The long term goals of this research are directed at elucidating the mechanisms involved in the biogenesis and assembly of the outer membrane in Gram-negative bacteria. It is anticipated that a knowledge of these processes in prokaryotic organisms will provide insights into general mechanisms involved in membrane biogenesis and assembly in eukaryotic cells and cell organelles. The specific research proposed here is concerned with determining the mechanism of biosynthesis of the enterobacterial common antigen (ECA). ECA is a cell surface antigen present in the outer membrane of all Gram-negative bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of N-acetyl-D-glucosamine (G1cNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-4,6- dideoxy-D-galactose (Fuc4NAc). These amino sugars form the repeating trisaccharide unit Greater than 4)-Beta-D-ManpNAcUA-(1 Greater than 4)-Alpha-D-Glcp-NAc-(1 Greater than 3)-D-Fucp4NAc-(1 Greater than. Although the parochial taxanomic distribution of this molecule suggests a function uniquely associated with Gram-negative enteric bacteria, nothing is known concerning either the function or mechanism of biosynthesis of theis molecule. We propose here experiments designed to define the initial steps in the pathway of ECA biosynthesis. These experiments will pursue preliminary observations which suggest that G1cNAc-pyrophosphorylundecaprenol (G1cNAc-PP-lipid) is an early intermediate in ECA synthesis. Accordingly, we will investigate the possibility that the initial trisaccharide repeat unit is synthesized by the sequential transfer of ManNAcUA and Fuc4NAc into G1cNAc-PP-lipid from the nucleotide sugars donors UDP-ManNAcUA and TDP-Fuc4NAc, respectively. Subsequent experiments will be directed at demonstrating the in vitro synthesis of lipid-linked heteropolysaccharide chains containing multiple repeat units, and we will also determine the mechanism of chain elongation. Additional experiments will be concerned with identifying the step at which ECA synthesis is blocked in rfe mutants of S. typhimurium. The proposed research will utilize techniques of molecular biology and biochemistry such as in vivo pulse-labeling, in vitro polysaccharide synthesis, and isolation, fractionation, and structural characterization of biosynthetic intermediates.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI021309-06
Application #
3131283
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-07-01
Project End
1994-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20852
Rick, P D; Hubbard, G L; Barr, K (1994) Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. J Bacteriol 176:2877-84
Barr, K; Rick, P D (1993) Physical map location of the rffC and rffA genes of Escherichia coli. J Bacteriol 175:5738-9
Sherry, B; Yarlett, N; Strupp, A et al. (1992) Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages. Proc Natl Acad Sci U S A 89:3511-5
Meier-Dieter, U; Barr, K; Starman, R et al. (1992) Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. J Biol Chem 267:746-53
Meier-Dieter, U; Starman, R; Barr, K et al. (1990) Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis. J Biol Chem 265:13490-7
Barr, K; Nunes-Edwards, P; Rick, P D (1989) In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen. J Bacteriol 171:1326-32
Rick, P D; Wolski, S; Barr, K et al. (1988) Accumulation of a lipid-linked intermediate involved in enterobacterial common antigen synthesis in Salmonella typhimurium mutants lacking dTDP-glucose pyrophosphorylase. J Bacteriol 170:4008-14
Barr, K; Ward, S; Meier-Dieter, U et al. (1988) Characterization of an Escherichia coli rff mutant defective in transfer of N-acetylmannosaminuronic acid (ManNAcA) from UDP-ManNAcA to a lipid-linked intermediate involved in enterobacterial common antigen synthesis. J Bacteriol 170:228-33
Barr, K; Rick, P D (1987) Biosynthesis of enterobacterial common antigen in Escherichia coli. In vitro synthesis of lipid-linked intermediates. J Biol Chem 262:7142-50
Rick, P D; Mayer, H; Neumeyer, B A et al. (1985) Biosynthesis of enterobacterial common antigen. J Bacteriol 162:494-503