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J Swings - One of the best experts on this subject based on the ideXlab platform.
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Vibrio hispanicus sp. nov., isolated from Artemia sp. and sea water in Spain.
International journal of systematic and evolutionary microbiology, 2004Co-Authors: B Gomez-gil, F L Thompson, C C Thompson, A Garcia-gasca, A Roque, J SwingsAbstract:Three Gram-negative, small, motile, rod-shaped bacteria were isolated from Artemia sp. and sea water in Barcelona, Spain, during 1990 and 1991. They were fermentative, oxidase-positive, sensitive to Vibriostatic agent O/129, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and grew in the absence of NaCl. They differed from phenotypically related species by their ability to grow at 4 degrees C and utilize L-rhamnose. Cloning of the 16S rRNA gene of the type strain produced two different 16S rRNA gene sequences, which differed by 15 bases (0.99%); comparison of these sequences with those deposited in GenBank showed close relationships with Vibrio proteolyticus (97.6% similarity), Vibrio diazotrophicus (97.9%), Vibrio campbellii (96.8%) and Vibrio alginolyticus (96.8%), among others. DNA-DNA hybridization levels with the closest phylogenetically related Vibrio species were
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Vibrio hispanicus sp. nov., isolated from Artemia sp. and sea water in Spain.
International Journal of Systematic and Evolutionary Microbiology, 2004Co-Authors: B Gomez-gil, F L Thompson, C C Thompson, A Garcia-gasca, A Roque, J SwingsAbstract:Three Gram-negative, small, motile, rod-shaped bacteria were isolated from Artemia sp. and sea water in Barcelona, Spain, during 1990 and 1991. They were fermentative, oxidase-positive, sensitive to Vibriostatic agent O/129, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and grew in the absence of NaCl. They differed from phenotypically related species by their ability to grow at 4 °C and utilize l-rhamnose. Cloning of the 16S rRNA gene of the type strain produced two different 16S rRNA gene sequences, which differed by 15 bases (0·99 %); comparison of these sequences with those deposited in GenBank showed close relationships with Vibrio proteolyticus (97·6 % similarity), Vibrio diazotrophicus (97·9 %), Vibrio campbellii (96·8 %) and Vibrio alginolyticus (96·8 %), among others. DNA–DNA hybridization levels with the closest phylogenetically related Vibrio species were
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Vibrio hispanicus sp nov isolated from artemia sp and sea water in spain
International Journal of Systematic and Evolutionary Microbiology, 2004Co-Authors: Bruno Gomezgil, F L Thompson, C C Thompson, A Roque, Alejandra Garciagasca, J SwingsAbstract:Three Gram-negative, small, motile, rod-shaped bacteria were isolated from Artemia sp. and sea water in Barcelona, Spain, during 1990 and 1991. They were fermentative, oxidase-positive, sensitive to Vibriostatic agent O/129, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and grew in the absence of NaCl. They differed from phenotypically related species by their ability to grow at 4 °C and utilize l-rhamnose. Cloning of the 16S rRNA gene of the type strain produced two different 16S rRNA gene sequences, which differed by 15 bases (0·99 %); comparison of these sequences with those deposited in GenBank showed close relationships with Vibrio proteolyticus (97·6 % similarity), Vibrio diazotrophicus (97·9 %), Vibrio campbellii (96·8 %) and Vibrio alginolyticus (96·8 %), among others. DNA–DNA hybridization levels with the closest phylogenetically related Vibrio species were <26·4 %. Sufficient evidence is provided to support the identity of the three strains analysed as members of a novel species of the genus Vibrio, for which the name Vibrio hispanicus sp. nov. is proposed, with the type strain LMG 13240T (=CAIM 525T=VIB 213T).
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Dominant intestinal microflora of sea bream and sea bass larvae, from two hatcheries, during larval development
Aquaculture, 1997Co-Authors: L Grisez, J Swings, J Reyniers, Ludo Verdonck, Frans OllevierAbstract:Copyright (c) 1997 Elsevier Science B.V. All rights reserved. The intestinal microflora of larval sea bream (Dicentrarchus labrax) and sea bass (Sparus aurata) from two marine hatcheries (Greece and Spain) was studied. Samples for bacteriological analysis were taken during feeding regimes of the larvae with rotifers and Artemia. A total of five production cycles was examined: three involved sea bream and two involved sea bass. When the larvae were fed with rotifers, the incidence of Vibrio anguillarum, Vibrio tubiashii and non-Vibrio groups was high. During feeding with Artemia, Vibrio alginolyticus, Vibrio proteolyticus, Vibrio harveyi and Vibrio natriegens were mainly isolated. During larval development, no dominant and persistent colonisation of the intestine by any given bacterial species was observed. Fluctuations in the composition of the dominant microflora appeared to reflect the bacterial composition of the ingested live feed. Selection towards the genus Vibrio was not observed until the larvae reached the end of the larval life stage. Two additional samples were taken during massive mortality outbreaks in bream larvae. In both samples, V. anguillarum was dominant as associated with the feeding with rotifers. The results suggest that disease outbreaks can occur when V. anguillarum dominates in the larval intestine. © 1997 Elsevier Science B.V.
