F6 M. tb knock out mutant Vs wildtype after 24hrs starvation in PBS Tyloxapol
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ABSTRACT: The sRNA F6 in M.tb is under the control of SigF, a non essential sigma factor known to be induced during nutrient starvation. We knocked out this sRNA in M. tb and subjected the strain to 24 hours starvation. RNA/RNA microarrays were then used in order to look at effects in gene expression between wildtype H37Rv and F6 knockout.
Project description:Tuberculosis is an infectious disease, with latent infection with Mycobacterium tuberculosis (M.tb) affecting 1/3 of humanity. It can remain quiescent for long time, making eradication very difficult. To do so, M.tb need to carefully orchestrate its gene expression to survive immune response and starvation but still be able to reactivate to enable transmission to new hosts. Here we used whole transcriptome deep sequencing to compare gene expression between wild type M.tb and a strain with its whiB6, a gene encoding a putative redox-sensing transcription factor, disrupted by a transposon. We found several genes associated with dormancy such as hspX, fdxA and narK2 upregulated in the transposon mutant, indicating that WhiB6 may be a repressor of such genes. The results suggest that WhiB6 may be a complement to the dosS/dosR system in regulating genes important for dormancy. Triplicate cultures of a mutant with its whiB6 gene disrupted by a transposon and wild-type M. tuberculosis CDC1551 were grown in 7H9 media. RNA was extracted and depleted from rRNA. Global gene expression was measured using AB SOLID RNA sequencing.
Project description:The Alphaproteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metal in these sites. It has been reported that C. crescentus responds to chromium exposure by altering expression of a large number of genes. In this work, we showed that the ECF sigma factor SigF is one of the regulatory proteins involved in the control of this transcriptional response. Microarray experiments from cells under dichromate stress showed that SigF controls a small regulon consisting of seven genes. Promoter analyses revealed that a conserved SigF-dependent sequence is located upstream of all but one gene of this regulon. Surprisingly, sigF itself was not found to be strongly auto-regulated under dichromate exposure. Furthermore, we showed that the second gene (CC3252) in the sigF operon acts as a negative regulator of SigF function, as overexpression of this putative membrane protein abolishes the transcriptional activation of sigF and its regulon under dichromate stress. Additionally, we found that substitution of one or both the conserved cysteines in the protein encoded by CC3252 (C131 and C181) affects the ability of this protein in maintaining expression of SigF-dependent genes at basal levels. Interestingly, we showed that the activation of SigF is not caused by reactive oxygen species generated as a consequence of dichromate exposure. Therefore, dichromate most probably may lead to conformational changes in the putative anti-sigma factor CC3252, releasing SigF to bind RNA polymerase core and drive transcription of its regulon. Three replicates perfomed with independent biological samples
Project description:The genome wide ChIP-on-chip analysis to identify the DNA binding sites for the M.tuberculosis sigma factor Rv3286c (SigF) SigF binding sites were determined by microarray analysis of anti-sigF immunoprecipitated DNA from H37Rv(ΔrsbW/sigF)::pmySigF compared to H37Rv(ΔrsbW/sigF)::pMY769 (both cultured with pristinamycin for 3 days). 4 biological replicates were performed.
Project description:RNA-seq was carried out to compare the transcriptomes of wild-type MG1655 E coli with mutant lacking the prominent sRNA, SdsR, in bacteria that had been exposed to long-term (24hr) nitrogen starvation in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of SdsR was to bacteria experiencing long-term nitrogen starvation.
Project description:This SuperSeries is composed of the following subset Series: GSE34919: Genome-wide Definition of the SigF Regulon in Mycobacterium tuberculosis (ChIP-chip) GSE34922: Genome-wide Definition of the SigF Regulon in Mycobacterium tuberculosis (Expression) Refer to individual Series
Project description:Comparing transciptional profile of a M. smegmatis mc2155 ÎsigF strain and wild type (control) in in vitro culture at two time points (exponential phase and stationary phase) for identification of SigF-dependent genes. Analysis of 2 time points: exponential and stationary phase. 4 biological replicates comparing expression of wild type (control) and ÎsigF strain incl. dye swaps for each time point. 3 technical replicates per array
Project description:Proteomics was carried out to compare the proteomes of wild-type MG1655 E coli with mutants lacking either prominent sRNA, SdsR, OxyS or ZbiJ, or lacking the ubiquitous RNA chaperone proteins hfq, in bacteria that had been exposure to long-term (24hr) nitrogen starvation in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of these three sRNA was to bacteria experiencing long-term nitrogen starvation.
Project description:The genome wide transcriptome analysis to identify the M.tuberculosis sigma factor Rv3286c (SigF) dependent transcripts as well as Pristimaycin induced transcripts Pristinamycin induced transcripts were identified by comparing the cDNA extracted from H37Rv::pMY769 with and without pristinamycin (3 biological replicates). SigF controlled transcripts were identified by comparing the cDNA extracted from H37Rv::pMYsigF with and without pristinamycin (4 biological replicates).
Project description:Microparticles (MP) are small membranous particles (100-1000 nm) released under normal steady-state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M.tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M.tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M.tb-infected (TBinf-MP) and uninfected human THP-1 monocytic cells. MP proteins were analysed by GeLC-MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf-MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4) and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP-1 cells to TBinf-MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M.tb infection.
Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression. Two color Experiment,Organism: Mycobacterium Tuberculosis, ilife Discoveries designed Custom Mycobacterium tuberculosis on 8x15k GE Microarray. Two-condition experiment, H37Rv vs. H37Rv/SL3. Biological replicates: 2 biological control H37RV replicates labelled with Cy3, 2 SL3 biological expressing replicates labelled with Cy5.