Knockout of hcp genes in Salmonella typhimurium by Red recombination system

Abstract:

[Background] Salmonella is an important food-borne pathogen causing disease such as gastroenteritis, typhoid fever and paratyphoid fever in humans and animals worldwide. The pathogenic mechanism is still unclear. Gene knockout plays an important role in studying the pathogenicity of Salmonella, however, currently it is time consuming and the success rate is very low. S. typhimurium has a Type VI secretion system (T6SS). The component Hemolysin-coregulated protein (Hcp) may play an important role in its pathogenicity. [Objective] To establish a quick and effective gene knock out system by knocking out the three Hcp encoding genes in S. typhimurium, thus to study the pathogenicity of Salmonella. [Methods] Kanamycin resistance gene fragments with homologous upstream and downstream sequences of hcp genes were amplified with pKD4 as template, and then were introduced into S. typhimurium which has Red recombination system enzyme. Then pCP20 were electroporated into the cells to delete the integrated kanamycin resistant gene from the recombinant bacteria. [Results] Both individual hcp genes and their recombinations were successfully knocked out from the genome of S. typhimurium. We also summarized some solutions to the problems we may encounter. [Conclusion] Red recombination system is a good method to knock out genes in S. typhimurium. We can improve the efficiency by optimizing the experimental conditions such as the length of the homologous fragment, the concentration of the PCR templates, the time point for L-arabinose addition and the culture temperature. It is a simple and efficient method and deserves to be popularized.