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Detection of a Putative TetR-Like Gene Related to Mycobacterium bovis BCG Growth in Cholesterol Using a gfp-Transposon Mutagenesis System.

Authors
  • Otal, Isabel1
  • Pérez-Herrán, Esther2
  • Garcia-Morales, Lazaro3
  • Menéndez, María C4
  • Gonzalez-Y-Merchand, Jorge A3
  • Martín, Carlos1
  • García, María J4
  • 1 Grupo de Genética de Micobacterias, Departamento de Microbiologia, Medicina Preventiva y Salud Pública, Universidad de ZaragozaZaragoza, Spain; Centros de Investigación Biomédica en Red Enfermedades Respiratorias, Instituto de Salud Carlos IIIMadrid, Spain; Instituto de Investigación Sanitaria AragónZaragoza, Spain. , (Spain)
  • 2 Grupo de Genética de Micobacterias, Departamento de Microbiologia, Medicina Preventiva y Salud Pública, Universidad de ZaragozaZaragoza, Spain; Diseases of the Developing World, GlaxoSmithKlineTres Cantos, Spain. , (Spain)
  • 3 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional Ciudad de Mexico, Mexico. , (Mexico)
  • 4 Departamento de Medicina Preventiva, Universidad Autónoma Madrid, Spain. , (Spain)
Type
Published Article
Journal
Frontiers in Microbiology
Publisher
Frontiers Media SA
Publication Date
Jan 01, 2017
Volume
8
Pages
315–315
Identifiers
DOI: 10.3389/fmicb.2017.00315
PMID: 28321208
Source
Medline
Keywords
License
Unknown

Abstract

In vitro transposition is a powerful genetic tool for identifying mycobacterial virulence genes and studying virulence factors in relation to the host. Transposon shuttle mutagenesis is a method for constructing stable insertions in the genome of different microorganisms including mycobacteria. Using an IS1096 derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly in Mycobacterium bovis BCG. Bacteria with a single copy of the gfp gene per chromosome from an M. bovis BCG::Tngfp library were analyzed and cells exhibiting high levels of fluorescence were detected by flow cytometry. Application of this approach allowed for the selection of a mutant, BCG_2177c::Tngfp (BCG-Tn), on the basis of high level of long-standing fluorescence at stationary phase. This BCG-Tn mutant showed some particular phenotypic features compared to the wild type strain, mainly during stationary phase, when cholesterol was used as a sole carbon source, thus supporting the relationships of the targeted gene with the regulation of cholesterol metabolism in this bacteria. This approach showed that Tngfp is a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria.

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