Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix

Abstract

Pseudomonas aeruginosa forms biofilms, which are cellular aggregates encased in an extracellular matrix. Molecular genetics studies of three common autoaggregative phenotypes, namely wrinkled colonies, pellicles, and solid-surface-associated biofilms, led to the identification of two loci, pel and psl, that are involved in the production of carbohydrate-rich components of the biofilm matrix. The pel gene cluster is involved in the production of a glucose-rich matrix material in P. aeruginosa strain PA14 (L. Friedman and R. Kolter, Mol. Microbiol. 51:675-690, 2004). Here we investigate the role of the pel gene cluster in P. aeruginosa strain ZK2870 and identify a second genetic locus, termed psl, involved in the production of a mannose-rich matrix material. The 11 predicted protein products of the psl genes are homologous to proteins involved in carbohydrate processing. P. aeruginosa is thus able to produce two distinct carbohydrate-rich matrix materials. Either carbohydrate-rich matrix component appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains PA14 and ZK2870.

FIG. 1.

Autoaggregative properties of P. aeruginosa strain ZK2870. P. aeruginosa strain ZK2870 forms wrinkled colonies (A) under conditions in which P. aeruginosa strain PA14 forms smooth flat colonies (B). The plates contained 1% agar, 10 g of tryptone/liter, 40 mg of Congo red/liter, and 15 mg of Coomassie brilliant blue/liter. Single colonies were grown at room temperature for 8 days.

FIG. 2.

Role of the pel locus in P. aeruginosa strain ZK2870. (A) Wild-type pellicle and Δpel mutant pellicle in strain ZK2870. (B) Close-up photos of the wild-type pellicle and the Δpel mutant pellicle after the pellicles were grown in the presence of Congo red (40 mg/liter), removed from the culture, and rinsed in water before being photographed. (C) Colonies derived from single cells grown at 22°C for 6 days on 0.5% agar, 10 g of tryptone/liter, 40 mg of Congo red/liter, and 15 mg of Coomassie brilliant blue/liter.

FIG. 3.

The psl locus. The 11 open reading frames are outlined in black and drawn to scale. The PA numbers and gene names are shown directly below the map. The locations of the transposon insertions in P. aeruginosa strain ZK2870 are indicated with triangles directly above the open reading frames. Open triangles, insertions in the wild-type strain; gray-filled triangles, insertions in the Δpel mutant background. The gray shading within the open reading frames indicates the genes that are missing from P. aeruginosa strain PA14. The stars indicate the in-frame deletions that we created in both the wild-type (open stars) and Δpel mutant (gray stars) backgrounds in P. aeruginosa strain ZK2870.

FIG. 4.

Colony morphology and pellicle formation in P. aeruginosa strain ZK2870. (A) Colonies derived from single cells were grown on 0.5% agar at room temperature on plates containing 10 g of tryptone/liter, 40 mg of Congo red/liter, and 15 mg of Coomassie brilliant blue/liter. The images labeled Δpsl and Δpsl Δpel show the ΔpslC and ΔpslD Δpel mutants, respectively. (B) Pellicles form in wild-type, Δpel, and Δpsl standing liquid cultures, but not in the Δpsl Δpel double mutant culture. The images shown are of ΔpslD and ΔpslD Δpel cultures and are similar to those for all other psl and psl pel mutants tested.

FIG. 5.

SSA biofilm formation in P. aeruginosa strains ZK2870 and PA14. The crystal violet assay was used to measure SSA biofilm formation at 37°C (A and B) and 22°C (C). (A) The initiation of biofilm formation was assayed by gentle washing of the standing cultures over time. (B and C) Mature biofilm formation was assayed by harsh running water washing of the vessels.

FIG. 6.

Comparison of carbohydrate contents of Δpel pellicle and pslI::Tn5 pellicle of P. aeruginosa strain ZK2870.