Development of a new recombineering system for <i>Edwardsiella</i> species.
The new recombineering system enhances genetic manipulation of Edwardsiella species, aiding research into pathogenesis and vaccine development.
Where it sits
this study against the rest of the glutathione (gsh) corpusSummary and findings
The study developed a recombineering system for Edwardsiella species, focusing on genome editing efficiency. The BAS_MS17 system showed the highest recombination efficiency, with improvements achieved by extending homology arms and adding specific proteins. The system successfully knocked out ten genes with high accuracy.
Abstract
<i>Edwardsiella</i> species are important aquaculture pathogens that also cause opportunistic infections in humans, necessitating efficient genome editing tools to study their pathogenesis and develop control strategies. In this study, we identified and characterized six endogenous recombinases pairs from <i>Edwardsiella</i> and its phages. Among these, the BAS_MS17 system exhibited the highest recombination efficiency in <i>E. piscicida</i> EIB202Δp. Extending homology arms from 150 bp to 200 bp improved editing efficiency by 2-fold, while the addition of Redg or Plug further enhanced recombination by 3-fold and 2.5-fold, respectively, without compromising accuracy (100%). More importantly, when applied to <i>E. piscicida</i> sdu12S, Redg or Plug improved the editing efficiency by 8-fold and 7-fold, respectively. Deletion of the phage-derived single-strand binding protein (SSB) reduced efficiency to 25% of the BAS_MS17 level, whereas expression of the endogenous RecA-family SSB (rSSB) increased recombinant yield by 5-fold, highlighting functional conservation. Furthermore, SSB proteins from heterologous hosts failed to enhance recombination efficiency. Using the optimized system, we successfully knocked out ten distinct genes, including virulence-associated loci, with editing accuracy exceeding 85%. Phenotypic analysis revealed that <i>luxR</i>, but not the other tested genes, contributes to biofilm formation. Virulence evaluation results showed that <i>aroA</i>, <i>fur</i>, and <i>hfq</i> are critical virulence-associated factors. Collectively, this streamlined recombineering system provides a simple, rapid, and efficient genetic tool for <i>Edwardsiella</i>, supporting mechanistic studies of virulence and the development of live attenuated vaccine candidates.
Background
Edwardsiella species are significant pathogens in aquaculture and can cause opportunistic infections in humans. Understanding their pathogenesis and developing control strategies require efficient genome editing tools. This study addresses the need for such tools by developing a recombineering system to facilitate genetic manipulation of Edwardsiella species.
Methods
The study identified and characterized six endogenous recombinase pairs from Edwardsiella and its phages. The BAS_MS17 system was tested in E. piscicida EIB202Δp, with homology arms extended from 150 bp to 200 bp. Additional proteins, Redg and Plug, were used to enhance recombination efficiency. The system's efficacy was also tested in E. piscicida sdu12S.
Results
The BAS_MS17 system demonstrated the highest recombination efficiency. Extending homology arms from 150 bp to 200 bp improved efficiency by 2-fold, while Redg and Plug enhanced it by 3-fold and 2.5-fold, respectively. Deleting the phage-derived SSB reduced efficiency to 25% of the BAS_MS17 level, while expressing the endogenous rSSB increased yield by 5-fold. The optimized system allowed successful knockout of ten genes with over 85% accuracy.
Interpretation
The study provides a significant advancement in genetic tools for Edwardsiella species, offering a reliable method for genome editing. While the improvements in recombination efficiency are notable, the clinical significance remains limited to research applications. The study's findings support mechanistic studies of virulence and potential vaccine development, though further validation in broader contexts is needed.
Key findings
- BAS_MS17 system exhibited highest recombination efficiency in E. piscicida EIB202Δp.
- Extending homology arms from 150 bp to 200 bp improved editing efficiency by 2-fold.
- Addition of Redg or Plug enhanced recombination by 3-fold and 2.5-fold, respectively.
- Deletion of phage-derived SSB reduced efficiency to 25% of BAS_MS17 level.
- Expression of endogenous rSSB increased recombinant yield by 5-fold.
Limitations
- Not reported in abstract.