Motomitsu Kitaoka - One of the best experts on this subject based on the ideXlab platform.
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A reducing-end-acting chitinase from Vibrio proteolyticus belonging to glycoside hydrolase family 19
Applied Microbiology and Biotechnology, 2008Co-Authors: Yuji Honda, Hajime Taniguchi, Motomitsu KitaokaAbstract:A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus ( chi19 ) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N -acetylated chitooligosaccharides, (GlcNAc)_ n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc)_ n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)_2 from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.
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chitobiose phosphorylase from Vibrio proteolyticus a member of glycosyl transferase family 36 has a clan gh l like alpha alpha 6 barrel fold
Structure, 2004Co-Authors: Masafumi Hidaka, Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi, Satoru Nirasawa, Takayoshi Wakagi, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into α-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO 4 . It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a β sandwich domain and an (α/α) 6 barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.
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Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.
Biochemical Journal, 2004Co-Authors: Yuji Honda, Motomitsu Kitaoka, Kiyoshi HayashiAbstract:A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.
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Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.
Structure, 2004Co-Authors: Masafumi Hidaka, Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi, Satoru Nirasawa, Takayoshi Wakagi, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into α-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO 4 . It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a β sandwich domain and an (α/α) 6 barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.
Peter Bossier - One of the best experts on this subject based on the ideXlab platform.
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Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against Vibriosis
Journal of Experimental Marine Biology and Ecology, 2009Co-Authors: Carlos R Rojas-garcía, Patrick Sorgeloos, Peter BossierAbstract:Abstract Three putative beneficial unicellular organisms, the marine bacterium Roseobacter sp, the yeast Saccharomyces cerevisiae mnn9 strain (SC-mnn9) and the microalga Tetraselmis suecica were cooked and offered separately as diets to developing germ-free (GF) Artemia larvae, in order to analyze their alimentary and protective effects. GF Artemia larvae were able to grow with cooked Tetraselmis and SC-mnn9 but failed with cooked Roseobacter. In spite of its high dietary quality, Tetraselmis failed to provide protection against Vibrio proteolyticus infection, while Roseobacter failed as food as well to provide protection. Cooked SC-mnn9 appeared to possess both values, dietary for growth and protective against Vibrio infection. GF Artemia larvae were apparently rapid adapted to dietary swaps; from yeast to algal and from algal to yeast. While the diets swap from algal or yeast, to bacterial diet appeared to be detrimental. Phenoloxidase-L (PO-L) and trypsin-L were used as biochemical indicators of defense and digestive functions, respectively. Developmental trypsin-L patterns were similar when fed on yeast and microalgae diets, suggesting a good digestive adaptation to plant or fungal substrates at early stages. On the contrary, diets swap or Vibrio infection affected PO-L and trypsin-L suggesting a sort of ‘alteration’ of digestive and defense functions.
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Cell wall deficient Saccharomyces cerevisiae strains as microbial diet for Artemia larvae: Protective effects against Vibriosis and participation of phenoloxidase
Journal of Experimental Marine Biology and Ecology, 2008Co-Authors: C.r. Rojas-garcia, Patrick Sorgeloos, A.f.m. Hasanuzzaman, Peter BossierAbstract:Thousands of Artemia larvae (1000 to 4800 ind bottles −1 ) were reared axenically with live and cooked cell wall deficient Saccharomyces cerevisiae (SC) strains as microbial diet to examine the protective effects against Vibriosis and the participation of phenoloxidase. Firstly, six different cell wall deficient SC strains (fks1, knr4, kre6, chs3, gas1 and mnn9) were compared for best larval growth and phenoloxidase-like (PO-L) responses after infection with Vibrio proteolyticus. Live SC-mnn9 and SC-gas1 provided the best (Pb0.05) dietary support for Artemia growth and induced higher PO-L (Pb0.05). Secondly, SC-mnn9, SC-gas1 and the SC-wild type (WT) were prepared as cooked microbial diet and were offered to germfree (GF) larvae. Growth, PO-L responses and survival after infection with Vibrio campbelii were higher (Pb0.05) in larvae fed with cooked SC-mnn9 and SC-gas1 compared to SC-WT. Cooked SC-WT was a poor diet for GF Artemia, nevertheless, a rapid diet swap to cooked SC-gas1 or SC mnn9 improved the PO-L and survival against V. campbelii which suggested that the protective effect of SC-mnn9 and SC-gas1 could also be effective in larvae that have been undernourished. Artemia's PO-L seemed to vary in accordance to growth status, health or disease caused by Vibriosis. This apparent capacity of Artemia to express PO-L under health and disease offers an interesting tool to explore its participation in the innate defense system of crustacean larval stages. © 2008 Published by Elsevier B.V.
