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Gene expression profling of Mycobacterium tuberculosis H37Rv and H37RvΔdosSΔdosT in 0.2% glucose/acetate under aerobic/hypoxic conditions

ABSTRACT: Gene expression profling of Mycobacterium tuberculosis H37Rv and H37RvΔdosSΔdosT in 0.2% glucose/acetate under aerobic/hypoxic conditions

PROVIDER: PRJNA482099 | ENA |

REPOSITORIES: ENA

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Gene expression profling of Mycobacterium tuberculosis H37Rv and H37RvΔdosSΔdosT in 0.2% glucose/acetate under aerobic/hypoxic conditions

Project description:The present study reports the gene expression data of Mycobacterium tuberculosis H37Rv and H37RvΔdosSΔdosT (DKO) grown on 0.2 % acetate/glucose under aerobic/hypoxic conditions. Acetate was reported to be present in granulomas of Mycobacterium tuberculosis infected guinea pigs which are also hypoxic. By exposing Mycobacterium tuberculosis H37Rv and H37RvΔdosSΔdosT to different combinations of granulomatous stresses (acetate/glucose and aerobic/hypoxic conditions) alongwith other experimental data, we were able to delineate a new signaling pathway that activates DevR (DosR) regulon through Acetyl phosphate. The presence of two pathways highlights the importance of targeting DevR and not DevS/DosT for intercepting DevRST signalling cascade.

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Project description:Gene expression microarray was performed using early log phase culture at 37 degree and 200 rpm followed by RNA extraction from M.tb H37Rv, HigB KO M.tb H37Rv and complemented Hig B KO M.tb H37Rv For gene expression profiling, total RNA was isolated from wild type H37Rv, higB KO mutant strain and complemented strains. The bacterial pellets for M.tb H37Rv wild type, M.tb H37Rv HigB KO mutant strain and complemented strain were resuspended in 1.0 ml TRIzol (Invitrogen Corporation, Carlsbad, CA); mRNA was extracted as per standard protocols and cleaned using RNAeasy columns (Qiagen GmbH, Hilden, Germany). Extracted mRNA (1 μg) was treated with DNase I using the Ambion DNA-free kit (Invitrogen Corporation, Carlsbad, CA) to remove genomic DNA contamination and quantified using Nanodrop 2000c spectrophotometer (Thermo Scientific, USA). The purity and integrity of RNA samples were assessed Agilent 2100 Bio analyzer (Agilent TechnologiesInc., USA). Further, 25ng of RNA was amplified and labeled using Low input Quick Amp WT Labeling kit (Agilent Technologies, USA). The labeled cRNA was purified using RNAeasy columns (Qiagen, USA) and total yields were quantified using Nanodrop 2000c spectrophotometer.

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