Variable-number tandem repeat (VNTRs) occur throughout the chromosome of Mycobacterium tuberculosis. Although these polymorphic VNTRs, also known as mycobacterial interspersed repetitive units (MIRUs), have proved to be useful tools in molecular epidemiology, their biological significance is less well understood. This study investigated the polymorphism of the VNTR 3690 locus located in the intergenic region between rv3304 and rv3303c (encoding the gplD2 and lpdA genes, respectively) and its possible function in the regulation of gene expression. The copy number of VNTR 3690 was found to vary among Indian clinical isolates of M. tuberculosis (one to twelve copies), M. tuberculosis H37Rv TMC102 (four copies), M. tuberculosis H37Ra (two to four copies), Mycobacterium bovis BCG (one copy). The expression of lpdA as measured by quantitative RT-PCR was 12-fold higher in M. tuberculosis H37Rv than in M. bovis BCG. Using a GFP reporter system in which the 5′-flanking region of lpdA was fused to the gfp gene, the effect of VNTRs on gene expression was measured in an M. bovis BCG host background by real-time PCR. Compared with one VNTR repeat, a 12.5-fold upregulation of GFP expression was found with a flanking region containing four VNTR 3690 repeats, indicating that there is a good correlation between VNTR copy number and transcription of lpdA.
AkhtarP.,
SrivastavaS.,
SrivastavaA.,
SrivastavaM.,
SrivastavaB. S.,
SrivastavaR.2006; Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice. Microbes Infect 8:2855–2862[CrossRef]
AmanoS.,
TatsumiK.,
TanabeN.,
KasaharaY.,
KurosuK.,
TakiguchiY.,
KasuyaY.,
KimuraS.,
KuriyamaT.2004; Polymorphism of the promoter region of prostacyclin synthase gene in chronic thromboembolic pulmonary hypertension. Respirology 9:184–189[CrossRef]
ArgyrouA.,
VettingM. W.,
BlanchardJ. S.2004; Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis
. J Biol Chem 279:52694–52702[CrossRef]
DebD. K.,
DahiyaP.,
SrivastavaK. K.,
SrivastavaR.,
SrivastavaB. S.2002; Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach. Tuberculosis (Edinb 82:175–182[CrossRef]
FabreM.,
KoeckJ. L.,
Le FlecheP.,
SimonF.,
HerveV.,
VergnaudG.,
PourcelC.2004; High genetic diversity revealed by variable-number tandem repeat genotyping and analysis of hsp65 gene polymorphism in a large collection of “ Mycobacterium canettii ” strains indicates that the Mycobacterium tuberculosis complex is a recently emerged clone of “ M. canettii ”. J Clin Microbiol 42:3248–3255[CrossRef]
FrothinghamR.,
Meeker-O'ConnellW. A.1998; Genetic diversity in the Mycobacterium tuberculosis complex based on variable number of tandem DNA repeats. Microbiology 144:1189–1196[CrossRef]
Le FlècheP.,
FabreM.,
DenoeudF.,
KoeckJ.,
VergnaudG.2002; High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol 2:37–49[CrossRef]
LivakK. J.,
SchmittgenT. D.2001; Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408[CrossRef]
SaxenaA.,
SrivasrtavaV.,
SrivastavaR.,
SrivastavaB. S.2008; Identification of genes of Mycobacterium tuberculosis upregulated during anaerobic persistence by fluorescence and kanamycin resistance selection. Tuberculosis (Edinb) 88:518–525[CrossRef]
SmittipatN.,
BillamasP.,
PalittapongarnpimM.,
Thong-onA.,
TemuM. M.,
ThanakijcharoenP.,
KarnkawinpongO.,
PalittapongarnpimP.2005; Polymorphism of variable number tandem repeats at multiple loci in Mycobacterium tuberculosis
. J Clin Microbiol 43:5034–5043[CrossRef]
Spire-Vayron de la MoureyreC.,
DebuysèreH.,
FazioF.,
SergentE.,
BernardC.,
SabbaghN.,
MarezD.,
Lo GuidiceJ. M.,
D'halluinJ. C.,
BrolyF.1999; Characterization of a variable number tandem repeat region in the thiopurine S -methyltransferase gene promoter. Pharmacogenetics 9:189–198
SreevatsanS.,
PanX.,
StockbauerK. E.,
ConnellN. D.,
KreiswirthB. N.,
WhittamT. S.,
MusserJ. M.1997; Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci U S A 94:9869–9874[CrossRef]
SrivastavaV.,
RouanetC.,
SrivastavaR.,
RamalingamB.,
LochtC.,
SrivastavaB. S.2007; Macrophage-specific M. tuberculosis genes identified by green fluorescent protein and kanamycin resistance selection. Microbiology 153:659–666[CrossRef]
SrivastavaV.,
JainA.,
SrivastavaB. S.,
SrivastavaR.2008; Selection of genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice. Tuberculosis (Edinb 88:171–177[CrossRef]
SupplyP.,
MagdalenaJ.,
HimpensS.,
LochtC.1997; Identification of novel intergenic repetitive units in a mycobacterial two-component system operon. Mol Microbiol 26:991–1003[CrossRef]
SupplyP.,
MazarsE.,
LesjeanS.,
VincentV.,
GicquelB.,
LochtC.2000; Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 36:762–771
SupplyP.,
LesjeanS.,
SavineE.,
KremerL.,
Van SoolingenD.,
LochtC.2001; Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 39:3563–3571[CrossRef]
ValwayS. E.,
SanchezM. P.,
ShinnickT. F.,
OrmeI.,
AgertonT.,
HoyD.,
JonesJ. S.,
WestmorelandH.,
OnoratoI. M.1998; An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis
. N Engl J Med 338:633–639[CrossRef]
Van BelkumA.,
SchererS.,
van LeeuwenW.,
WillemseD.,
van AlphenL.,
VerbrughH.1997; Variable number of tandem repeats in clinical strains of Haemophilus influenzae
. Infect Immun 65:5017–5027
WangG.,
MaierR. J.2004; An NADPH quinone reductase of Helicobacter pylori plays an important role in oxidative stress resistance and host colonization. Infect Immun 72:1391–1396[CrossRef]