Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length

Abstract

Lipopolysaccharide (LPS) from smooth strains of Pseudomonas aeruginosa 503, PAZ1, PA01715, PA01716, and Z61 was fractionated by gel filtration chromatography. LPS samples from the first four strains, all PAO1 derivatives, separated into three major size populations, whereas LPS from strain Z61, a Pac K799/WT mutant strain, separated into two size populations. When column fractions were applied to sodium dodecyl sulfate-polyacrylamide gels in their order of elution, molecules of decreasing size were resolved, and the ladder of molecules with different-length 0 antigens formed a diagonal across the gel. The LPS from the PAO1 derivatives contained two distinct sets of bands, distinguished on the gels as two sets of diagonals. The set of bands with the faster mobility, the B bands, was found in column fractions comprising the three major amino sugar-containing peaks. In the sample from strain 503, a fourth minor peak which contained B bands was resolved. The slower-moving set of bands, the A bands, were recovered in a minor peak. LPS from strain Z61 contained only one set of bands, with the higher-molecular-weight molecules eluting from the column in a volume similar to that of the B bands of the PAO1 strains. Analysis of the fractions of LPS from all strains indicated that less than 8% of the LPS molecules had a long, attached 0 antigen. Analysis of the peak that contained mainly A bands indicated a lack of reactive amino sugar and phosphate, although heptose and 2-keto-3-deoxyoctulosonic acid were detected. Reaction of isolated fractions with monoclonal antibody specific for the PA01 0-antigen side chain indicated that only the B bands from the PAO1 strains were antigenically reactive. The bands from strain Z61 showed no reactivity. The data suggest that the A and B bands from the PAO1 strains are antigenically distinct. We propose that PAO1 strains synthesize two types of molecules that are antigenically different.Lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is important in the structure (33, 37) and function (33,36) of this membrane. Structural microheterogeneity in several regions of LPS molecules from members of the family Enterobacteriaceae (2,14,22,37,38,48,51) and Pseudomonas aeruginosa strains (33, 57) has been demonstrated. Of the several methods used to separate the subclasses of LPS from individual strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20,24,45,48) and gel filtration (9, 26, 30, 32, 34, 48) are the best. Either of these two methods by themselves, however, may be insufficient to completely characterize the high-and low-molecular-weight fractions of LPS. Peterson and McGroarty (48) demonstrated that the SDS-PAGE of the column fractions of samples from Salmonella typhimurium, Salmonella minnesota, and Escherichia coli was instrumental in characterizing the various-sized fractions. Analysis of the isolated fractions allowed for the estimation of the average number of 0-antigen repeat units per LPS from each of the si...

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

“…LPS is a glycolipid composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. P. aeruginosa produces two forms of O antigen, which are the homopolymeric A band and the heteropolymeric B band (15,21). The gene clusters associated with the biosynthesis of Aband and B-band O antigen in P. aeruginosa have been well characterized (for reviews, see references 18 and 22).…”

Influence of Water Hardness on Silver Ion and Silver Nanoparticle Fate and Toxicity TowardNitrosomonas europaea

“…Gram-negative bacteria have a negative net surface charge due to the negatively charged functional groups of the lipopolysaccharide (LPS) layer (Costerton et al, 1974). Mg 2 + acts to stabilize bacterial membranes through the bridging of the negative charges of LPS functional groups, including phosphates, phosphatidic acid, and 2-keto-3-deozyoctulosonic acid (Schindler and Osborn, 1979;Rivera et al, 1988;Amro et al, 2000). Mg 2 + will also bind to other membrane constituents, including inorganic phosphates and teichoic acids (Heptinstall et al, 1970;Naumann et al, 1989;van Veen, 1997).…”

Common antigen lipopolysaccharide from Pseudomonas aeruginosa AK1401 as a receptor for bacteriophage A7

“…The SDS-polyacrylamide gel of the column fractions, when applied in order of elution, revealed two major size populations: the A-band or common antigen LPS and an 0-serotype-like LPS which is composed of core and lipid A with no or one 0-repeat unit (short-chain [SC] or B-band LPS; results not shown). As indicated in earlier studies (22,23), the SC-LPS fraction contains the majority of the molecules. The column elution profile confirmed earlier results which indicated that this strain is defective in the synthesis of 0 antigen (3).…”

“…The mixture was incubated with mild agitation at 20°C for 2, 3, 4, 5, and 24 h. An aliquot of 50 ,lI of the digested LPS was mixed with an equal volume of electrophoresis sample buffer (containing 4% SDS) and applied to an SDS-polyacrylamide gel. Electrophoresis was performed as previously described (22), and the gels were silver stained. The SDS-polyacrylamide gels of the digested samples showed that the polysaccharide chain of the A-band LPS was completely hydrolyzed by phage A7 within 2 h (Fig.…”