doi:10.3389/fmicb.2017.00315engOtal, I.Pérez-Herrán, E.Garcia-Morales, L.Menéndez, M. C.Gonzalez-y-Merchand, J. A.Martín, C.García, M. J.Detection of a putative TetR-like gene related to Mycobacterium bovis BCG growth in cholesterol using a gfp-transposon mutagenesis systemART-2017-98571In 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.2017http://zaguan.unizar.es/record/6099310.3389/fmicb.2017.00315http://zaguan.unizar.es/record/60993oai:zaguan.unizar.es:60993info:eu-repo/grantAgreement/ES/DGA/B25info:eu-repo/grantAgreement/ES/MEC/SAB2010-0102info:eu-repo/grantAgreement/ES/MINECO/BIO2014-5258PFRONTIERS IN MICROBIOLOGY 8 (2017), 315 [13 pp.]byhttp://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccess