TY - JOUR
T1 - Detection of a putative TetR-like gene related to Mycobacterium bovis BCG growth in cholesterol using a gfp-transposon mutagenesis system
AU - Otal, Isabel
AU - Pérez-Herrán, Esther
AU - Garcia-Morales, Lazaro
AU - Menéndez, María C.
AU - Gonzalez-y-Merchand, Jorge A.
AU - Martín, Carlos
AU - García, María J.
N1 - Publisher Copyright:
© 2017 Otal, Pérez-Herrán, Garcia-Morales, Menéndez, Gonzalez-y-Merchand, Martín and García.
PY - 2017/3/6
Y1 - 2017/3/6
N2 - 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.
AB - 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.
KW - BCG_2177c gene
KW - Cholesterol
KW - Gfp
KW - Mycobacterium bovis BCG
KW - TetR-family
KW - Transposon mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=85016609278&partnerID=8YFLogxK
U2 - 10.3389/fmicb.2017.00315
DO - 10.3389/fmicb.2017.00315
M3 - Artículo
C2 - 28321208
SN - 1664-302X
VL - 8
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
IS - MAR
M1 - 315
ER -