Oligosaccharide stores and internal core framework of LOS are proven.
This argues that in YeO3 the core oligosaccharide biosynthesis is not temperature-regulated.
oligosaccharide (LOS) biosynthesis loci ..
The bacterial lipid A biosynthesis protein, or lipid A biosynthesis (KDO)2-(lauroyl)-lipid IVA acyltransferase , transfers myristate or laurate, activated on ACP, to the lipid IVA moiety of (KDO)2-(lauroyl)-lipid IVA during lipopolysaccharide core biosynthesis .
This argues that in YeO3 the core oligosaccharide biosynthesis is not temperature-regulated.AB - The rfb region of Yersinia enterocolitica O:3 (YeO3) that determines the synthesis of the O-side chain of the lipopolysaccharide was cloned and expressed in Escherichia coli K12 previously.
Identification of lipo-oligosaccharide biosynthesis ..
Moreover by using a PCR-based screening of LOS gene content, ca. 80% of the LOS loci from more than 100 C. jejuni strains were assigned into these eight distinct locus classes (A through H) (). In the present study, we report the sequencing and characterization of 20 additional C. jejuni LOS outer core biosynthesis loci between waaM (Cj1134 in NCTC 11168) and waaV (Cj1146c in NCTC 11168), including 15 with an unknown LOS locus class. Based on gene content and organization, we identified 11 novel LOS locus classes, and each of these newly characterized LOS locus classes possessed regions with some similarity in gene content and organization, as observed in previously described classes (A through H), suggesting high levels of recombination within the locus (). In addition, we observed in these new classes many of the genetic mechanisms of variation previously observed with other C. jejuni LOS classes, including frameshift mutations and missense mutations. The importance of these findings with regard to the evolution of both recognition specificity and different LOS structures is also discussed.
The identification of the LOS biosynthesis locus in NCTC 11168 facilitated the cloning and characterization of LOS biosynthesis genes from other C. jejuni strains involved in the transfer of galactose, N-acetylgalactosamine, and sialic acid to the LOS outer core (, , ). The LOS biosynthesis region was identified as a hypervariable region within C. jejuni strains by whole-genome microarray analyses (, , , , , ), and sequencing of the LOS biosynthesis loci from several C. jejuni strains revealed differences in gene content and organization (, , , , ). In addition, comparisons of LOS structures and the corresponding DNA sequences of the LOS biosynthesis loci demonstrated that the structural diversity can be the consequence of either major or minor genetic differences at the LOS biosynthesis loci of the strains (). Eight LOS biosynthesis locus classes were defined previously based on major genetic differences, gene content, and organization. Three of these LOS locus classes—A, B, and C—encode genes responsible for the production of sialylated LOS that are ganglioside mimics (), while five other loci (D to H) lack a cst gene that encodes a sialic acid transferase (, , ). Sequence analysis also revealed minor genetic variation between C. jejuni strains that resulted in major LOS structural differences between strains that possessed the same LOS locus classes (, , , ). These minor genetic variations included (i) phase-variable homopolymeric tracts, (ii) gene inactivation by the deletion or insertion of a single base (without phase variation), (iii) missense or nonsense mutations leading to the inactivation of a glycosyltransferase, and (iv) single or multiple missense mutations leading to “allelic” glycosyltransferases (-, ).
jejuni LOS outer core biosynthesis ..
In the regions that differ from classes D and F, classes I, J, and S are structurally similar to each other in terms of gene order and gene content and provide evidence for a set of common insertion or deletion events. These classes diverge from classes D and F in that orf16 is deleted and replaced by five (class J) or six genes (classes I and S). Classes I and S contain six additional ORFs, and five of these (orf40, orf42, orf43, orf44, and orf45) show similarity to capsular biosynthesis genes from the HS:41 C. jejuni strain 176.83 (GenBank accession no. BX545857). The class J LOS locus also contains these five capsular biosynthesis ORFs. The orf40 encoding a glycosyltransferase has a putative length of 1,053 nt and shows 92% identity over a 643-nt span to HS41.29 from the HS:41 C. jejuni strain 176.83. The four additional HS:41-like ORFs (orf42, orf43, orf44, and orf45) encode a nucleotidyl-sugar pyranose mutase, a sugar epimerase, putative UDP-glucose 6-dehydrogenase, and nucleotidyl-sugar pyranose mutase, respectively. These genes are in the same order as the ORFs 41.24, 41.25, 41.26, and 41.27 from the Penner HS:41 capsular biosynthesis region of C. jejuni strain 176.83 (GenBank accession no. BX545857) (). Indeed, the 4,094-nt region spanning these four ORFs in classes I, J, and S shows 95% identity to the capsular region from the HS:41 strain 176.83, suggesting that the whole gene cassette (orf42, orf43, orf44, and orf45) was transferred together. Other than the HS:41 genes, the class I and S LOS loci possess a sixth ORF, orf41, that shows similarity at the amino acid level to a number of putative group 1 glycosyltransferases from other bacteria (pfam00535). Considering that the 5′ 279 nt of orf40j and orf41i(s) are 98% identical, it is likely that orf41 recombined into this region of orf40. Thus, it appears that an additional insertion event (orf41) gave rise to class S from class J. This six-gene cassette could then homologously recombine from class S to class D, giving rise to class I.
