Abstract
Biofilm development in Pseudomonas aeruginosa is in part dependent on a filamentous phage, Pf4, which contributes to biofilm maturation, cell death, dispersal and variant formation, e.g., small colony variants (SCVs). These biofilm phenotypes correlate with the conversion of the Pf4 phage into a superinfection (SI) variant that reinfects and kills the prophage carrying host, in contrast to other filamentous phage that normally replicate without killing their host. Here we have investigated the physiological cues and genes that may be responsible for this conversion. Flow through biofilms typically developed SI phage approximately days 4 or 5 of development and corresponded with dispersal. Starvation for carbon or nitrogen did not lead to the development of SI phage. In contrast, exposure of the biofilm to nitric oxide, H2O2 or the DNA damaging agent, mitomycin C, showed a trend of increased numbers of SI phage, suggesting that reactive oxygen or nitrogen species (RONS) played a role in the formation of SI phage. In support of this, mutation of oxyR, the major oxidative stress regulator in P. aeruginosa, resulted in higher level of and earlier superinfection compared to the wild-type (WT). Similarly, inactivation of mutS, a DNA mismatch repair gene, resulted in the early appearance of the SI phage and this was four log higher than the WT. In contrast, loss of recA, which is important for DNA repair and the SOS response, also resulted in a delayed and decreased production of SI phage. Treatments or mutations that increased superinfection also correlated with an increase in the production of morphotypic variants. The results suggest that the accumulation of RONS by the biofilm may result in DNA lesions in the Pf4 phage, leading to the formation of SI phage, which subsequently selects for morphotypic variants, such as SCVs.
Highlights
It is increasingly accepted that biofilms, or surface-attached communities, account for the majority of bacteria in the environment and that planktonic cells may be more relevant for the dissemination of cells between biofilm habitats (McDougald et al, 2012)
The results suggest that the accumulation of reactive oxygen or nitrogen species (RONS) by the biofilm may result in DNA lesions in the Pf4 phage, leading to the formation of SI phage, which subsequently selects for morphotypic variants, such as small colony variants (SCVs)
In P. aeruginosa PAO1 biofilms, the lysogenic Pf4 prophage converts into its SI form during the dispersal phase and can be detected by the ability of the SI phage to form plaques on lawns of the WT P. aeruginosa. This indicates that the SI phage can infect and kill P. aeruginosa, which is otherwise resistant to reinfection by the WT Pf4 phage
Summary
It is increasingly accepted that biofilms, or surface-attached communities, account for the majority of bacteria in the environment and that planktonic cells may be more relevant for the dissemination of cells between biofilm habitats (McDougald et al, 2012). The Pf4 phage, which has a genome comprised of 12 Kbp, is a member of the Inoviridae and is closely related to ssDNA phage such as M13 and fd. We have recently shown through deep sequencing of the biofilm dispersal population that the phage accumulates mutations at a significantly higher frequency than the rest of the PAO1 genome, suggesting that superinfection is linked to mutations in the Pf4 prophage (McElroy et al, 2014). The specific conditions that lead to SI are currently not known
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