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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan l-substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane-spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.
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  • 2
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: dTDP-D-glucose 4,6-dehydratase (RmlB) is the second of four enzymes involved in the dTDP-L-rhamnose pathway and catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The ultimate product of the pathway, dTDP-L-rhamnose, is the precursor of L-rhamnose, which is a key component of the cell wall of many pathogenic bacteria. RmlB from Salmonella enterica serovar Typhimurium has been overexpressed and purified, and crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with lithium sulfate as precipitant. Diffraction data have been obtained to a resolution of 2.8 Å on a single frozen RmlB crystal which belongs to space group P21, with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 Å, β = 131.53°. The asymmetric unit contains four monomers in the form of two RmlB dimers with a solvent content of 62%. A molecular-replacement solution has been obtained and the model is currently being refined.
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  • 3
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from α-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.
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  • 4
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7–56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filaments surface. Similarly, mAb 1–5.1 recognised an internal epitope shared only by serotypes H1 and H2. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating unitsOAc12/6→3)-β-d-Galf-(1 →3)-α- d-Galp-(1→d-Galactan I-OAc→3)-α-d-Galp-(1 →3)-β-d-Galp-(1→d-Galactan II. K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only d-galactan I-OAc. The 1H- and 13C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on d-galactan I and O-acetylation occurs only on the β- d-galactofuranose residues; 60% of the available β- d-galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to d-galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of d-galactan I. rfbKpo1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce d-galactan I. Six O1 strains were examined and all showed hybridization with rfbKpO1 probes under conditions of high stringency. Three serotype O2 strains produce d-galactan I and these strains also contained DNA sequences recognized by rfbKpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfbKpO8 region from K. pneumoniae serotype O8 was only recognized by rfbKpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.
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  • 6
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and ...
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  • 7
    ISSN: 1399-0047
    Source: Crystallography Journals Online : IUCR Backfile Archive 1948-2001
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP-L-rhamnose, is synthesized from α-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 Å. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 Å and α = β = 90, γ = 120°.
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic ( cps ) operons. Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart–RfaH antitermination mechanism, with the cps promoter being localized to a region immediately upstream of the JUMPStart sequence. A putative stem–loop structure located within the K30 cps cluster separates conserved genes with products that are required for surface expression of capsule from serotype-specific genes encoding enzymes for polymer repeat-unit synthesis and polymerization. This putative stem–loop structure significantly reduces transcription in a termination-probe vector and may contribute to differential expression of the cps genes. Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs ( r egulator of c apsule s ynthesis) proteins. However, neither overexpression of the transcriptional activator RcsB nor an rcsB :: aadA chromosomal insertion altered the level of transcription measured by cps::lacZ fusions. In the group 1 strains examined, an RcsAB box was found immediately upstream of galF , a gene involved in the production of sugar nucleotide precursors. Overexpression of RcsB was found to result in a threefold increase in transcription of a galF::lacZ chromosomal fusion. Moreover, overexpression of GalF gave rise to a two- to threefold increase in cell-free as well as cell-associated capsule, without affecting cps::lacZ activity. These results indicate that transcription of the E. coli group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive. However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing galF transcription and influencing the available pool of biosynthetic precursors.
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  • 9
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The waa locus on the chromosome of Salmonella enterica encodes enzymes involved in the assembly of the core oligosaccharide region of the lipopolysaccharide (LPS) molecule. To date, there are two known core structures in Salmonella, represented by serovars Typhimurium (subspecies I) and Arizonae (subspecies IIIA). The waa locus for serovar Typhimurium has been characterized. Here, the corresponding locus from serovar Arizonae is described, and the molecular basis for the distinctive structures is established. Eleven of the 13 open reading frames (ORFs) are shared by the two loci and encode conserved proteins of known function. Two polymorphic regions distinguish the waa loci. One involves the waaK gene, the product of which adds a terminal α-1,2-linked N-acetylglucosamine residue that characterizes the serovar Typhimurium core oligosaccharide. There is an extensive internal deletion within waaK of serovar Arizonae. The serovar Arizonae locus contains a novel ORF (waaH) between the waaB and waaP genes. Structural analyses and in vitro glycosyltransferase assays identified WaaH as the UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase responsible for the addition of the characteristic terminal glucose residue found in serovar Arizonae. Isolates comprising the Salmonella Reference Collections, SARC (representing the eight subspecies of S. enterica) and SARB (representing subspecies I), were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies identified additional waa locus polymorphisms, shedding light on the genetic basis for diversity in the LPS core oligosaccharides of Salmonella isolates and identifying potential sources of further novel LPS structures.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Many Escherichia coli strains are covered in a layer of surface-associated polysaccharide called the capsule. Capsular polysaccharides represent a major surface antigen, the K antigen, and more than 80 distinct K serotypes result from structural diversity in these polymers. However, not all capsules consist of K antigen. Some are due to production of an extensive layer of a polymer structurally identical to a lipopolysaccharide O antigen, but distinguished from lipopolysaccharide by the absence of terminal lipid A-core. Recent research has provided insight into the manner in which capsules are organized on the Gram-negative cell surface, the pathways used for their assembly, and the regulatory processes used to control their expression. A limited repertoire of capsule expression systems are available, despite the fact that the producing bacteria occupy a variety of ecological niches and possess diverse physiologies. All of the known capsule assembly systems seen in Gram-negative bacteria are represented in E. coli, as are the majority of the regulatory strategies. Escherichia coli therefore provides a variety of working models on which studies in other bacteria are (or can be) based. In this review, we present an overview of the current molecular and biochemical models for capsule expression in E. coli. By taking into account the organization of capsule gene clusters, details of the assembly pathway, and regulatory features that dictate capsule expression, we provide a new classification system that separates the known capsules of E. coli into four distinct groups.
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