Advancement and application of tet-regulation in Stenotrophomonas maltophilia

The Gram-negative bacterium Stenotrophomonas maltophilia is ubiquitously present in the environment and represents a potential threat to immunocompromised patients in healthcare settings. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. We have established tetracycline-dependent gene regulation (tet-regulation) in S. maltophilia en route to further advancement and application.
Our one-plasmid-based tet-regulation set up (doi: 10.1128/spectrum.01576-23) contains a chloramphenicol resistance marker, the tetR gene and three intertwined tet-promoters from transposon Tn10. Here, we replaced wt-tetR for a reverse tetR variant whose gene was codon adapted to an elevanted GC content. The dynamic range of the regulatory system was quantified using a super-folder gfp reporter and visualized by confocal laser scanning microscopy. Contrary to wt-TetR, cultivation in the presence of inducer anhydrotetracycline yielded reduced fluorescence compared to inducer-free conditions.
Next, we addressed four potentially essential S. maltophilia genes using wt-tet-regulation by either inducible plasmid-based antisense-RNA expression, or by upstream chromosomal integration of a tet-regulation unit into the genome of S. maltophilia K279a. Growth analyses in liquid medium with or without inducer permitted an assessment of gene essentiality.
Finally, the wt-tet-regulation unit was bracketed by 19 bp "Mosaic Elements" to allow for randomized integration into plasmid- or genomic DNA, exploiting hyperactive Tn5 transposase. In vitro transposition of the tet-insertional element (1633 bp) into pUC19 yielded plasmids with insertions at different positions.
We next plan to assemble ternary complexes of transposase and tet-insertional element (transposomes), for electroporation into S. maltophilia. Dependent on the yield of transformants, we can expect to identify conditional lethal mutant strains with essential genes under tet-control. These approaches may lead to the identification of potential targets for antiinfectives against S. maltophilia.