The region of the 81-176 chromosome containing the lgtF gene (CJJ81176_1152) has been described previously () (Table ). This open reading frame (ORF) encoded a predicted protein consisting of 515 amino acids with a predicted molecular mass of 61 kDa that showed 88% identity and 94% similarity to Cj1135 of C. jejuni NCTC 11168, which was annotated as a putative two-domain glycosyltransferase. In addition, the N-terminal amino acid sequence encoded by this ORF showed significant sequence similarity with the LgtF protein (33% identity and 54% similarity) of Haemophilus ducreyi. The lgtF gene of H. ducreyi has been shown to encode a β-1,4-glucosyltransferase involved in assembly of the inner core OS of LOS (). The C. jejuni 81-176 lgtF mutant was disrupted by insertional mutagenesis, as described in Materials and Methods. The positions and orientations of the random insertions of the cat gene within the clone were determined by sequence analysis. One insertion that occurred 757 bp from the translational start of lgtF in a nonpolar orientation was selected to generate C. jejuni mutants. Plasmid DNA from this cat insertion was used to electroporate C. jejuni 81-176 with selection for Cmr. Several Cmr transformants were confirmed by PCR in order to verify that the insert had integrated via a double crossover (data not shown).
Core oligosaccharide - Wikipedia
The core includes both the inner and outer core
Construction of this mutant confirmed the minimal LOS core biosynthesis gene content for bacterial viability ..
The O antigen is attached to the core oligosaccharide, ..
06/06/2015 · Characterization of lipooligosaccharide-biosynthetic loci of Campylobacter ..
ASMscience | Campylobacter jejuni Lip
acceptor on which the core oligosaccharide and LOS chains are assembled via ..
jejuni, the biosynthesis and genetic ..
Lipooligosaccharide (LOS) is the major constituent of the outer leaflet of the outer membranes of gram-negative bacteria such as Campylobacter jejuni. LOS consists of two covalently linked domains: (i) the lipid A moiety, a hydrophobic membrane anchor consisting of lipopolysaccharide, and (ii) a nonrepeating core oligosaccharide (OS) consisting of an inner core region and an outer core region. Based on structural studies, the Campylobacter inner core region of LOS has been shown to be highly conserved among C. jejuni serotypes (, ). This region consists of a single 3-deoxy--manno-octulosonic residue (Kdo) attached to the lipid A and two -glycero--manno-heptose (-Hep) residues attached to the Kdo. In addition, the inner core region of certain strains has been shown to contain a glucose residue and a phosphoethanolamine moiety attached to the first heptose residue. The outer core region of LOS of Campylobacter strains consists of various hexoses, N-acetylglucosamine, and N-acetylneuraminic acid (NeuNAc) (sialic acid), which have been shown to mimic human gangliosides (, , ). Sialylation of the LOS core of C. jejuni is thought to play a role in immune evasion and has been demonstrated to increase resistance to normal human serum (). However, this molecular mimicry can also result in an autoimmune response that can lead to Guillain-Barre syndrome, a serious postinfection paralysis (). It is believed that the Campylobacter core OS plays an important role in processes associated with pathogenesis of diarrheal disease, such as colonization and invasion of intestinal epithelial cells ().
Review: Lipopolysaccharide inner core oligosaccharide structure ..
We report isolation and characterization of Campylobacter jejuni 81-176 lgtF and galT lipooligosaccharide (LOS) core mutants. It has been suggested that the lgtF gene of C. jejuni encodes a two-domain glucosyltransferase that is responsible for the transfer of a β-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a β-1,2-glucose residue on Hep II. A site-specific mutation in the lgtF gene of C. jejuni 81-176 resulted in expression of a truncated LOS, and complementation of the mutant in trans restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a β-1,4-glucose on Hep I and a β-1,2-glucose on Hep II. Mutation of another gene, galT, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in C. jejuni 81-176, resulted in loss of the β-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core waaC mutant. These studies have important implications for the role of LOS in the pathogenesis of Campylobacter-mediated infection.
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