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non lethal heat shock protects gnotobiotic artemia franciscana larvae against virulent Vibrios
Fish & Shellfish Immunology, 2007Co-Authors: Yeong Yik Sung, Patrick Sorgeloos, Els J M Van Damme, Peter BossierAbstract:Abstract Brine shrimp Artemia were exposed under gnotobiotic conditions to a non-lethal heat shock (NLHS) from 28 to 32, 37 and 40 °C. Different recovery periods (2, 6, 12 and 24 h) and different heat-exposure times (15, 30, 45 and 60 min) were tested. After these NLHS, Artemia was subsequently challenged with Vibrio. Challenge tests were performed in stressed and unstressed nauplii at concentrations of 107 cells ml−1 of pathogenic bacteria, Vibrio campbellii and Vibrio proteolyticus. A NLHS with an optimal treatment of 37 °C for 30 min and a subsequent 6 h recovery period resulted in a cross-protection against pathogenic Vibrio. A 100% increase in the larval survival (P
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Use of microalgae and bacteria to enhance protection of gnotobiotic Artemia against different pathogens
Aquaculture, 2006Co-Authors: António Marques, Patrick Sorgeloos, Toi Huynh Thanh, Peter BossierAbstract:The present study investigates the use of microalgae, probiotic and dead bacteria in gnotobiotic Artemia to overcome the virulence of two pathogenic bacterial strains: Vibrio campbellii and Vibrio proteolyticus. For that purpose, two strains of the microalga Dunaliella tertiolecta (a medium- and a good-quality microalga) and two beneficial bacteria, selected from previous well-performing Artemia cultures, were provided to the brine shrimp cultured under gnotobiotic conditions. The daily supplementation with D. tertiolecta conferred full protection to Artemia towards both Vibrios and was apparently more efficient and stable than the use of probiotics and dead bacteria. Only when Artemia were cultured in sub-optimal conditions with microalgae (but not when fed ad libitum), the addition of probiotic bacteria was able to partially protect the animals against both pathogens. The contribution of dead bacteria to the protection of Artemia against both pathogens was more pronounced in animals cultured with poor-quality feeds.
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Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens
Journal of Experimental Marine Biology and Ecology, 2006Co-Authors: António Marques, Patrick Sorgeloos, Willy Verstraete, Toi Huynh Thanh, Jean Dhont, Peter BossierAbstract:To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound β-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios.
George E. Fox - One of the best experts on this subject based on the ideXlab platform.
