A mini‐Tn5‐derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram‐negative bacteria

Abstract

Significance and Impact of the Study: Transposon mutagenesis remains a powerful and inexpensive tool in bacterial functional genomics. We developed a transposon with multiple combined features that allow selection of inducible or expressed genes, real-time quantification of gene expression levels using promoter-less GFP and rapid cloning and identification of the inactivated gene. Our Tn5-derived transposon worked in mutagenizing bacteria in the Enterobacteriaceae, Pectobacteriaceae and Pseudomonaceae families, indicating a broad range application. Using the transposon, we generated a high-quality and saturated mutant library of Pectobacterium versatile. Our high efficiency transposon is customizable and has different options for delivery and antibiotic selection, which make it a promising tool for molecular studies.

Phage resistance of Salmonella enterica obtained by transposon Tn5‐mediated SefR gene silent mutation

“…Tn5 transposon is a compound transposon that can be inserted randomly into the genome of a target strain in the form of a fragment, resulting in a random insertion mutation [15]. Tn5 transposon system features strong randomness, high mutation frequency, and stable insertion fragments, and is widely used for discovering new genes, analyzing functions of genes, and constructing mutant libraries [16]. Although more and more new mechanisms are discovered and single mechanisms are studied in deeper detail, there is a lack of studies in which host bacteria with cross-resistance at a relatively small cost.…”