title: Comparative analysis of mycobacterial truncated hemoglobin promoters and the groEL2 promoter in free-living and intracellular mycobacteria.
abstract: The success of Mycobacterium tuberculosis depends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO(2) levels. Therefore, an effective detoxification system is required for the pathogen to persist in vivo. The genome of M. tuberculosis contains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by the glbO and glbN genes, respectively, important in the survival of M. tuberculosis in macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. Whereas glbN showed an early response to the oxidative and nitrosative stresses tested, glbO gave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested, groEL2 showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb and groEL2 mRNAs showed an initial upregulation at 24 h postinfection. The presence of the glbO protein imparted an increased survival to M. smegmatis in THP-1 differentiated macrophages compared to that imparted by the glbN and hsp65 proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival of M. tuberculosis in the hostile environment of the host.
authors: Department of Molecular Microbiology, Rajiv Gandhi Centre for Biotechnology, Trivandrum, India.
authors: Sunil V
authors: SV
authors: Joseph
authors: G K
authors: GK
authors: Madhavilatha
authors: R Ajay
authors: RA
authors: Kumar
authors: Sathish
authors: S
authors: Mundayoor
mesh_terms: D019677:Artificial Gene Fusion; D018834:Chaperonin 60 / Q000235:genetics*; D003600:Cytosol / Q000382:microbiology; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008264:Macrophages / Q000382:microbiology; D009161:Mycobacterium / Q000235:genetics*; D009603:Nitroso Compounds / Q000633:toxicity; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D013312:Stress, Physiological; D014158:Transcription, Genetic; D054793:Truncated Hemoglobins / Q000235:genetics*
chemical_list: D018834:Chaperonin 60; D009603:Nitroso Compounds; D016877:Oxidants; D054793:Truncated Hemoglobins; D049452:Green Fluorescent Proteins