In silico analysis of the regulatory region of the Yellowtail Kingfish and Zebrafish Kiss and Kiss receptor genes.

We have cloned and analysed the partial putative promoter sequences of the Yellowtail Kingfish (Seriola lalandi) Kiss2 and Kiss2r genes (380 and 420 bp, respectively). We obtained in silico 1.5 kb of the zebrafish (Danio rerio) Kiss1, Kiss2, Kiss1r and zfKiss2r sequences upstream of the putative transcriptional initiation site. Bioinformatic analysis revealed promoter regulatory elements including AP-1, Sp1, GR, ER, PR, AR, GATA-1, TTF-1, YY1 and C/EBP. These regulatory elements may mediate novel roles of the Kiss genes and their receptors in addition to their established role in reproductive function.

The Kiss2 receptor (Kiss2r) gene in Southern Bluefin Tuna, Thunnus maccoyii and in Yellowtail Kingfish, Seriola lalandi - functional analysis and isolation of transcript variants.

The kisspeptin system plays an essential role in reproductive function in vertebrates, particularly in the onset of puberty. We investigated the kisspeptin system in two Perciform teleosts, the Southern Bluefin Tuna (SBT; Thunnus maccoyii), and the Yellowtail Kingfish (YTK; Seriola lalandi), by characterising their kisspeptin 2 receptor (Kiss2r) genes. In addition to the full length Kiss2r cDNA sequences, we have isolated from SBT and YTK a transcript variant that retained an intron. We have further obtained three ytkKiss2r transcript variants that contained deletions. In vitro functional analysis of the full length SBT and YTK Kiss2r showed higher response to Kiss2-10 than to Kiss1-10, with stronger transduction via PKC than PKA. The full length ytkKiss2r and two deletion variants were differentially expressed in the brain of male, but not in female, juvenile YTK treated with increasing doses of Kiss2-10 peptide. In the gonads, the expression level of the ytkKiss2r transcripts did not vary significantly either in the male or female fish. This is the first time that transcript variants of the Kiss2r gene that contain deletions and show responsiveness to treatments with kisspeptin have been reported in any teleost.

Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragacea.

Sea anemones produce water-soluble toxins that have the ability to interact with cell membranes and form pores within them. The mechanism of pore formation is based on an initial binding step followed by oligomerization and membrane insertion. Although the final structure of the pore remains unclear, biochemical studies indicate that it consists of a tetramer with a functional radius of approximately 1.1 nm. Since four monomers seem to be insufficient to build a pore of this size, the currently accepted model suggests that lipids might also participate in its structure. In this work, the crystallization and preliminary crystallographic analysis of two crystal forms of fragaceatoxin C (FraC), a newly characterized actinoporin from Actinia fragacea, are described. The crystals diffracted up to 1.8 A resolution and the preliminary molecular-replacement solution supports an oligomeric structure of about 120 A in diameter.

A combined immunomagnetic separation and lateral flow method for a sensitive on-site detection of Bacillus anthracis spores--assessment in water and dairy products.

AIM: Combination of immunomagnetic separation (IMS) and lateral flow device (LFD) assays for the development of a sensitive, rapid, on-site methodology that enables concentration and detection of Bacillus anthracis spores in complex samples. METHODS AND RESULTS: The data presents the development of an optimized, 30 min, IMS assay, with about 95% capture of B. anthracis spores from different dairy products (n = 38). No cross reactivity was detected with typical milk flora and some closely related Bacilli. To enable direct application of the IMS captured spores on the LFD, spores were eluted from the bead-spore complex utilizing 95% (v/v) formamide-10 mmol l(-1) EDTA for 30 s in a microwave oven. Detached spores were analysed on LFD enabling detection within 10 min. The combined IMS-LFD methodology (40 min) demonstrates a 60-fold improvement in sensitivity, relative to samples that were applied directly on the LFD without the IMS concentrating step. CONCLUSIONS: The IMS-LFD method is a powerful platform, combining rapidity, specificity and efficiency for concentrating and detecting B. anthracis from water and milk contaminated samples. SIGNIFICANT AND IMPACT OF THE STUDY: The combination of IMS and LFD enhances the sensitivity and flexibility of B. anthracis spore detection from complex samples. This method can potentially be extended to other toxins and micro-organisms in a variety of matrices.

A plate holder for non-destructive testing of mesophase crystallization assays.

Here, we describe a goniometer holder to mount standard 96-well crystallization plates directly onto the goniometer head of an oscillation camera. This attachment was designed to check crystallization conditions straight from the crystallization plates under X-rays, and was proven to be useful for checking small crystals and solutions that destabilize monoolein-based lipidic cubic phase (LCP) crystallization experiments. A quick procedure for setting up LCP assays employing commercially available instruments is also reported.

