Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV traits such as increased exopolysaccharide production leading to antibiotic tolerance, altered metabolism, and reduced immunogenicity may contribute to increased persistence in biofilms and in the airways of CF lungs.Pseudomonas aeruginosa is responsible for chronic infections in the airways of cystic fibrosis (CF) patients (13). During the course of chronic infection, P. aeruginosa forms biofilms, which are thought to promote persistence by protecting the bacterium from antibiotics and host clearance. P. aeruginosa also undergoes phenotypic and genotypic diversification. A manifestation of this diversification is the appearance of colony morphology variants among CF sputum sample isolates. One clear example of this phenomenon, which has been termed "dissociative" behavior (42), is the appearance of mucoid colonies. Mucoidy is characterized by overproduction of the exopolysaccharide (EPS) alginate, a polymer of 1,4-␤-linked mannuronic acid and its epimer, guluronic acid (13). The appearance of mucoid colonies is thought to correlate with a downturn in the p...

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in nonmucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that eleven psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose, and l-rhamnose: false[→3false)−normalβ−normalD−Manp2↑1α−D−Manp−false(1→3false)−normalβ−normalD−Manp−false(1→3false)−normalα−normalL−Rhap−false(1→3false)−normalβ−normalD−Glcp−false(1→false]normaln− In addition we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose, and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

The ability to form biofilms in the airways of people suffering from cystic fibrosis is a critical element of Pseudomonas aeruginosa pathogenesis. The 15-gene psl operon encodes a putative polysaccharide that plays an important role in biofilm initiation in nonmucoid P. aeruginosa strains. Biofilm initiation by a P. aeruginosa PAO1 strain with disruption of pslA and pslB (⌬pslAB) was severely compromised, indicating that psl has a role in cell-surface interactions. In this study, we investigated the adherence properties of this ⌬pslAB mutant using biotic surfaces (epithelial cells and mucin-coated surfaces) and abiotic surfaces. Our results showed that psl is required for attachment to a variety of surfaces, independent of the carbon source. To study the potential roles of Psl apart from attachment, we generated a psl-inducible P. aeruginosa strain (⌬psl/p BAD -psl) by replacing the psl promoter region with araC-p BAD , so that expression of psl could be controlled by addition of arabinose. Analysis of biofilms formed by the ⌬psl/p BAD -psl strain indicated that expression of the psl operon is required to maintain the biofilm structure at steps postattachment. Overproduction of the Psl polysaccharide led to enhanced cell-surface and intercellular adhesion of P. aeruginosa. This translated into significant changes in the architecture of the biofilm. We propose that Psl has an important role in P. aeruginosa adhesion, which is critical for initiation and maintenance of the biofilm structure.Pseudomonas aeruginosa is an important opportunistic human pathogen that can cause life-threatening infections in cystic fibrosis (CF) patients and individuals with a compromised immune system. This environmental bacterium is capable of living planktonically or in surface-associated communities known as biofilms. P. aeruginosa biofilms can form on a variety of surfaces, including in mucus plugs of the CF lung and abiotic surfaces, such as contact lenses and catheters (8,15,30,36). Bacteria growing in biofilms are less susceptible to antimicrobial agents and are protected from the host immune response, giving rise to chronic infections that are notoriously difficult to eradicate (9,13,27). Bacteria growing in biofilms produce one or more extracellular polymeric matrices that act as a scaffold, holding the cells of the biofilm community together. Polysaccharides are key components of the biofilm matrix, as they contribute to the overall biofilm architecture and to the resistance of bacteria in biofilms (5,26,29).Biofilm development is a sequential process initiated by the attachment of planktonic cells to a surface, which is followed by formation of microcolonies and biofilm maturation in which individual bacteria, as well as the entire community, are embedded in a matrix composed of nucleic acid, protein, and polysaccharides (2,5,30,34). Two potential polysaccharide biosynthetic loci, psl (PA2231 to PA2245) and pel (PA3058 to PA3064) of P. aeruginosa have been identified as loci that play important roles in biofilm initiation an...