pTRIP, a novel integration plasmid for Listeria monocytogenes
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In the past decades, several tools to genetically modify the human pathogen Listeria monocytogenes were developed. Here, we constructed a new integrative plasmid system for L. monocytogenes named pTRIP, for tr eB insertion p lasmid. pTRIP is a vector which stably integrates into the treB locus of the wild type EGD-e. This locus encodes the sole trehalose-specific EIIB and EIIC component of a phosphotransferase system. Successful integration leads to the disruption of treB and thus, to an inability of the resulting L. monocytogenes strains to grow on trehalose as sole carbon source. Due to integration through double homologous recombination, it is the first integrative system which does not require antibiotic selection pressure. To assess functionality of the pTRIP system, prfA and its native promoter region were integrated into the treB locus of a Δ prfA strain. Complementation was confirmed in 78% of the isolated clones, indicating successful integration of prfA into the treB locus. We further constructed derivatives of pTRIP harboring the constitutive P p60 (pTRIP1) and the inducible P rha (pTRIP2) promoter to further expand application possibilities. Microscopic analyses confirmed the functionality of both promoter constructs and showed dose-dependent induction for P rha . pTRIP is an efficient tool for stable gene expression as well as functional studies and expands genetic modification possibilities for L. monocytogenes .
Importance
L. monocytogenes is an important human pathogen that can cause severe infections in at-risk group individuals such as elderlies or immunocompromised patients. Over the past decades, several genetic tools have been developed to study the physiology and pathogenicity of L. monocytogenes . Currently, the possibilities to genetically modify L. monocytogenes using integrative plasmids are limited as these plasmids use the same integration site. We constructed the integrative plasmid pTRIP that integrates into the treB locus via homologous recombination. This enables stable integration without the use of antibiotic selection pressure leaving flexibility for usage of additional plasmids such as pPL2 or pIMK3. The pTRIP system therefore expands the genetic toolbox for L. monocytogenes and will be useful for future studies.
