AUTHOR=Otal Isabel , Pérez-Herrán Esther , Garcia-Morales Lazaro , Menéndez María C. , Gonzalez-y-Merchand Jorge A. , Martín Carlos , García María J. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.00315

DOI=10.3389/fmicb.2017.00315

ISSN=1664-302X

ABSTRACT=<p><italic>In vitro</italic> 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 IS<italic>1096</italic> derivative, we have constructed the Tn<italic>gfp</italic>, a transposon containing a promoterless green fluorescent protein (<italic>gfp</italic>) gene. This transposon was able to transpose randomly in <italic>Mycobacterium bovis</italic> BCG. Bacteria with a single copy of the <italic>gfp</italic> gene per chromosome from an <italic>M. bovis</italic> 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::Tn<italic>gfp</italic> (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 Tn<italic>gfp</italic> is a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria.</p>