Candidates of trichocyst matrix proteins of the dinoflagellate <Emphasis Type="Italic">Oxyrrhis marina</Emphasis>

Trichocysts are a common cell organelle of ciliates and dinoflagellates. They are composed of trichocyst matrix proteins and have been intensely investigated and characterized in ciliates. Here, for the first time, data have been obtained for trichocyst matrix proteins of a dinoflagellate. A DELTA-BLAST search using 14 available and complete amino acid sequences of mature trichocyst matrix proteins of the ciliate Paramecium tetraurelia resulted in 16 hits for the dinoflagellate Oxyrrhis marina when the E values and bit values to be scored were <10^?4 and >40. They code for proteins with acidic pI values and exceeded the precursors of the trichocyst matrix proteins of the ciliate approximately twofold in length. The values calculated for coverage, identity, and positives ranged from 76 to 100, 21.5 to 28.3, and 44.9 to 53.9%, respectively. Protein conformation predictions indicate coiled-coil domains which are a common feature of mature ciliate trichocyst matrix proteins. As often several EST sequences of O. marina matched with a queried mature trichocyst matrix protein of P. tetraurelia, a multigene family can be assumed for trichocyst proteins in this dinophyte, too. Trichocyst-enriched fractions of O. marina were isolated and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. When samples were incubated with loading buffer without a reducing agent, the banding pattern was mainly composed of three regions in the range of >90, 75–60, and 50–35 kDa, with each region consisting of four to five bands. Tryptic in gel digestion of proteins excised from these three gel regions followed by mass spectrometry confirmed that up to 14 of the 16 predicted proteins were present within the trichocyst-enriched fractions. When the samples were reduced with either ß-mercaptoethanol or dithiothreitol, the proteins of the three regions disappeared almost completely and proteins in the range of 27 to 15 kDa became the dominating bands. Up to 12 of the predicted proteins were detected within these bands.     

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved. Received 27 July 2005; Revisions requested 24 August 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Characterization of a metagenome-derived halotolerant cellulase

Metagenomes of uncultured microorganisms represent a sheer unlimited resource for discovery of novel biocatalysts. Here, we report on the biochemical characterisation of a novel, soil metagenome-derived cellulase (endoglucanase), Cel5A. The deduced amino acid sequence of Cel5A was similar to a family 5, single domain cellulase with no distinct cellulose binding domain from Cellvibrio mixtus. The 1092bp ORF encoding Cel5A was overexpressed in Escherichia coli and the corresponding 42.1 kDa protein purified using three-step chromatography. The recombinant Cel5A protein was highly active against soluble cellulose substrates containing beta-1,4 linkages, such as lichenan and barley beta-glucan, and not active against insoluble cellulose. Glucose was not among the initial hydrolysis products, indicating an endo mode of action. Cel5A displayed a wide range of pH activity with a maximum at pH 6.5 and at least 60% activity at pH 5.5 and 9.0. The enzyme was highly stable at 40 degrees C for up to 11 days, and retained 86-87% activity after incubation with 3M NaCl, 3M RbCl or 4M KCl for 20 h. Cel5A was also active in the presence of diverse divalent cations, detergents and EDTA. This highly stable, salt and pH tolerant cellulase is an ideal candidate for industrial applications.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA.     

Genomic analysis reveals <Emphasis Type="Italic">Lactobacillus sanfranciscensis</Emphasis> as stable element in traditional sourdoughs

Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis is the predominant key bacterium in traditionally fermented sourdoughs.The genome of L. sanfranciscensis TMW 1.1304 isolated from an industrial sourdough fermentation was sequenced with a combined Sanger/454-pyrosequencing approach followed by gap closing by walking on fosmids. The sequencing data revealed a circular chromosomal sequence of 1,298,316 bp and two additional plasmids, pLS1 and pLS2, with sizes of 58,739 bp and 18,715 bp, which are predicted to encode 1,437, 63 and 19 orfs, respectively. The overall GC content of the chromosome is 34.71%. Several specific features appear to contribute to the ability of L. sanfranciscensis to outcompete other bacteria in the fermentation. L. sanfranciscensis contains the smallest genome within the lactobacilli and the highest density of ribosomal RNA operons per Mbp genome among all known genomes of free-living bacteria, which is important for the rapid growth characteristics of the organism. A high frequency of gene inactivation and elimination indicates a process of reductive evolution. The biosynthetic capacity for amino acids scarcely availably in cereals and exopolysaccharides reveal the molecular basis for an autochtonous sourdough organism with potential for further exploitation in functional foods. The presence of two CRISPR/cas loci versus a high number of transposable elements suggests recalcitrance to gene intrusion and high intrinsic genome plasticity.     

Prospecting for novel biocatalysts in a soil metagenome

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.     

TAP score: torsion angle propensity normalization applied to local protein structure evaluation

Background  

Experimentally determined protein structures may contain errors and require validation. Conformational criteria based on the Ramachandran plot are mainly used to distinguish bet ween distorted and adequately refined models. While the readily available criteria are sufficient to detect totally wrong structures, establishing the more subtle differences between plausible structures remains more challenging.