Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitiveLipopolysaccharide (LPS) is a key component of the outer membrane of gram-negative bacteria. It is comprised of three distinct regions: lipid A, the oligosaccharide core, and commonly a long-chain polysaccharide O antigen that causes a smooth phenotype. Lipid A is the most conserved part of LPS. It is connected to the core part, which links it to the O repeating units. In Escherichia coli, five different core structures (K-12 and R1 to R4) have been described (2, 18, 43). The O repeating units are highly polymorphic, and more than 190 serologically distinguished forms in E. coli are known today (35).The LPS core-encoding genes are located at a conserved position on the E. coli K-12 chromosomal map (81 to 82 min) (5). The wa‫ء‬ (formerly called rfa) gene clusters contain the genes which code for the enzymes required for the core assembly and consist of three operons (defined by the first genes in the operons; gmhD, waaQ, and waaA). Although the O-unitencoding gene cluster is extremely polymorphic in the E. coli species, it is localized at a conserved position on the E. coli K-12 chromosome between the galF and gnd genes (45.4 min) (5). These determinants consist of several sugar transferase-, epimerase-, and isomerase-encoding genes, the O-antigen flippase gene (wzx), the O-antigen polymerase gene (wzy [formerly called rfc]), as well as the genes coding for enzymes involved in carbohydrate biosynthesis pathways. Until now, several E. coli O-antigen-encoding gene clusters have been studied, e.g., those of serotypes O7, O111, O113, and O157 (32, 38, 54, 55). These gene clusters show no significant nucleotide homology with each other, with the exception of some common genes such as manC and manB. However, they contain a conserved range of predicted enzyme activities. The O6 antigen is widely distributed among pathogenic and nonpathogenic fecal E. coli isolates and is often found in uropathogenic E. coli strains. It is associated with R1-type core LPS and has not been investigated in detail so far.E. coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) is a nonpathogenic fecal isolate, which is used as a probiotic agent in medicine (7), mainly for treatment of various gastroenterological indications (23,26,30,33,41). This strain exhibits a serum-sensitive, semirough phenotype. Since colonies of this strain on agar plates show a special smooth-and-rough

Borrelia burgdorferi sensu lato is the causing agent of Lyme disease, an infectious disease frequently occurring in the United States, Europe, and Northern Asia. Currently, diagnosis of and vaccination strategies against this pathogen are exclusively based on proteinaceous structures. Here we report on a novel class of immunogenic glycolipids purified from B. burgdorferi sensu stricto B31. Employing a butanol/water extraction procedure with subsequent Bligh/Dyer extraction of the organic phase, thin layer chromatography analysis revealed the presence of three distinct glycolipids, which were chemically analyzed employing combined gas-liquid chromatography/mass spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and NMR. We identified acylated cholesteryl galactoside (ACG) next to cholesteryl galactoside and ␣-monogalactosyl-diacylglycerol. After extensive purification, the glycolipids investigated failed to cause proinflammatory responses in human cells transfected with human toll-like receptor (TLR)-2 or -4. However, we observed a marked recognition of ACG by sera derived from patients suffering from Lyme disease. These data indicate that newly described ACG is involved in developing host immunity during Lyme disease and thus may be useful for diagnosis and vaccination.

Background: Rough-type LPS of C. canimorsus is biologically active, whereas lipid A is not. Results: Purified C. canimorsus rough-type LPS could be analyzed in intact form by NMR. Conclusion: Inactive lipid A becomes active in case the core oligosaccharide is attached. Significance: This study provides evidence that lipid A is not the sole part of the LPS structure responsible for endotoxic activity.

Despite its importance for membrane stability and pathogenicity of mammalian pathogens, functions of the O-polysaccharide (OPS) of lipopolysaccharide (LPS) remain unclear in plant-associated bacteria. Genetic information about OPS biosynthesis in these bacteria is largely missing. Genome analysis of various plant-associated Pseudomonas strains revealed that one of the two known OPS biosynthesis clusters from Pseudomonas aeruginosa PAO1, the common polysaccharide antigen (CPA) gene cluster, is only conserved in some strains of the Pseudomonas fluorescens group. For the O-specific antigen (OSA) biosynthesis cluster, the putative genomic position could be identified, but orthologues of most functional important OSA biosynthesis enzymes could not be detected. Nevertheless, orthologues of the glycosyltransferase WbpL, required for initiation of CPA and OSA synthesis in P. aeruginosa PAO1, could be identified in the analysed Pseudomonas genomes. Knockout mutations of wbpL orthologues in Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas cichorii ATCC10857/DSM50259 (Pci) resulted in strains lacking the OPS. Infection experiments of Arabidopsis thaliana plants revealed a reduced entry into the leaf apoplast after spray inoculation and a reduced apoplastic amplification of Pst ∆wbpL. Stab and spray inoculation of lettuce (Lactuca sativa) leaves with Pci ∆wbpL causes reduced infection symptoms compared to the wild-type strain. Furthermore, swarming motility was reduced in ∆wbpL mutants of Pst and Pci. This might be a possible reason for reduced bacterial titres after surface inoculation and reduced bacterial amplification in the plant. Our results imply that the presence of lipopolysaccharide OPS is required for efficient host colonization and full virulence of plant-pathogenic Pseudomonas bacteria.

Bacterial strain or plasmid Relevant genotype or phenotype a Source or reference E. coli strains TXM319 Wildtype BL21 (DE3); E. coli B F Ϫ ompT hsdS B (r B Ϫ m B Ϫ ) gal dcm lon (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Lab stock TXM322 BL21 (DE3) arnA::