# Collaboration between a temperate phage and Pseudomonas aeruginosa quorum sensing constrains social cheats

## TL;DR

A temperate phage collaborates with Pseudomonas aeruginosa to prevent the spread of non-cooperative bacteria.

## Contribution

A new bacteriophage is shown to help Pseudomonas aeruginosa resist invasion by QS mutants.

## Key findings

- CI27 strain can constrain QS mutant emergence despite lacking cyanide synthesis genes.
- Phage RC3 specifically increases in QS mutants and protects against their invasion.
- Lysates from QS mutants show higher phage numbers and eventual massive lysis.

## Abstract

Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate cooperative behaviors. In the model strain PAO1, QS activates dozens of genes, including those for cyanide production and cyanide resistance. The induction of cyanide synthesis and resistance mechanisms serves to constrain the emergence of QS mutants. Strain CI27 does not possess the cyanide synthesis genes. Here, we ask if this isolate can constrain emergence of QS mutants. In daily transfer experiments, where PAO1 cyanide synthesis mutants are incapable of QS mutant restraint, we found that CI27 constrained emergence of QS mutants. Genome sequencing revealed that a temperate phage, which we call RC5, in evolved QS mutants had copied itself at new chromosomal locations. This led to investigations of the relationship between QS and temperate phages in CI27. We found seven prophage genomes in the CI27 chromosome (RC1–7), one of which occurred in two copies (RC3). Upon mitomycin C induction of CI27, we found four different phage genomes in lysates with RC7 predominating. The lysates of a QS mutant contained three times as many phages as lysates. Without mitomycin C induction, phage numbers in culture fluid from either the wild-type or a CI27 QS mutant were low, but upon daily transfer, phage numbers increased dramatically in the QS mutant. The increase was due to RC3 specifically, and after several transfers, the mutant cultures underwent a massive lysis. Further experiments indicated that RC3 protects P. aeruginosa from QS mutant invasion. Protection might result at least in part from the fact that LasR mutants are more susceptible to RC3 superinfection than the wild type.

Quorum sensing (QS) enables bacteria such as Pseudomonas aeruginosa to coordinate cooperative activities. How bacteria in cooperating groups can resist infiltration by non-cooperating variants is an emerging area of interest in sociobiology and molecular biology. There have been several recent reports on how QS and certain bacteriophage interact. In some strains of P. aeruginosa, QS can activate phage defense systems. At least one bacteriophage can repress P. aeruginosa QS. Here, we show that a previously undescribed bacteriophage can help cooperating groups of P. aeruginosa resist infiltration by non-cooperating QS mutants. This represents a mutualism in which both the bacteriophage and the P. aeruginosa host benefit at least under certain conditions.