Expression of the psl Operon in Pseudomonas aeruginosa PAO1 Biofilms: PslA Performs an Essential Function in Biofilm Formation

Abstract

The psl gene cluster, comprising 15 cotranscribed genes from Pseudomonas aeruginosa, was recently identified as being involved in exopolysaccharide biosynthesis and biofilm formation. In this study, we investigated the regulation of the psl gene cluster and the function of the first gene in this cluster, the pslA gene. PslA shows strong similarities to UDP-glucose lipid carriers. An isogenic marker-free pslA deletion mutant of P. aeruginosa PAO1 deficient in attachment and biofilm formation was used for complementation studies. The expression of only the pslA gene, comprising a coding region of 1,437 bp, restored the biofilm-forming phenotype of the wild type, indicating that PslA is required for biofilm formation by nonmucoid P. aeruginosa. The promoter region of the psl gene cluster, which encodes PslA-PslO, was identified by rapid amplification of cDNA 5 ends. Promoter assays using transcriptional fusions to lacZ and gfp indicated a constitutive expression of the psl cluster in planktonic cells and a highly regulated and localized expression in biofilms, respectively. Expression of the psl cluster in biofilms was almost exclusively found in the centers of microcolonies, as revealed by confocal laser scanning microscopy. These data suggest that constitutive expression of the psl operon enables efficient attachment to surfaces and that regulated localized psl operon expression is required for biofilm differentiation.

Analysis ofPseudomonas aeruginosaConditional Psl Variants Reveals Roles for the Psl Polysaccharide in Adhesion and Maintaining Biofilm Structure Postattachment

“…Previous studies showed that biofilm initiation by nonmucoid P. aeruginosa strains with mutations in the psl genes was deficient, most likely due to the reduced ability of these mutants to interact with cell surfaces (12,20,29,32). The attachment assays in these studies were performed with a rich medium, and it was unclear if Psl contributed to cell-surface interactions when bacteria were grown under other conditions.…”

“…Overhage and colleagues recently mapped the psl operon promoter 41 bp upstream of the pslA start codon (32). Their data also suggested that psl expression was localized to the centers of microcolonies within biofilms (32). In addition, Kirisits et al showed that the expression of psl and pel was elevated in variants isolated from aging P. aeruginosa PAO1 biofilms (22).…”

“…The psl cluster contains 15 cotranscribed genes (pslA to pslO) encoding proteins predicted to be involved in polysaccharide biosynthesis. Overhage and colleagues recently mapped the psl operon promoter 41 bp upstream of the pslA start codon (32). Their data also suggested that psl expression was localized to the centers of microcolonies within biofilms (32).…”

“…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 and formation in nonmucoid P. aeruginosa strains (11,12,20,22,29,32,41). The psl cluster contains 15 cotranscribed genes (pslA to pslO) encoding proteins predicted to be involved in polysaccharide biosynthesis.…”

Role of polysaccharides in Pseudomonas aeruginosa biofilm development

“…This is an area requiring future research. Overhage and Campisano et al demonstrated that pslA and pslD, which encode a putative UDP-glucose lipid carrier and exporter, respectively, are essential for biofilm formation in strain PAO1 [23,24]. They also demonstrated that while psl is constitutively expressed in planktonic cells, its expression is localized to the center of developing biofilm microcolonies [24].…”