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Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtpl, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzymesubstrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtpl and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound. doi: 10.1128/JB.00561-10

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.

O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-D-glucose (D-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-D-Qui4N and dTDP-D-Qui4Nac (the nucleotide-activated precursors of D-Qui4NGlyAc and D-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-D-Qui4N is synthesized from [alpha]-D-glucose-l-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-D-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-D-Qui4N into dTDP-D-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (Vio[A.sub.D7]) and E. coil O7 (Vio[A.sub.O7]) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-D-Qui4N and dTDP-D-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development.

Comparison of the O antigens of Shigella boydii types 10 and 6 by chemical analysis and nuclear magnetic resonance spectroscopy showed that their structures are similar, with the only difference being the presence or absence of D-ribofuranose, which is the immunodominant sugar in S. boydii type 10. In S. boydii type 6, a residue previously reported as [alpha]-D-GlcpA, was shown to be [beta]-D-GlcpA as in S. boydii type 10. S. boydii types 10 and 6 are reported not to cross-react serologically, and the role of D-ribofuranose in the specificity of S. boydii was confirmed by making a mutant of type 10 that lacked D-ribofuranose. However, S. boydii type 11, which has a D-ribofuranose but with different linkage does show cross-reaction with type 10. The O-antigen gene loci of S. boydii types 10 and 6 were shown to be virtually identical except that orf8 (wbaM), which was confirmed as the ribofuranosyltransferase gene, is interrupted by IS629 in type 6. Therefore, it is proposed that the O-antigen gene cluster of S. boydii type 6 was derived from type 10 by an IS element insertion.

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-n-glucose (GlcNAc), 2-acetamidoylaminn-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Sahnonella enterica serovar Touera and S. enterica subsp, arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes.

The structural and genetic organization of the Escherichia coli O52 O antigen was studied. As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E. coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-manno. heptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster of E. coli O52, which is located between the galF and grid genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coil Genes specific for E. coli O52 were also identified.

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coil constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coil and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gramnegative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected [sup.1]H, [sup.13]C and [sup.1]H, [sup.31]P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters.

Research has been conducted on lipopolysaccharide isolated from serum-sensitive Escherichia coli strain Nissle 1917. The structural analysis of this lipopolysaccharide has demonstrated that it contains a bisphosphorylated hexaacyl lipid A and a tetrasaccharide and that lipopolysaccharide is important for E. coli serum sensitivity and resistance.

Serological and immunochemical studies were conducted on lipopolysaccharides containing known O polysaccharides using monoclonal antibodies. Results from screening 800 strains representing 56 known Pseudomonas syringae pathovars indicated that strains of 15 pathovars belonged to serogroup O1 with majority showing the linear O polysaccharide structure.

Murine monoclonal antibodies reacting with Pseudomonas syringae lipopolysaccharide O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone were produced and utilized for immunochemical analysis and for serological classification of the bacteria. A total of 195 of 358 P. syringae strains tested were found to have a common epitope, 1a, and were classified into serogroup O1. Serotypes O2a and O2d were seen in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain.

Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-L-RhapIII-(1→2)- α-L-RhapII-(1→3)-α-L- RhapI-(1→3)-β-D-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on RhaI or/and RhaIII, and phosphorylation with phosphoethanolamine on RhaII or/ and RhaIII. Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SflI, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SflI prophage with a dysfunctional gtr locus for type II glucosylation. The 6-Oacetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri. [ABSTRACT FROM AUTHOR]

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-L-RhapIII-(1→2)-α-L-RhapII-(1→3)-α-L-RhapI-(1→3)-β-D-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on RhaI, and phosphorylation with phosphoethanolamine on RhaII or/and RhaIII. Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of RhaIII and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on RhaIII was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on RhaIII, revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants. [ABSTRACT FROM AUTHOR]

Characterizes a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide. Complementation of strain CS332; Localization and nucleotide sequence of lag-1 gene; Distribution of the lag-1 locus.

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol. [ABSTRACT FROM AUTHOR]

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzymesubstrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli O145 strains are emerging as causes of hemorrhagic colitis and hemolytic uremic syndrome. In this study, we present the structure of the E. coli O145 O antigen and the sequence of its gene cluster. The O145 antigen has repeat units containing three monosaccharide residues: 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamidoylamino-2,6-dideoxy-L-galactose, and N-acetylneuraminic acid. It is very closely related to Salmonella enterica serovar Touera and S. enterica subsp. arizonae O21 antigen. The E. coli O145 gene cluster is located between the JUMPStart sequence and the gnd gene and consists of 15 open reading frames. Putative genes for the synthesis of the O-antigen constituents, for sugar transferase, and for O-antigen processing were annotated based on sequence similarities and the presence of conserved regions. The putative genes located in the E. coli O145 O-antigen gene cluster accounted for all functions expected for synthesis of the structure. An E. coli O145 serogroup-specific PCR assay based on the genes wzx and wzy was also developed by screening E. coli and Shigella isolates of different serotypes. [ABSTRACT FROM AUTHOR]

