succession and pathogens. The C. florida population at the Ross Reserve declined by 50% between 1983 and 2000, with a survivorship of 24%. Analysis of 40 years of forest survey data showed that Quercus and Carya populations declined in importance, while Acer saccharum increased dramatically. This change in forest structure is consistent with successional changes occurring throughout the Midwest and can be attributed to suppression of disturbance. Cornus florida declined more sharply where A. saccharum increased. From 1983 to 1999, C. florida were less likely to survive if they were within 5 m of a A. saccharum. Light measurements showed that A. saccharum abundance correlated negatively with light available to C. florida, suggesting that increased shading by A. saccharum contributed to C. florida decline. The fungus, Discula destructiva causes the disease dogwood anthracnose that is associated with widespread decline of C. florida in the eastern United States. Tests for this pathogen in our study area were mostly negative. Other tests revealed that Armillaria root rot infected most C. florida, but this disease seemed to be a secondary effect of shading by A. saccharum. These results suggest that the lack of fire and other anthropogenic disturbances has resulted in an accelerated shift in dominance from Quercus and Carya to A. saccharum in the main canopy, and this shift, in turn, has resulted in increased shading of C. florida and its decline in previously more open Midwestern forests.     

Extended-Spectrum β-lactamase (ESBL) producing <Emphasis Type=\"Italic\">Enterobacter aerogenes</Emphasis> phenotypically misidentified as <Emphasis Type=\"Italic\">Klebsiella pneumoniae</Emphasis> or <Emphasis Type=\"Italic\">K. terrigena</Emphasis>

Background  

Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates.     

Effect of glycosylation on the biochemical properties of <Emphasis Type=\"Italic\">β</Emphasis>-xylosidases from <Emphasis Type=\"Italic\">Aspergillus versicolor</Emphasis>

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R_f. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.     

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.     

Morpho-histological study of somatic embryo-like structures in hypocotyl cultures of<Emphasis Type=\"Italic\"> Pelargonium</Emphasis> ×<Emphasis Type=\"Italic\"> hortorum</Emphasis> Bailey

Somatic embryo-like structures were produced from the hypocotyls of ten cultivars of Pelargonium × hortorum using the protocols of Marsolais et al. (1991; Can J Bot 69:1188–1193) and Slimmon et al. (1991; Plant Cell Rep 10:587–589) and their embryonic natures evaluated. Nine cultivars responded, and 937 structures were formed. Regeneration corresponded well with published data. The somatic embryo-like structures were globular- to leaf-shaped or similar to shoots. A root pole was never visible. Histological examinations confirmed the lack of bipolarity and revealed vascular connections to the explant in the more developed structures. Therefore, these structures cannot be classified as somatic embryos. The importance of these results is discussed in terms of evaluating published protocols for the propagation of these pelargoniums by somatic embryogenesis from hypocotyls.Communicated by H. Lörz     

Cytochrome-<Emphasis Type=\"Italic\">b</Emphasis> variation in <Emphasis Type=\"Italic\">Apis mellifera</Emphasis> samples and its association with COI–COII patterns

Five Mbo I (Mbo-A, Mbo-M, Mbo-C¹, Mbo-C² and Mbo-C³) and Hinf I (Hinf-1 to Hinf-5) patterns were observed in Apis mellifera samples after restriction of a 485 bp fragment of the mitochondrial cytochrome-b (cyt-b) gene. Associating the cyt-b Restriction fragment length polymorphism (RFLP) pattern of each sample to its respective previously established COI–COII (Dra I sites) pattern, five restriction patterns (Mbo-C¹, Mbo-C², Mbo-C³, Hinf-1 and Hinf-4) were observed in samples of maternal origin associated to the evolutionary branch C. No deletions or insertions were observed and the nucleotide substitution rate was estimated at 5.4%. Higher nucleotide diversity was observed among the branch C-haplotypes when compared with A and M lineages. Further studies are needed to confirm if the cyt-b + COI–COII haplotypes help to assign certain phylogeographic patterns to the branch C and to clarify phylogenetic relationships among A. mellifera subspecies.     

