Increase in rhamnolipid synthesis under iron-limiting conditions influences surface motility and biofilm formation in Pseudomonas aeruginosa

Abstract

Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility. In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.

FIG. 1.

Influence of iron on rhlI-gfp and rhlA-gfp expression in planktonic P. aeruginosa cultures. Wild-type P. aeruginosa PAO1 carrying an rhlA-lux (RT102) or rhlI-lux (RT103) promoter fusion was grown in BM2 medium, and expression of the rhlA and rhlI genes was assessed by measuring luminescence. (A) rhlA-lux measurements at different iron concentrations (results from measurements taken at 12 h are presented). Expression profiles for the rhlI-lux promoter fusion (B) and the rhlA-lux promoter fusion (C), grown with and without 5 μM iron, are shown. In all graphs, the promoter activity is given as relative luminescence units (RLU) per unit of growth (optical density at 595 nm). A promoterless lux-based vector control showed no luminescence (see Fig. S2 in the supplemental material).

FIG. 2.

Influence of rhamnolipids on twitching motility in P. aeruginosa. (A) Wild-type P. aeruginosa PAO1, its rhlI mutant (PAO1 rhlI), and the complemented rhlI mutant (PAO1 rhlI carrying the plasmid pGP003) were inoculated onto iron-replete (100 μM FeCl₃) or iron-limited BM2 glucose medium, with (+ RL) or without purified rhamnolipids (10 μg/ml), as indicated. (B) P. aeruginosa PAO1, its rhlAB mutant (PAO1 rhlAB), and the rhlAB mutant carrying the rhlAB-complementing vector (pRT2) were inoculated onto iron-replete or iron-limited BM2 glucose medium, with (+ RL) or without rhamnolipids, as indicated. (C) P. aeruginosa PAO1, a flagellum mutant (PAO1 fliM), and a type IV pilus mutant (PAO1 pilA) were inoculated onto iron-replete (100 μM FeCl₃) or iron-limited BM2 glucose medium. In all cases, plates were incubated at 37°C for 40 h, the twitching zones were stained, and their diameters were measured. Results shown represent the means ± standard deviations for a representative assay (16 individual twitching zones were measured per strain and/or growth condition on 2 separate plates) performed in duplicate.

FIG. 3.

Impact of rhlAB mutation on P. aeruginosa biofilm formation under iron-replete and iron-restricted conditions. Biofilms of wild-type P. aeruginosa PAO1 (top), the rhlAB mutant (PAO1 rhlAB) (middle), and the complemented rhlAB mutant (bottom), grown in flow cells for 4 days in iron-restricted or iron-replete medium as described in Materials and Methods, are shown. The three-dimensional (3-D) images presented were reconstructed from CSLM scans of 4-day-old biofilms. Each square is 25 μm on a side.

FIG. 4.

Influence of iron on rhlA-gfp expression in P. aeruginosa biofilms. GFP fluorescence was followed over a 5-day period (A to E) in wild-type P. aeruginosa carrying an rhlA-gfp promoter fusion (RT101) and grown in flow cells under iron-restricted (−Fe) or iron-replete (+Fe) conditions. Images of 3-D reconstructions (bottom images) and horizontal sections near the center of the biofilms (top images) are shown for all time points. (A) 1 day; (B) 2 days; (C) 3 days; (D) 4 days; (E) 5 days. On day 5 (E), the biofilm was stained with propidium iodide to visualize the entire biofilm structure. A horizontal view of the green channel alone is shown (top), and a horizontal section and 3-D reconstruction of the merged red and green channels are also presented (middle and bottom). For the 3-D images, the squares for panels A and B are 14 μm on a side, and those for panels C to E are 24 μm on a side. Bars, 10 μm (A and B) and 35 μm (C to E).

FIG. 5.

Quantification of rhlA expression in P. aeruginosa biofilms. rhlA expression was monitored over a 5-day period in P. aeruginosa biofilms grown under iron-restricted (solid squares) or iron-replete (solid triangles) conditions. The level of rhlA was measured by real-time PCR as described in Materials and Methods. Transcript levels are given in picograms, normalized to a genomic DNA standard.