Detection of a Putative TetR-Like Gene Related toMycobacterium bovisBCG Growth in Cholesterol Using agfp-Transposon Mutagenesis System.
In vitrotransposition 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 IS1096derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly inMycobacterium bovisBCG. Bacteria with a single copy of thegfpgene per chromosome from anM. bovisBCG::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 Tngfpis a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria.
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.
Isabel
I
Otal
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.
Esther
E
Pérez-Herrán
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional Ciudad de Mexico, Mexico.
Lazaro
L
Garcia-Morales
Departamento de Medicina Preventiva, Universidad Autónoma Madrid, Spain.
María C
MC
Menéndez
Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional Ciudad de Mexico, Mexico.
Jorge A
JA
Gonzalez-Y-Merchand
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.
Carlos
C
Martín
Departamento de Medicina Preventiva, Universidad Autónoma Madrid, Spain.
María J
MJ
García
BCG_2177c gene; Mycobacterium bovis BCG; TetR-family; cholesterol; gfp; transposon mutagenesis