The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage

Abstract

Mature Pseudomonas aeruginosa biofilms undergo specific developmental events. Using a bacteriophage mutant, generated by deletion of the entire filamentous Pf4 prophage, we show that the phage is essential for several stages of the biofilm life cycle and that it significantly contributes to the virulence of P. aeruginosa in vivo. Here, we show for the first time that biofilms of the Pf4 phage-deficient mutant did not develop hollow centres or undergo cell death, typical of the differentiation process of wild-type (WT) P. aeruginosa PAO1 biofilms. Furthermore, microcolonies of the Pf4 mutant were significantly smaller in size and less stable compared with the WT biofilm. Small colony variants (SCVs) were detectable in the dispersal population of the WT biofilm at the time of dispersal and cell death, whereas no SCVs were detected in the effluent of the Pf4 mutant biofilm. This study shows that at the time when cell death occurs in biofilms of the WT, the Pf4 phage converts into a superinfective form, which correlates with the appearance of variants in the dispersal population. Unexpectedly, mice infected with the Pf4 mutant survived significantly longer than those infected with its isogenic WT strain, showing that Pf4 contributes to the virulence of P. aeruginosa. Hence, a filamentous prophage is a major contributor to the life cycle and adaptive behaviour of P. aeruginosa and offers an explanation for the prevalence of phage in this organism.

Fig. 1

Construction of the Pf4 chromosomal deletion and location of primers. A) The genomic organization of the Pf4 prophage in P. aeruginosa PAO1 shown. The integration sites, attL and attR are shown, along with open reading frames, shown as thick arrows. Numbers above the arrows indicate gene numbers (adapted from (Webb et al, 2004)). Solid lines indicate amplification products and numbers indicate primers, as listed in Table 2. Small arrows indicate individual primers and are also numbered with reference to Table 2. B) Organisation of the ΔPf4 genomic region subsequent to deletion of the prophage. Black lines indicate the amplified 3′ and 5′ regions which are ligated to the FRT sites (hatched circles), recognised by the Flp recombinase, and the gentamycin resistance cassette (grey line, Gm^r). The figures are not drawn to scale.

Fig. 2

Detection of Pf4 phage in the supernatant of overnight cultures. Supernatants from overnight cultures of the P. aeruginosa PAO1 were spotted onto soft-agar lawns of the wild-type PAO1 (A), and the Pf4 mutant (B) and supernatants from overnight cultures of the Pf4 mutant were spotted onto either lawns of the wild-type PAO1 (C) or the ΔPf4 mutant (D).

Fig. 3

Biofilm formation by wild-type P. aeruginosa wild-type PAO1 and its isogenic ΔPf4 mutant. Confocal microscope images of BacLight, Live-Dead (Molecular Probes, USA) stained biofilms were collected on days 3, 5, and 7 for the wild-type and the ΔPf4 mutant. Magnification, 400X; Bars, 50 μm.

Fig. 4

The Pf4 mutant biofilm is less stable than the wild-type. Biofilms of P. aeruginosa PAO1 wild-type, a quorum sensing mutant, and the Pf4 mutant were formed in flow cells for 4 days in M9 minimal medium, at which time, they were treated with 0.01 % SDS (in M9 medium) under flow conditions for 2h at room temperature. Biofilms were stained with the BacLight Live-Dead staining reagents (Molecular Probes) and imaged using Confocal Scanning Laser Microscopy (CSLM). Images were analyzed to determined coverage in the X-Z plane (thickness) and normalized against the untreated controls, bars represent standard errors (A). CSLM images (X-Z plane) of the treated and untreated biofilms are shown in (B).

Fig. 5

Phage titre of biofilm effluents from P. aeruginosa PAO1 and the ΔPf4 mutant biofilms from days 1 to 5. Biofilm effluent was filtered through a 0.2 um filter to eliminate bacterial cells and serial dilutions were spotted onto soft agar lawns of either the PAO1 wild-type or the Pf4 mutant to quantify the number of phage in the effluent (pfu/ml). The phage titre of P. aeruginosa PAO1 biofilm effluent that were determined on the PAO1 lawn or the Pf4 mutant lawn are shown as black and grey bars, respectively.

Fig. 6

The Pf4 mutant is less virulent than the wild-type P. aeruginosa PAO1. BALB/c mice were infected with approximately 7 × 10⁷ cfu of either the wild-type PAO1 or the Pf4 mutant and were monitored over a period of 168 h to determine survival. Three independent experiments were performed and the results were pooled. Results are presented as the number of surviving mice at different time points post infection.