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 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.
Phillips
N J
NJ
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Miller
T J
TJ
Engstrom
J J
JJ
Melaugh
W
W
McLaughlin
R
R
Apicella
M A
MA
Gibson
B W
BW
D000215:Acylation; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics*; D008070:Lipopolysaccharides / Q000096:biosynthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D008070:Lipopolysaccharides