Lipopolysaccharides from Escherichia coli

In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69. 4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coli serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

The cell envelope is the first line of defense between a bacterium and the world-at-large. Often, the initial steps that determine the outcome of chemical warfare, bacteriophage infections, and battles with other bacteria or the immune system greatly depend on the structure and composition of the bacterial cell surface. One of the most studied bacterial surface molecules is the glycolipid known as lipopolysaccharide (LPS), which is produced by most Gram-negative bacteria. Much of the initial attention LPS received in the early 1900s was owed to its ability to stimulate the immune system, for which the glycolipid was commonly known as endotoxin. It was later discovered that LPS also creates a permeability barrier at the cell surface and is a main contributor to the innate resistance that Gram-negative bacteria display against many antimicrobials. Not surprisingly, these important properties of LPS have driven a vast and still prolific body of literature for more than a hundred years. LPS research has also led to pioneering studies in bacterial envelope biogenesis and physiology, mostly usingEscherichia coliandSalmonellaas model systems. In this review, we will focus on the fundamental knowledge we have gained from studies of the complex structure of the LPS molecule and the biochemical pathways for its synthesis, as well as the transport of LPS across the bacterial envelope and its assembly at the cell surface.

Previously, we reported the expression of chimeric lipopolysaccharides (LPS) in Escherichia coli strain JM109 (a K-12 strain) transformed with plasmids containing Haemophilus influenzae lipooligosaccharide synthesis genes (lsg) (Abu Kwaik, Y., McLaughlin, R. E., Apicella, M. A., and Spinola, S. M. (1991) Mol. Microbiol. 5, 2475-2480). In this current study, we have analyzed the O-deacylated LPS and free oligosaccharides from three transformants (designated pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7) by matrix-assisted laser desorption ionization, electrospray ionization, and tandem mass spectrometry techniques, along with composition and linkage analyses. These data show that the chimeric LPS consist of the complete E. coli LPS core structure glycosylated on the 7-position of the non-reducing terminal branch heptose with oligosaccharides from H. influenzae. In pGEMLOS-7, the disaccharide Gal1--> 3GlcNAc1--> is added, and in pGEMLOS-5, the structure is extended to Gal1-->4GlcNAc1-->3Gal1-->3GlcNAc1-->. PGEMLOS-5 LPS reacts positively with monoclonal antibody 3F11, an antibody that recognizes the terminal disaccharide of lacto-N-neotetraose. In pGEMLOS-4 LPS, the 3F11 epitope is apparently blocked by glycosylation on the 6-position of the terminal Gal with either Gal or GlcNAc. The biosynthesis of these chimeric LPS was found to be dependent on a functional wecA (formerly rfe) gene in E. coli. By using this carbohydrate expression system, we have been able to examine the functions of the lsg genes independent of the effects of other endogenous Haemophilus genes and expressed proteins.