# Prophage-encoded RexAB-type phage defense system in Pseudomonas putida

## TL;DR

This paper identifies a phage defense system encoded by prophages in Pseudomonas putida that protects the bacteria from superinfection.

## Contribution

The discovery of a RexAB-type phage immunity system encoded by prophage genes PP_5643 and PP_5644 in Pseudomonas putida.

## Key findings

- Prophages 1, 2, and 3 in Pseudomonas putida provide anti-phage activity against specific phages.
- RexBpp, a membrane protein, causes membrane damage and growth cessation in infected bacteria.

## Abstract

Temperate bacteriophages in their prophage form are abundant residents in bacterial genomes. As the fitness of a prophage depends on the welfare of its host, prophages often encode genes that enhance their host’s fitness, thereby also contributing to their own survival. One such strategy for mutual survival is to protect the host from superinfection by other phages. We recently showed that four cryptic prophages contribute to the phage resistance of Pseudomonas putida, a metabolically versatile and industrially promising model bacterium. Here, we show that prophages 1, 2, and 3 exhibit anti-phage activity against specific phages in our CEPEST collection. Focusing on P1-mediated phage defense, we uncover that P1 genes PP_5643 and PP_5644 encode a two-component RexAB-type phage immunity system. The RexApp is a DNA-binding protein that shares structural similarity with phage lambda RexA. RexBpp is a membrane protein that acts as a cell growth-suppressing effector when stimulated by RexApp overexpression, and probably by phage infection. The activation of RexBpp results in membrane damage, as evidenced by increased membrane permeability to SYTOX Green and propidium iodide, as well as leakage of cytoplasmic enzymes. Our data suggest that RexBpp causes growth cessation of infected bacteria and thus aborts the infection.

The online version contains supplementary material available at 10.1038/s41598-026-36734-5.