Unknown,Transcriptomics,Genomics,Proteomics

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Impact of mutations in rv2887, putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis.

ABSTRACT: This study was aimed towards understanding the consequences of mutating rv2887, a putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis. Since clones carrying mutations in this gene were found to be highly resistant to a potent mycobactericidal compound, 14, we also tested the impact of short-term exposure to this compound on gene expression in Mycobacterium tuberculosis. Three different strains, wild type H37Rv and the two clones resistant to compound 14, named 1A and 8A, were grown to log phase in 7H9 complete medium. The cells were diluted to an OD of 0.5 and split in to two equal parts, 1 arm received the vehicle (DMSO) and the other arm received compound 14 at a final concentration of 3.9 Î¼M. The exposure lasted 4 hours, at which point the bacterial pellet was collected and processed for RNA extraction using protocols described previously.

ORGANISM(S): Mycobacterium tuberculosis

SUBMITTER: Carl Nathan

PROVIDER: E-GEOD-77556 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. RNA-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. One wild type sample plus one isogenic phoP mutant sample

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Impact of mutations in rv2887, putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis.

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