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.     

Cloning and expression of <Emphasis Type=\"Italic\">β</Emphasis>-glucosidase gene from <Emphasis Type=\"Italic\">Bacillus licheniformis</Emphasis> into <Emphasis Type=\"Italic\">E. coli</Emphasis> BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. K _m and V _max were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy E_a, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pK_a1 and pK_a2 of the ionizable groups of active site residues involved in V_max were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.     

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.     

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.     

Starch-bound 2S proteins and kernel texture in einkorn, <Emphasis Type=\"Italic\">Triticum</Emphasis> <Emphasis Type=\"Italic\">monococcum</Emphasis> ssp <Emphasis Type=\"Italic\">monococcum</Emphasis>

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A ^m 1a and “valine-type” ETI encoded by allele Eti-A ^m 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm²/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.     

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     

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.     

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.     

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     

Protein production by<Emphasis Type=\"Italic\"> Escherichia</Emphasis><Emphasis Type=\"Italic\">coli</Emphasis> wild-type and <Emphasis Type=\"Italic\">ΔptsG</Emphasis> mutant strains with IPTG induction at the onset

During Escherichia coli growth on glucose, uptake exceeds the requirement of flux to precursors and the surplus is excreted as acetate. Beside the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. The deletion of ptsG, the gene coding for one of the components (IICB(Glc)) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced glucose consumption and acetate excretion. Induction of protein production at the onset of cultivation decreased growth rate and glucose consumption rate for both the WT and the mutant strains. The mutant strain produced beta-galactosidase at higher rates than the wild-type strain while directing more carbon into biomass and CO(2) and less into acetate.     

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.     

Infection of Barley by <Emphasis Type=\"Italic\">Ramularia</Emphasis> <Emphasis Type=\"Italic\">collo-cygni</Emphasis>: Scanning Electron Microscopic Investigations

Ramularia collo-cygni causes leaf spots on barley (Hordeum vulgare), a disease of growing economical importance. Scanning electron microscopy was used to study the life cycle of the fungus on barley during the vegetation period and in winter. The infectious stage started with conidium germination on the surface and the penetration into the leaf via the stomatal pore where the hyphae grew within the cells that became necrotic. The conidiophores emerged through the stomatal pore. On older leaves, however, they frequently emerged apart from it and the results suggested a pushing apart of adjacent cell walls of the epidermal cells. An assessment of the amount of conidium formation of one heavily infested barley plant resulted in 4.05 × 10⁶ conidia per plant. For the first time, conidiophores, conidium production and germination of conidia were also observed in winter on barley and on maize leaves.     

Indel patterns of the plastid DNA<Emphasis Type=\"Italic\"> trnL</Emphasis>–<Emphasis Type=\"Italic\">trnF</Emphasis> region within the genus<Emphasis Type=\"Italic\"> Poa</Emphasis> (Poaceae)

The use of insertion/deletion (indel) patterns from sequences of the trnL intron and trnL–F intergenic spacer (IGS) in finding plastid genome types of the genus Poa L. was studied. New sequences for 23 taxa (P. alpina, P. badensis, P. bulbosa, P. crassipes, P. molinerii, P. annua, P. chaixii, P. granitica, P. pratensis, P. sibirica, P. remota, P. botryoides, P. cenisia, P. compressa, P. laxa, P. margilicola, P. media, P. nemoralis, P. palustris, P. pannonica, P. pirinica, P. riphaea, and P. sejuncta) and 18 previously published sequences, which represent 11 of the 13 sections listed for Poa in Flora Europaea, were investigated. Collections were made primarily in central Europe. Indel patterns, despite sampling less than 0.7% of the plastid genome, produced four taxa groupings that were congruent with the major divisions obtained in intensive, previously published restriction-site studies. Insertion/deletion events in the trnL intron and trnL–trnF IGS were in nearly all cases unique to a single pattern group and thus provided almost no information about relationships among these groups. Indels did, however, provide a meaningful infrageneric classification criterion for Poa. They can serve as useful tools in studying relationships within this genus.     

Ciliate grazing on <Emphasis Type=\"Italic\">Nitrosomonas europaea</Emphasis> and <Emphasis Type=\"Italic\">Nitrospira</Emphasis><Emphasis Type=\"Italic\">moscoviensis</Emphasis>: Is selectivity a factor for the nitrogen cycle in natural aquatic systems?

Ciliated protists are important predators of bacteria in many aquatic habitats, including sediments. Since, many biochemical transformations within the nitrogen cycle are performed by bacteria, ciliates could have an indirect impact on the nitrogen cycle through selective grazing on nitrogen-transforming bacteria. As a case study, we examined ciliate grazing on nitrifying bacteria of the genera Nitrosomonas and Nitrospira. All experiments were designed as in vitro-experiments with cultures of different bacteria and ciliate species. The nitrifying bacteria used in our experiments were Nitrosomonas europaea [Winogradsky 1892] and Nitrospira moscoviensis [Ehrich 2001]. The ciliates comprised of four species that are known as efficient bacterivores and common members of the protist community in aquatic systems: Paramecium aurelia [Müller 1773], Euplotes octocarinatus [Carter 1972], Tetrahymena pyriformis [Ehrenberg 1830] and Cyclidium glaucoma [Müller 1786]. Our experimental approach, using a combination of DAPI and FISH staining, was successful in allowing the observation of ingestion of specific bacteria and their detection within ciliate food vacuoles. However, the ciliates in this study showed no significant selective grazing. No food preferences for a any bacterial taxon or any size class or morphotype were detected. Correlation with time between ciliate abundance and bacterial abundance or biovolume, using log transformed growth rates of ciliates and bacteria, showed no significant results. On the bacterial side, neither an active defence mechanism of the nitrifying bacteria against ciliate grazing, such as changes in morphology, nor competition for resources were observed. These results suggest that in our in vitro-experiments grazing by ciliates has no influence on abundance and growth of nitrifying bacteria and nitrification.     

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.     

Polyspecific associations of <Emphasis Type=\"Italic\">Cercopithecus campbelli</Emphasis> and <Emphasis Type=\"Italic\">C. petaurista</Emphasis> with <Emphasis Type=\"Italic\">C. diana</Emphasis>: what are the costs and benefits?

Polyspecific associations (PSA) are common in many African primate communities, including the diurnal primates at Taï Forest, Côte d’Ivoire. In this paper I use data on the PSA of two forest guenons, Campbell’s (Cercopithecus campbelli) and lesser spot-nosed monkeys (C. petaurista), with Diana monkeys (C. diana) and other primates to clarify interspecific relationships during 17 months including a 3-month low-fruit period. I analyzed association in relation to fruit availability and measured forest strata use for C. campbelli and C. petaurista when alone and in associations with and without C. diana. I also measured predator risk and reactions to potential predators. C. campbelli and C. petaurista had high association rates with C. diana monkeys, and fruit availability did not influence association rates. C. campbelli and C. petaurista used higher strata when in association with C. diana than when alone, but they used even higher strata when associated with other primates without C. diana. This suggested that C. diana competitively exclude C. campbelli and C. petaurista from higher strata. There were relatively large numbers of potential predators, and C. diana were usually the first callers to threatening stimuli, suggesting that antipredator benefits of association with C. diana outweighed the competitive costs. C. campbelli spent more time in association with C. diana than C. petaurista did and appeared to be more reliant on C. diana for antipredator benefits. C. petaurista were less reliant on C. diana because of a cryptic strategy and may have associated less in some months because of high chimpanzee (Pan troglodytes) presence.