Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Abstract

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.

FIG. 1.

(A) Schematic of the psl gene cluster from the annotated P. aeruginosa PAO1 genome. The psl gene cluster is located between opdE and bkdR and contains 15 putative ORFs (∼18.7 kb), annotated PA2231 to PA2245 (54). The ORFs of this cluster are tightly linked in the same orientation, which implies an operon structure. The position of the xylE aacC1 insertion in pslAB, as well as a map of cosmid pMO011305, which complements the WFPA60 mutation, is indicated. Black boxes indicate ORFs that share homology with proteins involved in polysaccharide synthesis, modification, or transport. (B) Schematic of the PA1381-PA1392 gene cluster from the annotated P. aeruginosa PAO1 genome (54). This gene cluster is located between aceK and cysC and contains 12 putative ORFs (∼16.9 kb). The ORFs of this cluster are tightly linked in the same orientation, which implies an operon structure. The position of the aacC1 insertion of strain MS2, replacing the PA1388-PA1391 cluster, is indicated. Black boxes indicate ORFs that share homology with proteins involved in polysaccharide synthesis, modification, or transport.

FIG. 2.

Biofilm formation by P. aeruginosa strains. (A) The biofilm formation of strains PAO1 (circles), WFPA50 (squares), and WFPA60/pMO011305 (triangles) was assayed at 2-h intervals in a static microtiter plate system (40). Surface-attached cells were stained with crystal violet, the stain was solubilized in ethanol, and the absorbance was analyzed at 540 nm. (B) Biofilm formation of PAO1 (black) and MS2 (white) at 10 h in a static microtiter plate system (40). Bacteria were cultured with the following carbon sources (0.4%): A, LB medium; B, LB medium with glucose; C, 10× glucose; D, succinate; E, glycerol; F, glutamate (F).

FIG. 3.

The pslAB mutant has no defects in LPS synthesis or motility. (A) LPS profiles of PAO1 (lane 5) or the following PAO1-derived strains: PAO1 algC (lane 2), PAO1 algC plus algC (lane 3), and WFPA60 (lane 4). LPS was extracted from these strains, separated by SDS-15% PAGE, and visualized by silver staining of the polyacrylamide gel. Lane 1 contains a protein standard. (B) Twitching motility assays of PAO1 AWO (pilA::tet) (spot 1), PAO1 (spot 2), and WFPA60 (spot 3). (C) Flagellum motility assays of WFPA50 (fliC::aacC1) (spot 1), PAO1 (spot 2), and WFPA60 (spot 3).

FIG. 4.

Qualitative analysis of PAO1 and WFPA60 biofilm populations. Strains harboring pMRP9 were inoculated into respective flow devices and assayed at various time points postinoculation up to 72 h. WFPA60 is deficient in biofilm formation under laminar flow growth conditions compared to PAO1.

FIG. 5.

Quantitative analysis of PAO1 and WFPA60 biofilm populations in silicone tubing continuous flow devices. Overnight cultures of each strain (∼10⁷ cells) were inoculated into a section of silicone tubing that had first been primed with medium. At designated endpoints (6, 12, 24, 36, and 48 h), the tubing was longitudinally sectioned and the biofilm-grown cells were harvested, resuspended, and enumerated by plate counts (see Materials and Methods). Data are expressed as the log CFU recovered at each time point examined and are the averages of three independent experiments.