MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial...

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and P_BAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 10⁵ distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.