Transcriptome wide mapping of Hfq mediated RNA-RNA interactions in E. coli (RIL-seq)
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ABSTRACT: Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
Project description:In this study we generated 5'P libraries in wt and RNAse III mutant strains, grown to exponential and stationary phases. Libraries that retain short RNA fragments were also generated in both growth phases. After sequencing by Illumina NextSeq 500 system, reads were mapped to E. coli genome NC_000913.3. By comparing the read start counts per position in the wt and mutant strain libraries we identified the cleavage sites of RNase III.
Project description:To study the regulatory outcome of Hfq-mediated sRNA-target interactions, we measured the change in gene expression following overexpression of each of five well-established sRNAs: GcvB, MicA, ArcZ, RyhB and CyaR. For each of the studied sRNAs we applied RNA-seq to two E. coli K-12 MG1655 strains: (1) a WT strain or a strain deleted of the sRNA gene; (2) a strain overexpressing the sRNA, either artificially from a plasmid or from the endogenous sRNA gene by changing the growth condition. For GcvB induction, the E. coli K-12 MG1655 Z1 gcvB::Cm, pZA12-gcvB and the E. coli K-12 MG1655 Z1 gcvB::Cm, pTP-011 strains were used. For MicA overexpression the E. coli K-12 MG1655lacIq pBRplac, pEF21-Hfq and the E. coli K-12 MG1655lacIq pMicA, pEF21-Hfq strains were used. For ArcZ induction the E. coli K-12 MG1655 Z1 arcZ::Cm, pZE12-ArcZ and the E. coli K-12 MG1655 Z1 arcZ::Cm, pJV300 strains were used. For RyhB overexpression the E. coli K-12 MG1655 ryhB::Cm and the E. coli K-12 MG1655 were used. For CyaR induction the E. coli K-12 MG1655 Z1 cyaR::Cm, pZE12-CyaR and the E. coli K-12 MG1655 Z1 cyaR::Cm, pJV300 strains were used. All E. coli strains used in this study were grown overnight in Luria Bertani (LB) medium at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB medium, and re-grown with shaking at 37 °C to exponential phase, for GcvB (OD600 = 0.3) and for RyhB (OD600 = 0.5), or to stationary phase, for ArcZ WT, ArcZ M1, ArcZ M2 and CyaR (OD600 = 1.0) and for MicA (grown for 6 hr). For induction of ArcZ and GcvB, IPTG was added (1mM, 20 min). MicA was constitutively expressed and further induced at the end of growth with IPTG (1mM, 20 min). For induction of RyhB, the iron chelator 2,2'-Dipyridyl was added (200 μM, 30 min).
Project description:E. coli ΔarcZ hfq-Flag strain was transformed with a WT arcZ, mut arcZC81G or a mut arcZC81T,T82G,G83A plasmids. Single colonies of the transformants were grown overnight in LB at 37 °C with shaking (200 r.p.m.). Cultures were diluted 100-fold in fresh LB re-grown with shaking at 37 °C to an optical density of OD600 = 1.0 and induced with IPTG (1mM, 20 min), RIL-seq experiments were preformed as described in Melamed et al 2018, each repeated three times.
Project description:We report that inhibition of rho by treatment with bicyclomycin, a potential inhibitor of rho affects H-NS binding to chromosome across most of the binding sites. For the current study, we have flag tagged H-NS by homologous recomination method. Cells were grown in the presence and absence of antibiotic bicylomyin. Chromatin immunoprecipitation was performed and anti flag antibody was used for immunoprecipitation. Immunoprecipitated samples both from BCM- and BCM + conditions were sequenced using HiSeq 1000 model. Input DNA was used as control for this experiment which was also sequenced. The 50-mer reads obtained were then mapped to Escherichia coli Mg1655 K-12 genome. Further analysis was carried out to calculate the ChIP signal score. We observed a drop in ChIP signal for bcm+ condition as compared to bcm-. ChIP-seq data generated by Hiseq 1000 model of samples with bcm+ and bcm- in biological duplicates along with Input DNA as control
Project description:Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available. Study using total RNA recovered from five conditions.
Project description:Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics from CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS system approached 1-µL loading capacity, 90-min separation window and high peak capacity (~280) for separation of Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. About 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins were identified from an Escherichia coli lysate by single-shot CZE-MS/MS with spectrum-level false discovery rate (FDR) less than 1%. The number of proteoforms is over three times higher than that from previous single-shot CZE-MS/MS.
Project description:RNAseq was used to map transcription start sites globally in wild type Escherichia coli. Total RNA was isolated from cells grown in LB media until exponential phase.
Project description:Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli using the Rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we appliedanalyzed metabolome and proteome of E.coli deletion mutants., and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR.