# The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin–Antitoxin System

## TL;DR

A plasmid in the fish pathogen Yersinia ruckeri is shown to transfer between bacteria and stay stable using a toxin-antitoxin system, though its role in disease remains unclear.

## Contribution

The first characterization of a HigBA toxin–antitoxin system in Yersinia ruckeri and demonstration of pYR4 conjugation.

## Key findings

- The pYR4 plasmid is conjugative and stabilized by a functional HigBA toxin–antitoxin system.
- Loss of pYR4 does not affect bacterial growth, twitching motility, or virulence in an insect larva model.
- pYR4 does not encode a growth burden for Y. ruckeri and may be important for its evolutionary trajectory.

## Abstract

The bacterium Yersinia ruckeri causes enteric redmouth disease, which affects salmonid fish, including farmed fish such as Atlantic salmon and rainbow trout, leading to significant commercial losses. In this study, we investigated a plasmid (an independently replicating DNA molecule) called pYR4 that is suspected of aiding Y. ruckeri in causing disease by producing adhesive structures known as type 4 pili. We identified a toxin–antitoxin locus on the plasmid, higBA, which we hypothesized might prevent loss of the plasmid from the bacterial cell through “addiction” to the antitoxin HigA. HigA is quickly degraded if the plasmid is lost, whereas the toxin HigB is stable and leads to cell death in the absence of HigA. We were able to demonstrate that HigBA is a functional toxin–antitoxin system and that stabilizes pYR4. We further showed that pYR4 can transfer between bacteria by a process known as conjugation. However, loss of the pYR4 plasmid did not have an effect in a simple insect larva infection model. Further work will have to be undertaken to evaluate the role of pYR4 in enteric redmouth disease. These results will help in understanding and ultimately managing a serious disease of commercially important fish.

The bacterium Yersinia ruckeri causes enteric redmouth disease in salmonids and hence has substantial economic implications for the farmed fish industry. The Norwegian Y. ruckeri outbreak isolate NVH_3758 carries a relatively uncharacterized plasmid, pYR4, which encodes both type 4 pili and a type 4 secretion system. In this study, we demonstrate that pYR4 does not impose a growth burden on the Y. ruckeri host bacterium, nor does the plasmid contribute to twitching motility (an indicator of type 4 pilus function) or virulence in a Galleria mellonella larval model of infection. However, we show that pYR4 is conjugative. We also reveal, through mutagenesis, that pYR4 encodes a functional post-segregational killing system, HigBA, that is responsible for plasmid maintenance within Y. ruckeri. This is the first toxin–antitoxin system to be characterized for this organism. Whilst further work is needed to elucidate the virulence role of pYR4 and whether it contributes to bacterial disease under non-laboratory conditions, our results suggest that the plasmid possesses substantial stability and transfer mechanisms that imply importance within the organism. These results add to our understanding of the mobile genetic elements and evolutionary trajectory of Y. ruckeri as an important commercial pathogen, with consequences for human food production.