Induction of the filamentous prophage, Pf4, is conditionally regulated by PA3572 (difP) in Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, often associated with airway infections in the lungs of people with cystic fibrosis (CF). Biofilms of many P. aeruginosa strains, such as PAO1, produce copious quantities of filamentous Pf4 phage. High titres of Pf4 phage are associated with a decline in lung function in people living with CF. We previously showed that genes encoding Pf4 and also an unlinked hypothetical protein, PA3572, form part of the core biofilm-associated transcriptome in PAO1. Here, using reporter gene assays, we confirm that PA3572 (which we designate difP – d epresses i nduction of f ilamentous P f4 prophage) is strongly induced in biofilms of PAO1. Transcriptomic analysis of a difP deletion mutant revealed elevated expression of several Pf4 ORFs, as well as elevated expression of the cell envelope stress-associated protein, CpxP. These modulations were confirmed by quantitative reverse transcription-PCR, indicating that DifP functions to depress Pf4 gene expression. Consistent with this, Pf4 phage titres were elevated in a Δ difP mutant, whereas over-expression of difP depressed Pf4 titres and Pf4 gene expression. This effect of difP was abolished in a cpxP mutant, indicating that DifP-dependent regulation of Pf4 production is likely linked with cell envelope stress sensing. Taken together and by contrast with most other regulators of Pf4 identified to date (which promote Pf4 production in biofilms), our data indicate that DifP plays a role in restraining Pf4 production in PAO1 biofilms.