The structural and genetic organization of the Escherichia coli 052 O antigen was studied. As identified ny sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen or E. coli 052 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy. D-mannoheptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster orE. coli 052, which is located between the galF and grid genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that 052 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coli. Genes specific for E. coil 052 were also identified. [ABSTRACT FROM AUTHOR]

Shigella is an important human pathogen. It is generally agreed that Shigella and Escherichia coli constitute a single species; the only exception is Shigella boydii type 13, which is more distantly related to E. coli and other Shigella forms and seems to represent another species. This gives S. boydii type 13 an important status in evolution. O antigen is the polysaccharide part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and plays an important role in pathogenicity. The chemical structure and genetic organization of the S. boydii type 13 O antigen were investigated. The O polysaccharide was found to be acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. The structure of the linear pentasaccharide phosphate repeating unit (O unit) was established by nuclear magnetic resonance spectroscopy, including two-dimensional COSY, TOCSY, ROESY, and H-detected ¹H,[sup 13]C and ¹H,[sup 31]P HMQC experiments, along with chemical methods. The O antigen gene cluster of S. boydii type 13 was located and sequenced. Genes for synthesis of UDP-2-acetamido-2,6-dideoxy-L-glucose and genes that encode putative sugar transferases, O unit flippase, and O antigen polymerase were identified. Seven genes were found to be specific to S. boydii type 13. The S. boydii type 13 O antigen gene cluster has higher levels of sequence similarity with Vibrio cholerae gene clusters and may be evolutionarily related to these gene clusters. [ABSTRACT FROM AUTHOR]

O-antigen variation due to the presence of different types of sugars and sugar linkages is important for the survival of bacteria threatened by host immune systems. The O antigens of Shigella dysenteriae type 7 and Escherichia coli O7 contain 4-(N-acetylglycyl)amino-4,6-dideoxy-D-glucose (D-Qui4NGlyAc) and 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), respectively, which are sugars not often found in studied polysaccharides. In this study, we characterized the biosynthetic pathways for dTDP-D-Qui4N and dTDP-D-Qui4NAc (the nucleotide-activated precursors of D-Qui4NGlyAc and D-Qui4NAc in O antigens). Predicted genes involved in the synthesis of the two sugars were cloned, and the gene products were overexpressed and purified as His-tagged fusion proteins. In vitro enzymatic reactions were carried out using the purified proteins, and the reaction products were analyzed by capillary electrophoresis, electrospray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. It is shown that in S. dysenteriae type 7 and E. coli O7, dTDP-D-Qui4N is synthesized from α-D-glucose-1-phosphate in three reaction steps catalyzed by glucose-1-phosphate thymidyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), and dTDP-4-keto-6-deoxy-D-glucose aminotransferase (VioA). An additional acetyltransferase (VioB) catalyzes the conversion of dTDP-D-Qui4N into dTDP-D-Qui4NAc in E. coli O7. Kinetic parameters and some other properties of VioA and VioB are described and differences between VioA proteins from S. dysenteriae type 7 (VioAD7) and E. coli O7 (VioAO7) discussed. To our knowledge, this is the first time that functions of VioA and VioB have been biochemically characterized. This study provides valuable enzyme sources for the production of dTDP-D-Qui4N and dTDP-D-Qui4NAc, which are potentially useful in the pharmaceutical industry for drug development. [ABSTRACT FROM AUTHOR]

Shigella flexneri O-antigen is an important and highly variable cell component presented on the outer leaflet of the outer membrane. Most Shigella flexneri bacteria share an O-antigen backbone composed of →2)-α-L-Rhap(III)-(1→2)-α-L-Rhap(II)-(1→3)-α-L-Rhap(I)-(1→3)-β-D-GlcpNAc-(1→ repeats, which can be modified by adding various chemical groups to different sugars, giving rise to diverse O-antigen structures and, correspondingly, to various serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha(I) or/and Rha(III), and phosphorylation with phosphoethanolamine on Rha(II) or/and Rha(III). Recently, a new O-antigen modification, namely, O-acetylation at position 6 of N-acetylglucosamine (GlcNAc), has been identified in S. flexneri serotypes 2a, 3a, Y, and Yv. In this study, the genetic basis of the 6-O-acetylation of GlcNAc in S. flexneri was elucidated. An O-acyltransferase gene designated oacD was found to be responsible for this modification. The oacD gene is carried on serotype-converting bacteriophage SfII, which is integrated into the host chromosome by lysogeny to form a prophage responsible for the evolvement of serotype 2 of S. flexneri. The OacD-mediated 6-O-acetylation also occurs in some other S. flexneri serotypes that carry a cryptic SfII prophage with a dysfunctional gtr locus for type II glucosylation. The 6-O-acetylation on GlcNAc confers to the host a novel O-antigen epitope, provisionally named O-factor 10. These findings enhance our understanding of the mechanisms of the O-antigen variation and enable further studies to understand the contribution of the O-acetylation to the antigenicity and pathogenicity of S. flexneri., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)