Characterization of chimeric lipopolysaccharides from Escherichia coli strain JM109 transformed with lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae

previously, we reported the expression of chimeric lipopolysaccharides (LPS ) in Escherichia coli strain JM109 (a K-12 strain) transformed with plasmid s containing Haemophilus influenzae lipooligosaccharide synthesis genes (ls g) (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 analyze d the O-deacylated LPS and free oligosaccharides from three transformants ( designated pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7) by matrix-assisted laser de sorption ionization, electrospray ionization, and tandem mass spectrometry techniques, along with composition and linkage analyses. These data show th at the chimeric LPS consist of the complete E, coli LPS core structure glyc osylated 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 mono clonal antibody 3F11, an antibody that recognizes the terminal disaccharide of lacto-N-neotetraose. In pGEMLOS-4 LPS, the 3F11 epitope is apparently b locked by glycosylation on the 6-position of the terminal Gal with either G al or GlcNAc, The biosynthesis of these chimeric LPS was found to be depend ent on a functional wecA (formerly rfe) gene in E. coli, By using this carb ohydrate expression system, we have been able to examine the functions of t he lsg genes independent of the effects of other endogenous Haemophilus gen es and expressed proteins.