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GE: Defining 5S rRNA structure space: Point mutation data can be used to predict the phenotype of multi-change variants. Mol Biol Evol 2011. doi:10.1186/1471-2148-11-218 Cite this article as: Lu and Fox: Resurrection of an ancestral 5S rRNA. BMC Evol
2016Co-Authors: Madhavi Nayar, George E. FoxAbstract:A portion of the 5S ribosomal RNA (rRNA) structure space in the vicinity of the Vibrio proteolyticus 5S rRNA sequence is explored in detail with the intention of establishing principles that will allow a priori prediction of which sequences would be valid members of a particular RNA structure space. Four hundred and one sequence variants differing from the V. proteolyticus 5S rRNA wild-type sequence in 1–7 positions were characterized using an in vivo assay system. Most significantly, it was found that in general, the phenotypic effects of single changes were independent of the phenotypic effect of a second change. As a result, it was possible to use the new data in conjunction with results from prior studies of the same RNA to develop ‘‘truth tables’ ’ to predict which multiple change variants would be functional and which would be nonfunctional. The actual phenotype of 93.8 % of the multichange variants studied was consistent with the predictions made using truth tables thereby providing for perhaps the first time an upper limit estimate of how frequent unexpected interactions are. It was also observed that single changes at positions involved in secondary structure were no more likely to be invalid than changes in other regions. In particular, internal changes in long standard stems were in fact almost always tolerated. Changes at positions that were hypervariable in the context of an alignment of related sequences were, as expected, usually found to be valid. However, the potential validity of changes that were idiosyncratic to a single lineage of related sequences when placed in the V. proteolyticus 5S rRNA context was unpredictable. Key words: 5S rRNA, sequence space, mutagenesis, neutral network, adaptive landscape
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Defining 5S rRNA Structure Space: Point Mutation Data Can Be Used to Predict the Phenotype of Multichange Variants
Molecular Biology and Evolution, 2011Co-Authors: Madhavi Nayar, George E. FoxAbstract:A portion of the 5S ribosomal RNA (rRNA) structure space in the vicinity of the Vibrio proteolyticus 5S rRNA sequence is explored in detail with the intention of establishing principles that will allow a priori prediction of which sequences would be valid members of a particular RNA structure space. Four hundred and one sequence variants differing from the V. proteolyticus 5S rRNA wild-type sequence in 1–7 positions were characterized using an in vivo assay system. Most significantly, it was found that in general, the phenotypic effects of single changes were independent of the phenotypic effect of a second change. As a result, it was possible to use the new data in conjunction with results from prior studies of the same RNA to develop ‘‘truth tables’’ to predict which multiple change variants would be functional and which would be nonfunctional. The actual phenotype of 93.8% of the multichange variants studied was consistent with the predictions made using truth tables thereby providing for perhaps the first time an upper limit estimate of how frequent unexpected interactions are. It was also observed that single changes at positions involved in secondary structure were no more likely to be invalid than changes in other regions. In particular, internal changes in long standard stems were in fact almost always tolerated. Changes at positions that were hypervariable in the context of an alignment of related sequences were, as expected, usually found to be valid. However, the potential validity of changes that were idiosyncratic to a single lineage of related sequences when placed in the V. proteolyticus 5S rRNA context was unpredictable.
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DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli
BMC Biotechnology, 2010Co-Authors: Yamei Liu, Richard C. Willson, George W. Jackson, Victor G. Stepanov, Ulrich Strych, George E. FoxAbstract:Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli , the chimeric RNA (3× pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.
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Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green.
Journal of Molecular Recognition, 2009Co-Authors: Xing Zhang, George E. Fox, Ajish Potty, George W. Jackson, Victor G. Stepanov, Andrew Tang, Yamei Liu, Katerina Kourentzi, Ulrich Strych, Richard C. WillsonAbstract:In previous work, Vibrio proteolyticus 5S rRNA was shown to stabilize 13–50 nucleotide “guest” RNA sequences for expression in Escherichia coli. The expressed chimeric RNAs accumulated to high levels in E. coli without being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera produced in vitro by transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF “ribosomal RNA aptamer.” Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer libraries in vivo. Copyright © 2009 John Wiley & Sons, Ltd.
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Common 5S rRNA Variants Are Likely To Be Accepted in Many Sequence Contexts
Journal of Molecular Evolution, 2003Co-Authors: Zhengdong Zhang, Lisa M. D'souza, Youn-hyung M. Lee, George E. FoxAbstract:Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.
Yuji Honda - One of the best experts on this subject based on the ideXlab platform.
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A reducing-end-acting chitinase from Vibrio proteolyticus belonging to glycoside hydrolase family 19
Applied Microbiology and Biotechnology, 2008Co-Authors: Yuji Honda, Hajime Taniguchi, Motomitsu KitaokaAbstract:A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus ( chi19 ) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N -acetylated chitooligosaccharides, (GlcNAc)_ n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc)_ n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)_2 from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.
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chitobiose phosphorylase from Vibrio proteolyticus a member of glycosyl transferase family 36 has a clan gh l like alpha alpha 6 barrel fold
Structure, 2004Co-Authors: Masafumi Hidaka, Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi, Satoru Nirasawa, Takayoshi Wakagi, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into α-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO 4 . It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a β sandwich domain and an (α/α) 6 barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.
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Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.
Biochemical Journal, 2004Co-Authors: Yuji Honda, Motomitsu Kitaoka, Kiyoshi HayashiAbstract:A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.
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Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.
Structure, 2004Co-Authors: Masafumi Hidaka, Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi, Satoru Nirasawa, Takayoshi Wakagi, Hirofumi Shoun, Shinya FushinobuAbstract:Abstract Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into α-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO 4 . It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a β sandwich domain and an (α/α) 6 barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.
