b Present address: Microiology and Immunoiology Department, School of Medicine, Queens University of Belfast, Grosvenor Road, Belfast BT12 6BN, Ireland.
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.
ArrudaS., BomfimG., KnightsR., Huima-ByronT.,
RileyL. W.1993; Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science 261:1454–1457[CrossRef]
BarkerL. P., BrooksD. M.,
SmallP. L. C.1998; The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein. Mol Microbiol 29:1167–1177[CrossRef]
BerthetF. X., LagranderieM., GounonP.9 other authors1998; Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene. Science 282:759–762[CrossRef]
ColeS. T., BroschR., ParkhillJ.39 other authors1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544[CrossRef]
CollinsD. M.,
GicquelB.2000; Genetics of mycobacterial virulence. In Molecular Genetics of Mycobacteria pp 265–278 Edited by
HatfullG. F.JacobsW. R.
Washington, DC: American Society for Microbiology;
CollinsD. M., KawakamiR. P., DelisleG. W., PascopellaL., BloomB. R.,
JacobsW. R.1995; Mutation of the principal σ factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 92:8036–8040[CrossRef]
DellagostinO. A., EspositoG., EalesL.-J., DaleJ. W.,
McFaddenJ.1995; Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology 141:1785–1792[CrossRef]
DussurgetO., RodriguezM.,
SmithI.1996; An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative-stress response. Mol Microbiol 22:535–544[CrossRef]
GoldB., RodriguezG. M., MarrasS. A. E., PentecostM.,
SmithI.2001; The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. Mol Microbiol 42:851–865
GomezM.,
SmithI.2000; Determinants of mycobacterial gene expression. In Molecular Genetics of Mycobacteria pp 111–129 Edited by
HatfullG. F.JacobsW.
Washington, DC: American Society for Microbiology;
GrahamJ. E.,
Clark-CurtissJ. E.1999; Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc Natl Acad Sci USA 96:11554–11559[CrossRef]
ManabeY. C., SaviolaB. J., SunL., MurphyJ. R.,
BishaiW. R.1999; Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor. Proc Natl Acad Sci USA 96:12844–12848[CrossRef]
McKinneyJ. D.7 other authorsHoner zu BentrupK.,
Muñoz-EliasE. J.2000; Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735–738[CrossRef]
RanesM. G., RauzierJ., LagranderieM., GheorghiuM.,
GicquelB.1990; Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum : construction of a ‘mini’ Mycobacterium – Escherichia coli shuttle vector. J Bacteriol 172:2793–2797
SchmittM. P., PredichM., DoukhanL., SmithI.,
HolmesR. K.1995; Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae . Infect Immun 63:4284–4289
SunL., vanderSpekJ.,
MurphyJ. R.1998; Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR. Proc Natl Acad Sci USA 95:14985–14990[CrossRef]
TimmJ., GomezM.,
SmithI.1999; Gene expression and regulation. In Mycobacteria: Molecular Biology and Virulence pp 59–92 Edited by
RatledgeC.DaleJ. W.
Oxford: Blackwell Science;
TriccasJ. A., BerthetF.-X., PelicicV.,
GicquelB.1999; Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells. Microbiology 145:2923–2930
TyagiA. K., Das GuptaS. K.,
JainS.2000; Gene expression: reporter gene technologies. In Molecular Genetics of Mycobacteria pp 131–147 Edited by
HatfullG. F.JacobsW. R.
Washington, DC: American Society for Microbiology;
ViaL. E., CurcicR., MuddM. H., DhandayuthapaniS., UlmerR. J.,
DereticV.1996; Elements of signal transduction in Mycobacterium tuberculosis : in vitro phosphorylation and in vivo expression of the response regulator MtrA. J Bacteriol 178:3314–3321