MiniTn7 Transposon insertion into Variovorax isolates to generate tagged strains for horizontal gene transfer studies.

Three strains of Variovorax were utilized in this study, including the type strain of Variovorax paradoxus (ATCC 17713), a quorum quenching isolate (VAI-C), and Variovorax NFACC26, were genetically modified to analyze the effects of inserting the transposon to the bacterial genome. An introduced Tn7 transposon is inserted by electroporation along with a helper plasmid into each strain. The transposon is predicted to recombine into the genome at a specific location in the main chromosome. The insertion cassette incorporates an antibiotic resistance marker and fluorescent reporter gene for screening and selection purposes. The strains were also tested using a microdilution approach to determine the minimum inhibitory concentration of each antibiotic utilized. This information is subsequently used for selection of transformants. The pTNS3 and mini-Tn7-DsRed were electroporated into the strains. The helper plasmid pTNS3 carries genes used to express the transposition machinery. The mini-Tn7DsRed plasmid cannot replicate in Variovorax,so the antibiotic and fluorescent reporter genes are only able to be maintained by transposon insertion. The chromosomal incorporation was confirmed by sequencing the genome and identifying the Tn7 insertions. These foundational assays and tools institute a platform for future functional studies and genetic manipulation in Variovorax paradoxus ATCC 17713, NFACC26, and VAI-C allowing for examination of interspecies interactions.