32 P]lipid IV A , at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mM CaCl 2 , which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mM CaCl 2 suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS. Lipopolysaccharide (LPS)1 is the major constituent of the outer leaflet of the outer membranes of Gram-negative bacteria (1-5). LPS consists of three covalently linked domains. These are: 1) the lipid A moiety, a glucosamine-based phospholipid that serves as the hydrophobic membrane anchor of LPS; 2) the core region, a nonrepeating oligosaccharide decorated with several phosphate-containing substituents; and 3) the O-antigen, a distal repeating oligosaccharide (Fig.

The polysaccharide capsule (CPS) of Campylobacter jejuni is the major serodeterminant of the Penner serotyping scheme. There are 47 Penner serotypes of C. jejuni, 22 of which fall into complexes of related serotypes. A multiplex PCR method for determination of capsule types of Campylobacter jejuni which is simpler and more affordable than classical Penner typing was developed. Primers specific for each capsule type were designed on the basis of a database of gene sequences from the variable capsule loci of 8 strains of major serotypes sequenced in this study and 10 published sequences of other serotypes. DNA sequence analysis revealed a mosaic nature of the capsule loci, suggesting reassortment of genes by horizontal transfer, and demonstrated a high degree of conservation of genes within Penner complexes. The multiplex PCR can distinguish 17 individual serotypes in two PCRs with sensitivities and specificities ranging from 90 to 100% using 244 strains of known Penner type.

Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first L-glycero-D-manno-heptose residue to 3-deoxy-D-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.

A waaF mutant of Campylobacter jejuni 81-176 showed decreased invasion of INT407 cells in vitro and increased sensitivity to some antibiotics compared to what was seen with the wild-type strain.

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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 ␤-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a ␤-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 ␤-1,4-glucose on Hep I and a ␤-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 ␤-(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.

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo 2 -[4- Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

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