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Paul Messner - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus
Journal of Biological Chemistry, 2008Co-Authors: Kerstin Steiner, Rene Novotny, Christina Schaffer, Andreas Hofinger, Daniel B Werz, Kristof Zarschler, Peter H Seeberger, Paul Kosma, Paul MessnerAbstract:The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.
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novel biocatalysts based on s layer self assembly of Geobacillus stearothermophilus nrs 2004 3a a nanobiotechnological approach
Small, 2007Co-Authors: Christina Schaffer, Rene Novotny, Uwe B Sleytr, Seta Kupcu, Sonja Zayni, Andrea Scheberl, Jacqueline Friedmann, Paul MessnerAbstract:The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.
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functional characterization of the initiation enzyme of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus nrs 2004 3a
Journal of Bacteriology, 2007Co-Authors: Kerstin Steiner, Rene Novotny, Kinnari B Patel, Evgenij Vinogradov, Chris Whitfield, Miguel A Valvano, Paul Messner, Christina SchafferAbstract:The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.
Christina Schaffer - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus
Journal of Biological Chemistry, 2008Co-Authors: Kerstin Steiner, Rene Novotny, Christina Schaffer, Andreas Hofinger, Daniel B Werz, Kristof Zarschler, Peter H Seeberger, Paul Kosma, Paul MessnerAbstract:The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.
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novel biocatalysts based on s layer self assembly of Geobacillus stearothermophilus nrs 2004 3a a nanobiotechnological approach
Small, 2007Co-Authors: Christina Schaffer, Rene Novotny, Uwe B Sleytr, Seta Kupcu, Sonja Zayni, Andrea Scheberl, Jacqueline Friedmann, Paul MessnerAbstract:The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.
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functional characterization of the initiation enzyme of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus nrs 2004 3a
Journal of Bacteriology, 2007Co-Authors: Kerstin Steiner, Rene Novotny, Kinnari B Patel, Evgenij Vinogradov, Chris Whitfield, Miguel A Valvano, Paul Messner, Christina SchafferAbstract:The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.
Makari Yamasaki - One of the best experts on this subject based on the ideXlab platform.
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inactivation of food poisoning bacteria and Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment
Food Control, 2009Co-Authors: Soichi Furukawa, Taisuke Watanabe, Junichi Hirata, Tetsuya Koyama, Hirokazu Ogihara, Naoki Narisawa, Makari YamasakiAbstract:Inactivation of the vegetative foodborne pathogens by high pressure carbon dioxide treatment (HPCT) was investigated. Pseudomonas aeruginosa IFO3445, Aeromonas hydrophila IFO13286, Salmonella enteritidis JCM3313, Salmonella typhimurium IID1000, Yersinia enterocolitica JCM1677, Escherichia coli O157:H7 ATCC35150, E. coli O157:H7 ATCC43889, Staphylococcus aureus FDA209P and Listeria monocytogenes 1/2a were used in this study. In all cases, HPCT at 35 °C, 10 MPa for 1 min reduced initial numbers by approximately 6 logs. In addition HPCT inactivation of 9 pathogenic strains of Bacillus cereus (mixture of spores and vegetative cells) was investigated. HPCT at 75 °C, 10 MPa for 120 min resulted in significant inactivation in all the strains. The effect of HPCT (30 MPa at 95 °C for up to 120 min) on the inactivation of Geobacillus stearothermophilus spores in the presence of sodium chloride (3% and 6% w/v), glucose (6% and 12% w/v) and ethanol (10% and 20% v/v) was also studied. Both sodium chloride and glucose had a protective effect and the level of inactivation was reduced. The effect was in proportion to the solute concentration. However, 10% and 20% ethanol did not significantly affect the level of inactivation.
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inactivation of Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment
Applied and Environmental Microbiology, 2003Co-Authors: Taisuke Watanabe, Junichi Hirata, Tetsuya Koyama, Soichi Furukawa, Hirokazu Ogihara, Makari YamasakiAbstract:High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95°C was more effective than treatment at 95°C alone.
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inactivation of Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment
Applied and Environmental Microbiology, 2003Co-Authors: Taisuke Watanabe, Junichi Hirata, Tetsuya Koyama, Soichi Furukawa, Hirokazu Ogihara, Makari YamasakiAbstract:High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.
Rene Novotny - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus
Journal of Biological Chemistry, 2008Co-Authors: Kerstin Steiner, Rene Novotny, Christina Schaffer, Andreas Hofinger, Daniel B Werz, Kristof Zarschler, Peter H Seeberger, Paul Kosma, Paul MessnerAbstract:The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.
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novel biocatalysts based on s layer self assembly of Geobacillus stearothermophilus nrs 2004 3a a nanobiotechnological approach
Small, 2007Co-Authors: Christina Schaffer, Rene Novotny, Uwe B Sleytr, Seta Kupcu, Sonja Zayni, Andrea Scheberl, Jacqueline Friedmann, Paul MessnerAbstract:The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.
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functional characterization of the initiation enzyme of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus nrs 2004 3a
Journal of Bacteriology, 2007Co-Authors: Kerstin Steiner, Rene Novotny, Kinnari B Patel, Evgenij Vinogradov, Chris Whitfield, Miguel A Valvano, Paul Messner, Christina SchafferAbstract:The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.
Kerstin Steiner - One of the best experts on this subject based on the ideXlab platform.
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molecular basis of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus
Journal of Biological Chemistry, 2008Co-Authors: Kerstin Steiner, Rene Novotny, Christina Schaffer, Andreas Hofinger, Daniel B Werz, Kristof Zarschler, Peter H Seeberger, Paul Kosma, Paul MessnerAbstract:The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.
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functional characterization of the initiation enzyme of s layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus nrs 2004 3a
Journal of Bacteriology, 2007Co-Authors: Kerstin Steiner, Rene Novotny, Kinnari B Patel, Evgenij Vinogradov, Chris Whitfield, Miguel A Valvano, Paul Messner, Christina SchafferAbstract:The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.