Introduction of a constitutive red fluorescent marker gene into four Variovorax strains using the Tn7 transposon.

Variovorax paradoxus has garnered much interest in environmental microbiology for its ability to degrade organic compounds and key role in the rhizosphere. Despite its important role in microbial communities, genetic manipulation of Variovorax has been proven difficult, but the development of genetically engineered strains is significantly promising for bioremediation applications. The aim of our research group was to create stable mini-Tn7 insertions into several Variovorax strains to express the red fluorescent protein DsRed along with a gene for gentamicin resistance. The Tn7 insertion will facilitate our study of horizontal gene transfer in these specific strains. Four strains were used in this study, MF004, MF295, MF278, and MF375, all of which were isolated from the Arabidopsis rhizosphere. We performed minimum inhibitory concentration experiments on the MF004 and MF295 strains to determine the lowest antibiotic concentration of Kanamycin and Gentamicin to inhibit microbial growth. We introduced the mTn7-DsRed into our strains by electroporation along with a helper plasmid, and then selected for insertion on gentamicin plates. Potential insertion strains were restreaked and then examined by fluorescence microscopy. Red fluorescence was observed in colonies and single cells, indicating successful integration of the transposon. The same experimental approach was also used to introduce the Tn7 into MF278 and MF375. We used the Wizard Genomic DNA Purification Kit to extract the genomic DNA from the bacteria, which will be used for genomic DNA sequencing to verify the insertion site. Preliminary experiments to determine bacterial competence and conjugation are also reported here.