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Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

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 pla...

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Autores principales: Brodmann, Maj, Heilig, Rosalie, Broz, Petr, Basler, Marek
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128221/
https://www.ncbi.nlm.nih.gov/pubmed/30234022
http://dx.doi.org/10.3389/fcimb.2018.00284
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author Brodmann, Maj
Heilig, Rosalie
Broz, Petr
Basler, Marek
author_facet Brodmann, Maj
Heilig, Rosalie
Broz, Petr
Basler, Marek
author_sort Brodmann, Maj
collection PubMed
description 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 ΔiglF, ΔiglI, and ΔiglC in-vitro and pFNMB1 to complement ΔiglI mutant in bone marrow derived macrophages.
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spelling pubmed-61282212018-09-19 Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida Brodmann, Maj Heilig, Rosalie Broz, Petr Basler, Marek Front Cell Infect Microbiol Cellular and Infection Microbiology 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 ΔiglF, ΔiglI, and ΔiglC in-vitro and pFNMB1 to complement ΔiglI mutant in bone marrow derived macrophages. Frontiers Media S.A. 2018-08-31 /pmc/articles/PMC6128221/ /pubmed/30234022 http://dx.doi.org/10.3389/fcimb.2018.00284 Text en Copyright © 2018 Brodmann, Heilig, Broz and Basler. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Brodmann, Maj
Heilig, Rosalie
Broz, Petr
Basler, Marek
Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title_full Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title_fullStr Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title_full_unstemmed Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title_short Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida
title_sort mobilizable plasmids for tunable gene expression in francisella novicida
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128221/
https://www.ncbi.nlm.nih.gov/pubmed/30234022
http://dx.doi.org/10.3389/fcimb.2018.00284
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