Comprehensive Mapping of EZ-Tn5 Transposon Insertion Sites in Pseudomonas argentinensis SA190 Using RATE-PCR.

Abstract

Transposon mutagenesis is a powerful tool for investigating gene function in bacteria, particularly in newly discovered species. In this study, we applied the hyperactive EZ-Tn5 transposase system to Pseudomonas argentinensis SA190, an endophytic bacterium known for enhancing plant resilience under drought stress. By leveraging the random amplification of transposon ends (RATE)-PCR method, we successfully mapped the insertion sites of the transposon within the SA190 genome. This approach enabled the precise identification of disrupted genes, offering insights into their roles in bacterial function and interaction with host plants. Our comprehensive protocol, including competent cell preparation, transformation, and insertion site mapping, provides a reliable framework for future studies aiming to explore gene function through mutagenesis. Key features • The use of the hyperactive EZ-Tn5 transposase system ensures efficient and detectable random mutagenesis across the Pseudomonas argentinensis SA190 genome, facilitating comprehensive gene disruption studies. • The technique is employed to identify and map the transposon insertion sites, allowing for precise determination of gene function and its impact on bacterial phenotypes. • This method enables the exploration of a broad range of gene functions within SA190, particularly those involved in plant growth promotion and stress tolerance. • This method can be readily adapted to generate mutant libraries in other bacterial species, emphasizing its transferability.