title: 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.
authors.affiliation: The School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Rice
authors.forename: Chuan Hao
authors.initials: CH
authors.lastname: Tan
authors.forename: Per Jensen
authors.initials: PJ
authors.lastname: Mikkelsen
authors.forename: Vanderlene
authors.initials: V
authors.lastname: Kung
authors.forename: Jerry
authors.initials: J
authors.lastname: Woo
authors.forename: Martin
authors.initials: M
authors.lastname: Tay
authors.forename: Alan
authors.initials: A
authors.lastname: Hauser
authors.forename: Diane
authors.initials: D
authors.lastname: McDougald
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Webb
authors.forename: Staffan
authors.initials: S
authors.lastname: Kjelleberg
mesh_terms: D000818:Animals; D018441:Biofilms / Q000254:growth & development*; D023061:Gene Order; D051379:Mice; D008807:Mice, Inbred BALB C; D039002:Prophages / Q000235:genetics / Q000502:physiology*; D011552:Pseudomonas Infections / Q000382:microbiology; D017105:Pseudomonas Phages / Q000235:genetics / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000472:pathogenicity / Q000502:physiology* / Q000821:virology; D017384:Sequence Deletion; D016019:Survival Analysis; D010948:Viral Plaque Assay; D014774:Virulence