Development and implementation of a single-chain Fv antibody for specific detection of Bacillus anthracis spores.

A single-chain Fv (scFv) antibody was developed and applied for efficient and specific detection of Bacillus anthracis spores. The antibody was isolated from a phage display library prepared from spleens of mice immunized with a water-soluble extract of the outer membrane of the B. anthracis spore (exosporium). The library (7 x 10(6) PFU) was biopanned against live, native B. anthracis ATCC delta14185 spores suspended in solution, resulting in the isolation of a unique soluble scFv antibody. The antibody was affinity purified and its affinity constant (3 x 10(8) +/- 1 x 10(8) M(-1)) determined via flow cytometry (FCM). Preliminary characterization of scFv specificity indicated that the scFv antibody does not cross-react with representatives of some phylogenetically related Bacillus spores. The potential use of scFv antibodies in detection platforms was demonstrated by the successful application of the soluble purified scFv antibody in enzyme-linked immunosorbent assays, immunofluorescence assays, and FCM.

Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase.

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.

Design and production of active cellulosome chimeras. Selective incorporation of dockerin-containing enzymes into defined functional complexes.

Defined chimeric cellulosomes were produced in which selected enzymes were incorporated in specific locations within a multicomponent complex. The molecular building blocks of this approach are based on complementary protein modules from the cellulosomes of two clostridia, Clostridium thermocellum and Clostridium cellulolyticum, wherein cellulolytic enzymes are incorporated into the complexes by means of high-affinity species-specific cohesin-dockerin interactions. To construct the desired complexes, a series of chimeric scaffoldins was prepared by recombinant means. The scaffoldin chimeras were designed to include two cohesin modules from the different species, optionally connected to a cellulose-binding domain. The two divergent cohesins exhibited distinct specificities such that each recognized selectively and bound strongly to its dockerin counterpart. Using this strategy, appropriate dockerin-containing enzymes could be assembled precisely and by design into a desired complex. Compared with the mixture of free cellulases, the resultant cellulosome chimeras exhibited enhanced synergistic action on crystalline cellulose.

Cohesin-dockerin interaction in cellulosome assembly: a single hydroxyl group of a dockerin domain distinguishes between nonrecognition and high affinity recognition.

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of this high affinity interaction, although additional residues were also implicated. In the present communication, we examine further the contributing factors for the recognition of a dockerin by a cohesin domain between the respective cellulosomal systems of Clostridium thermocellum and Clostridium cellulolyticum. In this context, the four confirmed residues were analyzed for their individual effect on selectivity. In addition, other dockerin residues were discerned that could conceivably contribute to the interaction, and the suspected residues were similarly modified by site-directed mutagenesis. The results indicate that mutation of a single residue from threonine to leucine at a given position of the C. thermocellum dockerin differentiates between its nonrecognition and high affinity recognition (K(a) approximately 10(9) m(-1)) by a cohesin from C. cellulolyticum. This suggests that the presence or absence of a single decisive hydroxyl group is critical to the observed biorecognition. This study further implicates additional residues as secondary determinants in the specificity of interaction, because interconversion of selected residues reduced intraspecies self-recognition by at least three orders of magnitude. Nevertheless, as the latter mutageneses served to reduce but not annul the cohesin-dockerin interaction within this species, it follows that other subtle alterations play a comparatively minor role in the recognition between these two modules.

Overproduction and characterization of seleno-methionine xylanase T-6.

The extracellular xylanase from Bacillus stearothermophilus T-6 is a thermostable alkaline tolerant enzyme that was found to bleach pulp optimally at pH 9 and 65 degrees C, and was successfully used in a large-scale bio-bleaching mill trial. In an attempt to obtain a heavy atom derivative suitable for complete X-ray analysis, xylanase T-6 was labeled biosynthetically with seleno-methionine, resulting in a 'built-in' array of atoms with specific X-ray anomalous scattering signal. Optimization of growth conditions resulted in over 0.8 g of homogeneous seleno-methionine xylanase T-6 per liter culture. The seleno-methionine enzyme was shown to be fully active and produced single crystals suitable for complete multiple wavelength anomalous diffraction (MAD) structural analysis.

Cohesin-dockerin recognition in cellulosome assembly: experiment versus hypothesis.

The cohesin-dockerin interaction provides the basis for incorporation of the individual enzymatic subunits into the cellulosome complex. In a previous article (Pagés et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the approximately 70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was examined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohesin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional residues may also contribute to the specificity of the interaction. Proteins 2000;39:170-177.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6.

Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6. Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 degrees C and was used successfully on an industrial-scale mill trial. To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli. The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized. The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step. Over 2 g soluble and active enzyme per 1 culture were achieved.