pTRIP, a novel integration plasmid for Listeria monocytogenes
- 1Georg-August-Universitat Gottingen Abteilung fur Allgemeine Mikrobiologie
Abstract
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 treB insertion plasmid. 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 {Delta}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 Pp60 (pTRIP1) and the inducible Prha (pTRIP2) promoter to further expand application possibilities. Microscopic analyses confirmed the functionality of both promoter constructs and showed dose-dependent induction for Prha. pTRIP is an efficient tool for stable gene expression as well as functional studies and expands genetic modification possibilities for L. monocytogenes.
Related
- Novel, highly divergent clones in Listeria monocytogenes serotype 4b in North America: Sublineages 782 and 1039, members of the hypervirulent clonal complex 2
- No two are the same: Identification and characterization of novel hyperactive PrfA* variants in Listeria monocytogenes
- Combined effects of heat and rhamnolipids against Listeria monocytogenes: Influence of water activity and pH.
- Atypical genomic features among Listeria monocytogenes strains isolated from the pig manure management chain
- Impaired adaptation to smoke-derived phenolic compounds in Listeria monocytogenes CC204 from smoked salmon and trout
BibTeX
@misc{schuler2026ptrip,
title = {{pTRIP, a novel integration plasmid for Listeria monocytogenes}},
author = {J. Schüler and J. Rismondo},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.11.750939},
url = {https://www.biorxiv.org/content/10.64898/2026.09.11.750939v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - pTRIP, a novel integration plasmid for Listeria monocytogenes
AU - J. Schüler
AU - J. Rismondo
PY - 2026
DA - 2026/09/11
PB - bioRxiv
DO - 10.64898/2026.09.11.750939
UR - https://www.biorxiv.org/content/10.64898/2026.09.11.750939v1
AB - 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 treB insertion plasmid. 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 {Delta}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 Pp60 (pTRIP1) and the inducible Prha (pTRIP2) promoter to further expand application possibilities. Microscopic analyses confirmed the functionality of both promoter constructs and showed dose-dependent induction for Prha. pTRIP is an efficient tool for stable gene expression as well as functional studies and expands genetic modification possibilities for L. monocytogenes.
ER -