The repressor C protein, Pf4r, controls superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 filamentous phage and regulates host gene expression

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homolog of the P2 phage repressor C. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show that repressor C (Pf4r) is the minimal factor for immunity against reinfection by Pf4 possibly through Pf4r binding to its putative promoter region, and that Pf4r also functions as a transcriptional regulator for expression of host genes. A binding motif for Pf4r was also identified. In wild type P. aeruginosa and Pfr4 complemented Pf4 deficient mutant strains, virulence factor related genes including phenazine and type VI secretion system effectors were upregulated, potentially explaining the reduced virulence of Pf4-deficient P. aeruginosa PAO1. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by MALS analysis where the Pf4r* protein only shows monomer formation. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development. For the ChIPseq, we aim to identify the potential regulon and DNA binding motif of Pf4r. For the RNAseq, we examine if Pf4r causes the differential expression of any genes to see if it also regulates other genes in the PAO1 genome.