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 (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.

Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation.

Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10(-3) substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern short-term, within-population bacterial diversification.