Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Abstract

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunism: pathogen Pseudomonas aeruginosa. The PsI polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in non-mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, psIACDEFGHIJKL, are required for PsI 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: [→ 3)-β-D-Manp-(1 → 3)-β-D-Manp-(1→ 3)-2 ↑ 1 α-D-Manp α-L-Rhap-(1 → 3)-β-D-Glcp-(1 →]n-In addition, we identified the sugar nucleotide precursors involved in PsI generation and demonstrated the requirement for GDP-D-mannose, UDP-D-glucose and dTDP-L-rhamnose in PsI production and surface attachment. Finally, genetic analyses revealed that wbpW restored PsI 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 PsI proteins and potential roles for PsI in the biology and pathogenesis of P. aeruginosa. © 2009 Blackwell Publishing Ltd.