The supermucoid Pseudomonas aeruginosa strain PDO300⌬alg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.Alginate is an important virulence factor for Pseudomonas aeruginosa, and the conversion of nonmucoid strains to alginate-overproducing mucoid strains early after the infection of cystic fibrosis patients is associated with a decline of pulmonary function and survival rate (11,13). Alginate functions as extracellular matrix material, enabling the formation of differentiated biofilms in which the diffusion of clinical antibiotics is decreased and the embedded cells are protected against human antibacterial defense mechanisms (9, 12). Although alginate is not required for P. aeruginosa biofilm formation (15), previous studies have provided evidence that it plays a role in the formation of thick and three-dimensional biofilms (5, 9). To further investigate the impact of alginate on attachment and biofilm architecture, we used a recently generated supermucoid strain, PDO300⌬alg8(pBBR1MCS-5:alg8) (14). This strain showed about 15-fold alginate overproduction compared to alginate-producing mucoid P. aeruginosa. The gene alg8 encodes the proposed catalytic subunit of alginate polymerase and is essential for alginate biosynthesis (14).Quantitative analysis of attachment and biofilm formation. The attachment characteristics of the supermucoid strain PDO300⌬alg8(pBBR1MCS-5:alg8) were compared with those of the wild-type strain PAO1 and the mucoid strain PDO300 (an isogenic mucA22 mutant of PAO1) (8) and its alginatenegative isogenic alg8 deletion mutant (14). A modification of the solid-surface assay (SSA) (10) was used to assess attachment in microtiter plates after incubation for 2, 4, and 6 h. Stationary cultures at 37°C in Luria-Bertani medium (containing gentamicin at 300 g/ml when appropriate) were adjusted to an optical density at 600 nm of 0.05, and 100-l aliquots were added to one column (8 wells) of each of five replicate 96-well tissue culture plates. After incubation at 37°C for the respective times, nonadherent bacteria were washed off by filling the wells three times with sterile water and then removing the well contents with gentle suction. Plates were then air dried, and adherent bacteria were stained with 100 l of 0.1% (wt/vol) crystal violet for 20 min at room temperature. The crystal violet was removed by washing as described above and dissolved in 100 l dimethyl sulfoxide. After 20 min, the absorbance at 595 nm was measured. The data presented here are the averages of results from three independent experiments with eight replicates each. The results showed excellent intra-assay and interassay reproducibility, with minimal background. During the early attachment phase (2 to 4 h), the nonmucoid s...