Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development

Abstract

A current question in biofilm research is whether biofilm-specific genetic processes can lead to differentiation in physiology and function among biofilm cells. In Pseudomonas aeruginosa, phenotypic variants which exhibit a small-colony phenotype on agar media and a markedly accelerated pattern of biofilm development compared to that of the parental strain are often isolated from biofilms. We grew P. aeruginosa biofilms in glass flow cell reactors and observed that the emergence of small-colony variants (SCVs) in the effluent runoff from the biofilms correlated with the emergence of plaque-forming Pf1-like filamentous phage (designated Pf4) from the biofilm. Because several recent studies have shown that bacteriophage genes are among the most highly upregulated groups of genes during biofilm development, we investigated whether Pf4 plays a role in SCV formation during P. aeruginosa biofilm development. We carried out immunoelectron microscopy using anti-Pf4 antibodies and observed that SCV cells, but not parental-type cells, exhibited high densities of Pf4 filaments on the cell surface and that these filaments were often tightly interwoven into complex latticeworks surrounding the cells. Moreover, infection of P. aeruginosa planktonic cultures with Pf4 caused the emergence of SCVs within the culture. These SCVs exhibited enhanced attachment, accelerated biofilm development, and large regions of dead and lysed cells inside microcolonies in a manner identical to that of SCVs obtained from biofilms. We concluded that Pf4 can mediate phenotypic variation in P. aeruginosa biofilms. We also performed partial sequencing and analysis of the Pf4 replicative form and identified a number of open reading frames not previously recognized in the genome of P. aeruginosa, including a putative postsegregational killing operon.

FIG. 1.

Wild-type colonies and SCVs derived from effluent runoff from a 7-day old biofilm (a) and a P. aeruginosa overnight planktonic culture infected with the Pf4 bacteriophage (SCVs only) (b). Bars = 3 mm.

FIG. 2.

Transmission electron microscopy and immunogold labeling with anti-Pf4 antibodies, showing high densities of filamentous bacteriophages on the cell surface of SCVs. (a) Wild-type (WT) P. aeruginosa cell, showing a single flagellum. (b) P. aeruginosa SCV7 cell with anti-Pf4 antibodies. (c) Higher magnification of Pf4 filaments tightly woven together. (d) ΔpilA mutant of P. aeruginosa, showing similar Pf4 filament production on the cell surface.

FIG. 3.

Adhesion of wild-type, ΔpilA, and SCVs to wells of tissue culture plates. WT, wild type; OD600, optical density at 600 nm.

FIG. 4.

Biofilms formed by SCVs have enhanced microcolony formation and large regions containing dead cells inside microcolonies. Five-day-old P. aeruginosa biofilms were stained with the BacLight LIVE/DEAD stain. The biofilms were inoculated with wild-type (a), SCV7 (b), and Pf4-infected (c) cells. Bars = 50 μm.

FIG. 5.

Comparison of the Pf4 genome with the genome of Pf1. Genes are indicated as follows: blue, homologous genes found in both Pf1 and Pf4; red, genes occurring only in Pf4; green, new genes or ORFs identified in this study that occur only in Pf4; grey, genes found only in Pf1 and not in Pf4. The numbers above the Pf1 genes are the ORF numbers as presented in the published genome sequence of Pf1 (24). The numbers below the Pf4 genes are gene numbers from the published P. aeruginosa genome sequence (54).