Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution

Abstract

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1(serotype 05) and IATS 06 (serotype 06), generated isogenic mutants from them, and examined the distribution of LPS on the surface ofthese organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31-to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype 06 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A-B-), rd7513 (A-B-), and R5 (an IATS 06-derived rough mutant; A-B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining.When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.Lipopolysaccharide (LPS) is anchored on the outer leaflets of the outer membranes of gram-negative bacteria via the lipid A moiety, with the core and 0-antigen polysaccharide regions extending outward from the cell. LPS possesses biologically important properties, such as adjuvant activities that are stimulatory to the immune systems of animal hosts and endotoxic effects that are deleterious to the health of the host (31). It can also act as a receptor for phage, as a physical barrier mediating interactions between a bacterium and its environment, and as a defense against hostile host molecules such as complement.In Pseudomonas aeruginosa, LPS is heterogeneous in size because of variations in the amount of 0-side-chain polymer substitution on the core and in the types of side-chain sugars. Several recent reports have indicated that most P. aeruginosa strains can simultaneously synthesize an A-band form and a B-band form of LPS (1,25,32,33). A-band LPS, a common antigen of uncharged sugars, is composed primarily of a1-->2-and al->3-linked D-rhamnose, whereas B-band LPS is a highly charged 0-antigen-containing LPS ...

Ultrastructure and chemical composition of the sheath of Leptothrix discophora SP-6

“…5 in reference 27). Whether this material represents a true capsule or longchain lipopolysaccharide, which has been shown to be preserved by freeze substitution (34), is unknown.…”

High-Resolution Visualization ofPseudomonas aeruginosaPAO1 Biofilms by Freeze-Substitution Transmission Electron Microscopy

“…In gram-negative bacteria, the periplasm is preserved as a so-called periplasmic gel and the chromosomal material of the nucleoid is dispersed throughout the cytoplasm (27,28). Remarkably, the freeze-substitution method has even preserved the O-side chains of the lipopolysaccharide (LPS) on the outer face of the outer membrane of Pseudomonas aeruginosa (41). Even the cell walls of gram-positive cells show more detail; the walls of Bacillus subtilis revealed structural aspects of cell wall turnover (24) and mycobacterial walls showed more accurate distribution of their complex polymeric networks (54,55).…”

“…Its presence affects the attachment of bacteria to a surface (49) and some of the chemical constituents identified in PAO1's EPS are similar to those in the O-side chain as well as in the core oligosaccharide (70 A-band has a relatively simple, uncharged O-side chain consisting of ϳ10 to 20 repeating units of a D-rhamnose trimer (2). This neutrally charged polymer is difficult to stain and visualize using TEM (41). The B-band O-side chain is serotype specific and is much more complex.…”

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

“…In Gram-negative pathogens, the principle surface polymers are capsular polysaccharides (CPSs) 2 and lipopolysaccharide (LPS) O-antigens. CPS and LPS are virulence determinants, typically providing protective functions.…”

“…The O-antigen extends from the cell surface to form a discrete layer (2,3). The population of LPS molecules isolated from a growing culture is heterogeneous in size, evident in characteristic ladder patterns in SDS-PAGE profiles (4 -6).…”