Pseudomonas aenrginosa PA01 possesses two distinct lipopolysaccharide (LPS)O-polysaccharide species, A-and B-band LPS, the relative expression of which appears to be under environmental control. In an attempt to identify the influence these LPS types have on surface characteristics and adhesion, we examined the surface hydrophobicity and surface charge of P. aemginosa PA01 ( 0 5 serotype) and its isogenic LPS derivatives which possessed A+B-, A-B+ and A-B-LPS. The surface characteristics of the strains affected their ability to adhere to hydrophilic (glass) and hydrophobic (polystyrene) surfaces. Cells possessing only A-band LPS demonstrated the highest surface hydrophobicity, followed by the strain lacking both A-and B-band LPS. The presence of B-band LPS resulted in a more hydrophilic surface. Strains lacking B-band LPS (A+B-and A-B-) had more electronegative surfaces than those possessing B-band LPS (A+B+ and A-B+), with cells lacking both A-and B-band LPS showing the highest surface electronegativity. These data suggest that the main surface-chargedetermining groups reside in the core region of the LPS molecule. Cells with the lowest surface hydrophobicity and lowest surface charge (A+B+, A-B+) adhered to glass the most efficiently, implying a role for electrostatic interaction, whereas adhesion to polystyrene mirrored the relative hydrophobicities of the strains (A+B-> A-B-> A+B+ > A-B+). It is postulated that phenotypic variation in the relative expression of A-and B-band LPS may be a mechanism by which P. aeruginosa can alter its overall surface characteristics in such a way as to influence adhesion and favour survival.N1G 2W1

The surfaces of bacteria are highly interactive with their environment. Whether the bacterium is Gram-negative or Gram-positive, most surfaces are charged at neutral pH because of the ionization of the reactive chemical groups which stud them. Since prokaryotes have a high surface area-to-volume ratio, this can have surprising ramifications. For example, many bacteria can concentrate dilute environmental metals on their surfaces and initiate the development of fine-grained minerals. In natural environments, it is not unusual to find such bacteria closely associated with the minerals which they have helped develop. Bacteria can be free-living (planktonic), but in most natural ecosystems they prefer to grow on interfaces as biofilms; supposedly to take advantage of the nutrient concentrative effect of the interface, although there must also be gained some protective value against predators and toxic agents. Using a Pseudomonas aeruginosa model system, we have determined that lipopolysaccharide is important in the initial attachment of this Gram-negative bacterium to interfaces and that this surface moiety subtly changes during biofilm formation. Using this same model system, we have also discovered that there is a natural tendency for Gram-negative bacteria to concentrate and package periplasmic components into membrane vesicles which bleb-off the surface. Since some of these components (e.g., peptidoglycan hydrolases) can degrade other surrounding cells, the vesicles could be predatory; i.e., a natural system by which neighboring bacteria are targeted and lysed, thereby liberating additional nutrients to the microbial community. This obviously would be of benefit to vesicle-producing bacteria living in biofilms containing mixed microbial populations.

Pseudomonas aeruginosa is ubiquitous in the environment and is also an important human opportunistic pathogen. As such, this organism must be able to readily adapt to widely varying nutrient levels and temperatures. The thermoadaptation of P. aeruginosa is complex but appears to involve alterations to both the lipid A and O-antigen components of lipopolysaccharide (LPS) (9, 15). In common with many other gram-negative species, growth at higher-than-optimal temperatures prompts a decrease in the proportion of high-molecularweight (high-MW) O antigen present on the surfaces of cells (1,9,15,18). It is now recognized that the majority of P. aeruginosa strains (including PAO1) possess the ability to produce two distinct O-polysaccharide types (12,19,20). The high-MW B band defines the serotype of the strain, with the PAO1 (O5 serotype) subunit consisting of two uronic acid derivatives and one N-acetylfucosamine residue (8). The A band contains shorter chains composed primarily of a polymer of ␣132-, ␣133-, ␣133-linked D-rhamnose and low levels of 2-keto-3-deoxyoctulosonic acid (2). With monoclonal antibodies (MAbs) to these LPS types (11, 12), we have been able to further investigate the temperature dependency of A-band and B-band polysaccharide expression in P. aeruginosa. P. aeruginosa PAO1 (H103) was used throughout the present study (4). Cells were cultured in tryptone soy broth at 15, 25, 35, or 45ЊC with shaking (150 rpm) to stationary phase (ca. 16 h), and 1 ml of culture was then transferred to fresh medium that was maintained at 15, 25, 35, or 45ЊC. Incubation was continued, and the cells were harvested when the optical density at 470 nm (OD 470 ) of the culture reached ca. 1.0. Before preparation for LPS analysis, samples were standardized with respect to protein by using a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) such that each 10 l of sample contained 50 g of total cell protein before digestion. LPS was prepared by the proteinase K method described by Hitchcock and Brown (5) and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% (wt/vol) acrylamide gels (13). The separated LPS bands were silver stained according to the method described by Tsai and Frasch (21); however, bands were developed with Bio-Rad silver stain developer (Bio-Rad Laboratories, Hercules, Calif.) as described by Petter et al. (17). When required, separated LPS samples were transferred from SDS-PAGE gels to nitrocellulose at 100 V for 1 h, were blocked with 3% (wt/vol) skimmed milk, and were reacted with MAb MF 15-4 (specific for B-band serotype O5 LPS [11]) or MAb N1F 10 (specific for A-band LPS [12]). The bands were visualized with goat antimouse antibodies conjugated to horseradish peroxidase, which was developed with 4-chloro-1-naphthol.For agglutination testing, a thick washed-cell suspension was prepared and 40 l was placed on a glass microscope slide. A 40-l drop of MF 15-4 was added to the suspension, and the slide was rocked back and forth for 5 min at room tempera...