Abstract
Background
Glaesserella parasuis (G. parasuis) is a significant pathogen causing Glässer’s disease and substantial financial losses in swine production systems. However, studies on its gene function and pathogenesis are limited due to the lack of a broad marker-free knockout system. Currently, researchers mainly use antibiotic-resistant marked knockout mutants. The expression of antibiotic-resistance genes in bacterial cells can cause polar termination and unpredictable effects. Although there have been precedents in G. parasuis using gene targeting to construct marker-free mutant strains, this method is time-consuming, labor-intensive, and has an extremely low success rate. Therefore, there is an urgent need for an efficient and feasible marker-free genetic manipulation method for G. parasuis strains.
Results
In this study, we developed an optimized marker-free knockout system for G. parasuis based on a temperature-sensitive vector. By culturing the transformants at alternating temperatures of 37°C and 30°C, we used this system to knock out the KanR cassette from the marked mutant JS0135ΔnanH::KanR. To verify that temperature changes were key to this method, we successfully knocked out the nanH and apd genes in the CF7066 strain using this system. Subsequently, we found that knocking out the nanH gene severely reduces the growth vitality of the strains, while knocking out the apd gene can improve the adhesion rate of the strains. Finally, we observed that the expression of recombinant genes in the transformants was higher at 30℃ than at 37℃, but there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in wild strains. This difference may be due to a higher copy number of target plasmids at 30℃, leading to the upregulation of the expression of recombinant genes.
Conclusions
Overall, this newly developed gene knockout system for G. parasuis could be a valuable tool for the study of this organism.