Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

Détails

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Etat: Public
Version: Final published version
ID Serval
serval:BIB_F65C630A1941
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
Périodique
Frontiers in Cellular and Infection Microbiology
Auteur(s)
Brodmann M., Heilig R., Broz P., Basler M.
ISSN
2235-2988
Statut éditorial
Publié
Date de publication
2018
Peer-reviewed
Oui
Volume
8
Pages
282
Langue
anglais
Résumé
Francisella tularensis is the causative agent of the life-threatening disease tularemia. However, the molecular tools to study Francisella are limited. Especially, expression plasmids are sparse and difficult to use, as they are unstable and prone to spontaneous loss. Most Francisella expression plasmids lack inducible promoters making it difficult to control gene expression levels. In addition, available expression plasmids are mainly designed for F. tularensis, however, genetic differences including restriction-modification systems impede the use of these plasmids in F. novicida, which is often used as a model organism to study Francisella pathogenesis. Here we report construction and characterization of two mobilizable plasmids (pFNMB1 and pFNMB2) designed for regulated gene expression in F. novicida. pFNMB plasmids contain a tetracycline inducible promoter to control gene expression levels and oriT for RP4 mediated mobilization. We show that both plasmids are stably maintained in bacteria for more than 40 generations over 4 days of culturing in the absence of selection against plasmid loss. Expression levels are dependent on anhydrotetracycline concentration and homogeneous in a bacterial population. pFNMB1 and pFNMB2 plasmids differ in the sequence between promoter and translation start site and thus allow to reach different maximum levels of protein expression. We used pFNMB1 and pFNMB2 for complementation of Francisella Pathogenicity Island mutants Delta iglF, Delta iglI, and Delta iglC in-vitro and pFNMB1 to complement Delta iglI mutant in bone marrow derivedmacrophages.
Mots-clé
Francisella novicida, expression plasmid, conjugation, ATc inducible, complementation, type VI secretion system, bacterial mutagenesis, tularemia
Web of science
Open Access
Oui
Création de la notice
13/09/2018 8:38
Dernière modification de la notice
20/08/2019 17:22
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