lipid-a and tricine

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Topics: Bacteriological Techniques; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Glycine; Lipid A; Lipopolysaccharides; Molecular Probe Techniques; O Antigens; Silver Staining; Sugar Acids; Sulfhydryl Compounds

Topics: Detergents; Electrophoresis, Polyacrylamide Gel; Glycine; Gram-Negative Bacteria; Lipid A; Lipopolysaccharides; Microchemistry; Molecular Weight; Oligosaccharides; Silver Staining; Solubility; Solvents

Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity.

Topics: Amino Acid Sequence; Antibodies; Bacterial Proteins; Binding Sites; Bordetella bronchiseptica; Carbohydrate Sequence; Carboxylic Ester Hydrolases; Centrifugation; Cloning, Molecular; Disaccharides; DNA; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gas Chromatography-Mass Spectrometry; Glycine; Histidine; Immunoblotting; Lipid A; Lipids; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Plasmids; Pseudomonas aeruginosa; Salmonella enterica; Sequence Homology, Amino Acid; Serine; Spectrometry, Mass, Electrospray Ionization