LPS quantitation procedures.

Abstract

Lipopolysaccharide is the predominant component of the Gram-negative cell wall occupying the outer leaflet of the outer membrane of Pseudomonas aeruginosa. Wild-type bacteria produce smooth LPS composed of lipid A, core oligosaccharide, and long O-antigen polysaccharide. In contrast, mutant bacteria defective in LPS biosynthesis produce rough LPS lacking the long O-antigen side chains. LPS is also a major virulence factor and proven to be crucial for full elaboration of other virulence factors and for a range of cellular functions. In order to determine the relationship between LPS and other cellular functions, a means to measure changes in the quantities of LPS being produced under certain growth/environmental conditions is important. Hence, the objective of this chapter is to provide readers with the methodologies for analyzing LPS of P. aeruginosa both qualitatively and quantitatively. As a prerequisite to quantifying LPS, one must be able to isolate LPS from the cell envelope; therefore, Subheading 2.1 is devoted to describing several standard LPS preparation methods. This is followed by Subheading 2.2, which deals with a number of practical methods for analyzing and/or quantifying whole-molecule LPS or assays for quantifying specific sugar constituents that are present within P. aeruginosa LPS. The methods described herein should be broadly applicable to the studying of LPS of other pseudomonads as well as Burkholderia species.