Project description:Expression profiles of wild-type and SgrR mutant E. coli strains under aMG and 2-DG-induced stress. Expression profiles of E. coli overexpressing SgrS sRNA.
Project description:Expression profiles of wild-type and SgrR mutant E. coli strains under aMG and 2-DG-induced stress. Expression profiles of E. coli overexpressing SgrS sRNA. Illumina RNA-Seq of total RNA extracted from wild-type, SgrR/SgrS mutant and SgrS overexpressing E. coli strains grown in different conditions.
Project description:Mature tRNA pools were measured using an adaptation of YAMAT-seq (Shigematsu et al., 2017; doi:10.1093/nar/gkx005 ) and further described in (Ayan et al., 2020; doi:10.7554/eLife.57947) in 10 strain-medium combinations (all strains dervied from the model bacterium E. coli MG1655). The aim of the experiment was to investigate the effect of reducing tRNA gene copy number on mature tRNA pools in rich and poor media.
Project description:Promoter recognition by bacterial RNA polymerase is mediated by ? subunits, which assemble transiently to RNA polymerase core enzyme (E) during transcription initiation. ? subunits drive transcription of specific sets of genes by allowing RNA polymerase to interact with different promoter sequences. However, ?70, the housekeeping ? subunit, and ?S, an alternative ? subunit mainly active during slow growth and in response to cellular stresses, appear to recognize almost identical promoter sequences, raising the question of how promoter selectivity is achieved in the bacterial cell. To identify?sequence determinants for selective promoter recognition, we performed a run-off/microarray experiment (ROMA): in vitro transcription experiments were carried out with RNA polymerase saturated either with ?70 (E?70) or with ?S (E?S) using the whole Escherichia coli genome as DNA template, and transcript levels were determined by microarray analysis. We found that several genes associated with bacterial growth (e.g., ribosomal operons) were transcribed more efficiently by E?70. In contrast, E?S transcribed preferentially genes involved in stress responses, secondary metabolism, as well as regulatory RNAs and intergenic regions with yet unknown function. Genes preferentially recognized in vitro by E?S showed reduced expression in a??S-deficient mutant strain of E. coli. Sequence comparison of E?70- versus E?S –dependent promoters confirms that the presence of a -35 sequence and the relative location of UP elements affect promoter interaction with either form of RNA polymerase, and suggests that a G/C bias in the -2/+1 nucleotides would favour efficient promoter recognition by E?70. We have performed in vitro transcription experiments with either E?70 or E?S, using the whole E. coli genome as template, to identify promoter regions selectively recognized by two forms of RNA polymerase by using E.coli genome 2.0 GeneChips.
Project description:Promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble transiently to RNA polymerase core enzyme (E) during transcription initiation. σ subunits drive transcription of specific sets of genes by allowing RNA polymerase to interact with different promoter sequences. However, σ70, the housekeeping σ subunit, and σS, an alternative σ subunit mainly active during slow growth and in response to cellular stresses, appear to recognize almost identical promoter sequences, raising the question of how promoter selectivity is achieved in the bacterial cell. To identify sequence determinants for selective promoter recognition, we performed a run-off/microarray experiment (ROMA): in vitro transcription experiments were carried out with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template, and transcript levels were determined by microarray analysis. We found that several genes associated with bacterial growth (e.g., ribosomal operons) were transcribed more efficiently by Eσ70. In contrast, EσS transcribed preferentially genes involved in stress responses, secondary metabolism, as well as regulatory RNAs and intergenic regions with yet unknown function. Genes preferentially recognized in vitro by EσS showed reduced expression in EσS -deficient mutant strain of E. coli. Sequence comparison of Eσ70- versus EσS –dependent promoters confirms that the presence of a -35 sequence and the relative location of UP elements affect promoter interaction with either form of RNA polymerase, and suggests that a G/C bias in the -2/+1 nucleotides would favour efficient promoter recognition by Eσ70.
Project description:Mapping the occupancy of FNR, HNS, and IHF throughout the genome of Escherchia coli MG1655 K-12 using an affinity purified antibody under anerobic growth conditions. We also mapped the binding of the ß subunit of RNA Polymerase under both aerobic and anaerobic growth conditions. As a control, we also performed ChIP-chip on FNR in a ∆fnr mutant strain of Escherchia coli MG1655 K-12. We also examined FNR immunoprecipitation at various FNR concentrations using IPTG and Ptac::fnr (PK8263). The ∆hns/∆stpA strains were also used. Descirbed in the manuscript Genome-scale Analysis of E. coli FNR Reveals the Complexity of Bacterial Regulon Structure
Project description:Transcriptome profiles were analyzed using the samples taken at the exponential and stationary phases during the cultivation of REL606 and MG1655 in LB medium. At the exponential growth phase, most highly expressed genes of B were those for replication, translation, or nucleotide transport and metabolism, while many of the K-12 genes were involved in cell motility, transcription, carbohydrate transport, or energy production. At the stationary phase, many of the genes highly expressed in B were for transport and metabolism of various amino acids and carbohydrates, whereas those in K-12 had functions related to cell motility, ribosomal subunit protein production, or energy generation. Many genes in REL606 and MG1655 showed highly distinct expression levels irrespective of growth conditions. Highly expressed genes in REL606 included those encoding enzymes for biosynthesis of L-arginine (argAGDECBHI) and branched-chain amino acid (ilvGMEDA), and those encoding a subunit of the L-arginine transporter (artJ), cytochrome b562 (cybC), subunits of the histidine ABC transporter (hisPJ), cytotoxins (hokED), outer membrane porin (ompF), L-arginine decarboxylase (speA), and cell division inhibitor (sulA). Highly expressed genes in MG1655 included those for chemotaxis (cheZYRWA, tap, trg, tsr), Lon protease (lon), C4-dicarboxylate-sensing histidine kinase (dcuS), chaperones (clpB, dnaK, groES, htpG, ibpA), the major subunit of type 1 fimbriae (fimA), a regulator of flagellar biosynthesis (flhC), glycerol-3-phosphate-dehydrogenase (glpABCD), glycerophosphoryl diester phosphodiesterase (glpQ), glycerol-3-phosphate transporter (glpT), hydrogenase 2 (hybCBO), outer membrane porins (nmpC, ompA, ompC), and galactitol transport and metabolism (gatYZC).