title: Use of an arrayed promoter-probe library for the identification of macrophage-regulated genes in Mycobacterium tuberculosis.
abstract: The survival of Mycobacterium tuberculosis within the human host after infection, especially within macrophages, is likely to require the activation of a number of mycobacterial genes. To identify such genes, a promoter-probe library was constructed in which fragments of M. tuberculosis H37Rv DNA were inserted upstream of a lacZ reporter gene, using an Escherichia coli-mycobacterial shuttle vector. Mycobacterium bovis Bacille Calmette-Guérin (BCG) was subsequently transformed with this library and 4800 BCG clones were arrayed in a 96-well microtitre format, enabling the testing of individual clones for promoter activity under a variety of conditions. From preliminary screening, 41 clones were selected that exhibited upregulation of lacZ expression when subjected to acidified sodium nitrite. Subsequent sequence analyses identified 26 of these clones as containing potential promoters. After measuring lacZ expression in BCG clones recovered from a THP-1 macrophage cell line, three genes were selected for assessment of their expression in M. tuberculosis during macrophage infection, by real-time RT-PCR. Two of these genes, Rv1265 (with unknown function) and Rv2711 (encoding the iron-dependent repressor protein IdeR), showed evidence of being upregulated within macrophages.
authors: School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK1.
authors: Russell J
authors: RJ
authors: Hobson
authors: School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK1.
authors: Alan J A
authors: AJA
authors: McBride
authors: GlaxoSmithKline Research and Development, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK2.
authors: Karen E
authors: KE
authors: Kempsell
authors: School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK1.
authors: Jeremy W
authors: JW
authors: Dale
mesh_terms: D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D015698:Genomic Library; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D009163:Mycobacterium bovis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D001616:beta-Galactosidase