Analysis of 5S rDNA changes in synthetic allopolyploids <Emphasis Type=\"Italic\">Triticum</Emphasis> × <Emphasis Type=\"Italic\">Aegilops</Emphasis>

Changes of 5S rDNA at the early stage of allopolyploidization were investigated in three synthetic allopolyploids: Aegilops sharonensis × Ae. umbellulata (2n = 28), Triticum urartu × Ae. tauschii (2n = 28), and T. dicoccoides × Ae. tauschii (2n = 42). Fluorescent in situ hybridization (FISH) revealed quantitative changes affecting separate loci of one of the parental genomes in S₃ plants of each hybrid combination. Southern hybridization with genomic DNA of the allopolyploid T. urartu × Ae. tauschii (TMU38 × TQ27) revealed a lower intensity of signals from Ae. tauschii fragments compared with those derived from T. urartu. This confirmed the signal reduction revealed for chromosome 1D of this hybrid by FISH. Neither Southern hybridization nor PCR testing of 5–15 plants of the S₂-S₃ generations revealed an appearance of new 5S rDNA fragments or a complete disappearance of parental fragments from the allopolyploids under study. No changes were found by aligning nine 5S rDNA sequences of the allopolyploid TMU38 × TQ27 with corresponding sequences of the parental species. The similarity between one of the synthetic allopolyploids examined and a natural allopolyploid with the same genome composition points to an early formation of the 5S rDNA organization unique for each allopolyploid.     

<Emphasis Type="Italic">Prunus serotina</Emphasis> unleashed: invader dominance after 70 years of forest development

Propagule pressure and disturbance have both been found to facilitate invasion. Therefore, knowledge on the history of introduction and disturbance is vital for understanding an invasion process, and research should focus on areas in which the invasive species has not been deliberately introduced or managed to study unconfounded colonization patterns. Comparing the outcome of such spontaneous colonization processes for different ecosystems might provide a useful framework for setting management priorities for invasive species that enter new, uninvaded areas. We focused on the 70-year spontaneous spread of the invasive tree species Prunus serotina in a pine forest in the Netherlands. To reconstruct the invasion pattern, we combined historical maps, tree ring analysis, spatially explicit tree inventory data, seed density data, and regeneration data for both native and non-native species. Prunus serotina was the only species that showed successful regeneration: the species was present throughout the forest in the tree, shrub, and herb layer. Native species were not able to outgrow the seedling stage. Our data demonstrate that P. serotina is a gap-dependent species with high seed production that builds up a seedling bank. We also compared the results of this study with a similar study on P. serotina colonization in a deciduous forest in Belgium, where P. serotina invasion was not successful. The sharp contrast between the outcomes of the two invasion processes shows the importance of studying an invasive species and the recipient ecosystem jointly and made us raise the hypothesis that herbivore pressure may facilitate P. serotina invasion.     

A chemical-genetic approach to elucidate protein kinase function <Emphasis Type=\"Italic\">in planta</Emphasis>

The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.     

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.     

Is the <Emphasis Type=\"Italic\">Mnr</Emphasis> locus of Triticeae species the same as the <Emphasis Type=\"Italic\">Ndh</Emphasis> and <Emphasis Type=\"Italic\">Dia</Emphasis> loci?

The menadione reductase (MNR), the nicotinamide adenine dinucleotide dehydrogenase (NDH) and diaphorase (DIA) isozymes were studied in the allohexaploid Triticum aestivum cv ”Chinese Spring” and in five diploid Triticeae species. The Mnr1, Ndh3 and Dia1 loci were located on the chromosome arms 3AL, 3BL and 3DL of T. aestivum, respectively. These loci were also located on the 3H chromosome of Hordeum vulgare cv ”Betzes”, the 3L chromosome of Aegilops longissima and the 6RL chromosome arm of Secale cereale cv ”Imperial”. The chromosomal location results together with the segregation studies support a tetrameric behaviour of the MNR1, NDH3 and DIA1 isozymes. The Ndh1 and Dia3 loci were located on homoeologous group 4 showing a monomeric behaviour. The chromosomal locations and linkage data of the Mnr, Ndh and Dia loci suggest that Mnr1=Ndh3=Dia1; Ndh1=Dia3 and Ndh2=Dia2. Received: 3 June 2001 / Accepted: 11 July 2001     

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.     

Karyotype reshufflings of <Emphasis Type=\"Italic\">Festuca pratensis</Emphasis> × <Emphasis Type=\"Italic\">Lolium perenne</Emphasis> hybrids

Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F₁ F. pratensis × L. perenne plants and generated F₂-F₉ progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown.     

Expression of an endoglucanase–cellobiohydrolase fusion protein in <Emphasis Type=\"Italic\">Saccharomyces cerevisiae,Yarrowia lipolytica,</Emphasis> and <Emphasis Type=\"Italic\">Lipomyces starkeyi</Emphasis>

The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.