The genes and factors that drive the conversion of the Pseudomonas aeruginosa Pf4 prophage into the superinfective form

Prophage have been identified in most sequenced bacterial genomes, however the effects of prophage genes on the host are poorly understood. Bacteriophage have been shown to play a role in the cell death phenomenon observed during biofilm development of Pseudomonas aeruginosa PAO1. The filamentous Pf4 prophage is important for mediating cell death within microcolonies, dispersal events and variant formation during biofilm development. Further, these effects were shown to result from the establishment of a superinfective phage phenotype. These observations have demonstrated the importance of phage activity in bacterial biofilms and their effects leading to biofilm variant formation and bacterial adaptation. However, little is known about the genetic mechanisms and triggers that lead to the conversion from lysogenic to the superinfective Pf4 phage. The aim of this study was to determine the factors and genes that induce the conversion of the lysogenic Pf4 phage into its lytic, superinfective form during P. aeruginosa PAO1 biofilm development.Here, the mechanisms in the induction into the superinfective phage and the gene responsible for variant formation in the biofilm were demonstrated.Two morphotypic variantswere isolated during the cell death and dispersal phase of the biofilm. These variants, the small colony variant SCV2 and spreader variant S4, carrya superinfective Pf4 phage and exhibited changes in motility and biofilm formation. Genetic analysis of these variants identified mutations within the immunity region of the Pf4 prophage genome. The mutations were identified to lie within the putative repressor c gene of the prophage and the putative promoter of the gene. Moreover, meta-genomic sequencing of pre- and post- dispersal biofilm populations identified the same mutations. The majority of the mutations were within the prophage genome at a frequency of up to 79% in contrast to mutations atless than 7% within the PAO1 genome. Collectively, these results suggest a role of the repressor C in superinfective phage conversion and strong selection for mutations within the immunity region of the phage to facilitate adaptation to biofilm lifecycle in the presence of phage infection.Variant formation is a common trait during biofilm development, as a result of genetic changes in the biofilm community. Furthermore, it has been observed that the appearance of variants from the biofilm correlated with the occurrence of the conversion into the superinfective phage. Environmental stresses have previously shown to cause phenotypic variants and were here tested for induction of the superinfective phage of PAO1 biofilm. Reactive oxygen and nitrogen species have been shown to accumulate within the biofilm microcolonies. Biofilms exposed to oxidative stress induced the conversion of the superinfective phage. Phenotypic variants are a commonly isolated cystic fibrosis patients suffering from lung infection, and have shown to exhibit mutator phenotypes with lost of mut genes of the mismatch repair system. DNA damaging agent mitomycin C and mutS mutant biofilms have also shown to induce superinfective phage. These results have indicated that the oxidative stress and DNA damage are triggers to induce mutations within the biofilm resulting in the conversion into the superinfective form. Interestingly, the OxyR oxidative stress response regulator binds within the repressor c gene, suggesting a potential role of OxyR in the conversion of the superinfective phage.Biofilms have been estimated to be associated with 80% of chronic bacterial infections and have been a challenge for treatment and therapy. While the mechanisms contributing to the conversion into the superinfective Pf4 phage remains elusive, this study has demonstrated the complexity involved in the study of phage infection in biofilms. Phage genes play a major role in the ability of the phage to cause infection against the host and provide resistance for host against infection. The proposed modelinvolves oxidative stress induced mutations within the repressor C region of the prophage andlead to selection for variants that are resistant against superinfective phage. Thus, biofilm variants carrying the superinfective Pf4 phage persist within the biofilm.As P. aeruginosa is known to be a pathogenic bacterium and is involved in several biofilm-related diseases such as chronic infections and cystic fibrosis, it is important to understand the mechanism of biofilm development and to further improve the current treatments conducted

Environmental cues and genes involved in establishment of the superinfective Pf4 phage of Pseudomonas aeruginosa

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. These biofilm phenotypes correlate with the conversion of the Pf4 phage into a superinfective 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 superinfective phage around day 4 or 5 of development and corresponded with dispersal. Starvation for carbon or nitrogen did not lead to the development of superinfective phage. In contrast, exposure of the biofilm to nitric oxide, H2O2 or the DNA damaging agent, mitomycin C, reproducibly led to an increase in the superinfective phage, suggesting that reactive oxygen or nitrogen species (RONS) played a role in the formation of superinfective phage. In support of this, an oxyR mutant, the major oxidative stress regulator in P. aeruginosa, displayed significantly higher and earlier superinfection than the wild-type. Similarly, inactivation of mutS, a DNA mismatch repair gene, resulted in an early and a four log increase in the amount of superinfective phage generated by the biofilm. In contrast, loss of recA, important for DNA repair and SOS response, also resulted in a delayed and decreased production of superinfective 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 superinfective phage, which subsequently selects for morphotypic variants, such as small colony variants