Project description:In this study we have combined RNA-seq analysis of genome-wide transcriptional start sites with regular RNA-seq to study the transcriptional landscape of Mycobacterium tuberculosis during exponential culture and growth arrest using a starvation model where exponentially growing cells are incubated in PBS for 24 hours. Three independent biological replicates were used in this experiment.
Project description:Mycobacterium tuberculosis is an intracellular human pathogen with the ability to resist and adapt to many adverse conditions it encounters upon infection. Among these, overcoming the production of nitric oxide by macrophages could be key for M. tuberculosis success. We have challenged M. tuberculosis with a sub-lethal concentration of nitric oxide and followed the transcriptomic response through RNA-seq for 48 hours.
Project description:The global diversity of Mycobacterium tuberculosis comprises at least seven lineages, each with its distinct geographic distribution. The aim of this experiment was to perform a comparative analysis of two of these lineages: Lineage 1 and Lineage 2. The former is found around the rim of the Indian ocean and in south-east Asia, while the latter is widely spread throughout Asia and shows an increasing global spread. We have chosen three fully drug susceptible clincal isolates to represent each of the two lineages. We performed RNAseq analysis on rRNA depleted samples isolated from cultures during mid-log phase. Each strain was measured in triplicate.
Project description:The experiment was designed to infer the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through expression analysis. The approach relied on: 1. Tracking the expression changes occurring as a result of the rifampicin-resistance conferring mutation Ser450Leu in RpoB and subsequent gain of compensatory mutation Leu516Pro in RpoC. The hypothesis was that any cost-incurring expressional changes would be reversed in the presence of compensatory mutations. The strains in this set were described before here: PMID 22179134. 2. Comparing the impact of the same rifampicin-resistance mutation (RpoB Ser450Leu) in five different genetic backgrounds. Here the comparison was solely between RpoB Ser450Leu and their cognate drug susceptible wild type ancestor. One of the strain pairs is the same as in point 1. above.
Project description:RNA-seq analysis of total RNA was conducted on wild-type and ΔsubI Mycobacterium tuberculosis (H37Rv strain) to uncover transcriptomic adaptations resulting from the deletion of subI, the gene encoding the sulfate-binding subunit of the SubI-CysTWA sulfate transporter.
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. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. 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). Our prediction suggested that Mcr7 might regulate tatC at the post-transcriptional level by occlusion of the RBS and the consequent translational arrest. Consequently, we studied the secretome from exponentially grown cultures of strain H37Rv, its phoP mutant and a phoP complemented mutant by in-depth proteomics. The results are consistent with a regulatory model involving PhoP, Mcr7 and tatC mRNA since the absence of Mcr7 in the phoP mutant would result in more efficient TatC translation and therefore increased secretion.
Project description:RNase J1 is the first nuclease with 5’-3’ exonuclease activity in bacteria and plays an important role in the maturation and degradation of mRNA. RNase J1 could also play a role in transcription termination of aberrant complexes. RNase J1 could bind to nascent RNA in such complexes, degrade the nascent RNA, and upon catching up with RNA polymerase (RNAP) dissociate the complex. Similar model was showed in eukaryotes. We did ChIP-seq to confirm our hypothesis.
Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. smallRNA-Seq in mouse and rooster testes
Project description:Mitochondrial heteroplasmy, the presence of more than one mtDNA variant in a cell or individual is not as uncommon as previously thought. It is mostly due to the high mutation rate of the mtDNA and limited repair mechanisms present in the mitochondrion. The phenomenon has been studied mostly in human samples and in medical contexts. Heteroplasmy has also been researched in other species in fields such as forensics or genetic foot printing, but these studies usually focused on contained families within closely related species. Here we describe a large cross-species evaluation of heteroplasmy in mammals. We employed a novel approach to detect mitochondrial heteroplasmy in both novel and previously reported ChIP-sequencing datasets, which include concomitant mitochondrial DNA sequenced in the experiment. Here, we report novel ChIP-seq experiments for H3K4me1 and CEBPA across mammals, as well as some H3K4me3, H3K27ac and total histone H3 experiments. Most of the reported CEBPA experiments are good quality pull-downs, however the quality of many of the other experiments reported here has not been interrogated in detail. Whereas this does not affect the investigation of mitochondrial DNA pollution for the purposes of this study, both H3K4me1 and total histone H3 ChIP-seq datasets were often sequenced to relatively low depth and showed low ChIP enrichment compared to the other antibodies.
Project description:In response to host-generated stresses, Mycobacterium tuberculosis (Mtb) reprograms its physiology in myriad ways to establish and maintain an infection, yet the signals that underlie this transformation are not well defined. The abundant toxin-antitoxin (TA) systems harbored in the Mtb genome, including eleven in the mazEF family, are thought to act as stress sensors, yet their roles are largely unknown. Although TA systems from other bacteria are generally thought to impart reversible growth arrest in response to stress, the exquisite specificity of Mtb tRNase toxins instead portends a more nuanced role. Here, we used a proteomics approach to track de novo protein synthesis to uncover molecular events initiated by the Mtb MazF-mt9 toxin (MazF7, Rv2063A). First, we documented striking enrichment of enzymes and transporters derived from the contiguous 36 gene region for phthiocerol dimycocerosate (PDIM) synthesis without an accompanying increase in PDIM lipid production. This paradox was reconciled by concomitant downregulation of proteins comprising the Mce1 transporter (imports host fatty acids), cholesterol breakdown, and β oxidation enzymes (limiting the PDIM precursor methylmalonyl-CoA). Thus, increased catalytic efficiency of the PDIM pathway appears to offset substrate starvation to ensure adequate production of PDIMs essential for Mtb early immune escape and virulence. Finally, isocitrate lyase 1 levels also increased, which in this context are expected to primarily catalyze the glyoxylate shunt to sustain central carbon metabolism while minimizing carbon loss. These exacting proteomic signatures are paralleled within the bedaquiline-treated Mtb transcriptome, highlighting a critical role for MazF-mt9 in orchestrating Mtb stress survival.