title: Activity of mycobacterial promoters during intracellular and extracellular growth.
abstract: pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.
authors.forename: Odir A
authors.initials: OA
authors.lastname: Dellagostin
authors.forename: Giovanna
authors.initials: G
authors.lastname: Esposito
authors.forename: Lesley-Jane
authors.initials: LJ
authors.lastname: Eales
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Dale
authors.forename: JohnJoe
authors.initials: J
authors.lastname: McFadden
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics; D016384:Consensus Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C067864:18 kDa protein, Mycobacterium leprae; D001426:Bacterial Proteins; D018834:Chaperonin 60; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase