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Simple Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram‐negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid‐state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N‐termini facing the extracellular surface, and a second with the N‐termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process. YadA is an essential virulence factor of Yersinia enterocolitica and Yersinia pseudotuberculosis. It is a well‐studied member of the trimeric autotransporter adhesins (TAAs). TAAs are exported to the bacterial cell surface by a peculiar mechanism called "Type Vc secretion". In this work, we set out to understand the molecular mechanism of type Vc secretion, and found evidence of a hairpin intermediate that initiates transport. [ABSTRACT FROM AUTHOR]