Intracellular survival is critical to establish infection with M. tuberculosis. Understanding how M. tuberculosis survives inside macrophages will provide insight into the pathogenesis of the bacterium. sigmaE, an extracytoplasmic sigma factor of M. tuberculosis, is transcribed more during human macrophage infection than during infection of human type II pneumocytes or during growth in broth culture. A sigmaE mutant is attenuated for survival in human and murine macrophages. Identifying sigmaE-regulated genes may indicate how M. tuberculosis survives inside macrophages.;A genetic approach was developed termed I-TRAPS, identification of transcriptional regulator a ctivated promoter sequences, to identify sigmaE-regulated promoters of M. tuberculosis . I-TRAPS is based on the fact that some genes will be differentially expressed in the presence and absence of a transcriptional regulator. A library of M. tuberculosis genomic DNA was created in a promoter-trap plasmid containing a promoterless kanamycin-resistance gene and xylE gene. In M. smegmatis, all promoters were selected from the library by selection on kanamycin in the presence of overexpressed M. tuberculosis sigmaE. Plasmid DNA from the pool of kanamycin-resistant colonies was transformed into a sigmaE mutant strain of M. smegmatis. M. tuberculosis sigma E-dependent promoters were differentiated from other promoters by screening for expression of the xylE gene. 360 catechol 2,3 dioxygenase negative transformants were identified. Sequencing of 48 of these clones identified 28 putative promoters according to the location of each upstream of known open reading frames.;Using semi-quantitative RT-PCR analysis, gene expression was studied in M. tuberculosis bacteria overexpressing sigma E and was compared to expression in wild type bacteria to determine if transcription of the genes downstream of putative sigmaE-induced promoters was sigmaE-induced. Gene expression was also studied during oxidative and detergent stress to determine if the sigmaE -induced genes are expressed under conditions when the sigE gene is expressed. Gene expression was studied in a sigma E mutant strain of M. tuberculosis treated with SDS and compared to expression in wild type bacteria treated with SDS to confirm sigma E-dependent expression of the genes. Using RT-PCR analysis, 9 clusters of genes, resembling putative operons, are sigmaE-induced. The putative sigmaE consensus sequence was found upstream of 6 sigmaE-induced genes.
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