Determinants of heat resistance in Escherichia coli AW1.7
Ontology highlight
ABSTRACT: Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked AW1.7 or GGG10 cultures was performed to compare gene expression of these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR (q-PCR). DnaK, 30S and 50S risobomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7.
Project description:Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked AW1.7 or GGG10 cultures was performed to compare gene expression of these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR (q-PCR). DnaK, 30S and 50S risobomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50M-BM-0C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Gene expression of a heat resistant strain, E. coli AW1.7, was compared to gene expression in a heat sensitive strain, E coli GGG10. RNA was isolated from late exponential cultures, or from late exponential cells heat-shocked by exposure to 50M-BM-0C for 15 min. Three independent biological repeats were analyzed, and technical repeats (dye-swap) were performed for two of three biological repeats.
Project description:In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to dissociation of 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.
Project description:Distribution of topoisomerase I (EcTopoI) on genomic sites was studied for exponentially growing Escherichia coli in different conditions using ChIP-Seq.
Project description:The heat shock response is critical for organisms to survive at a high temperature. Heterologous expression of eukaryotic molecular chaperons protects Escherichia coli against heat stress. Here we report that expression of the plant E3 ligase BnTR1 significantly increase the thermotolerance of Escherichia coli. Different from eukaryotic chaperones, BnTR1 post-transcriptionally regulates the heat shock factor σ32 though zinc fingers of the RING domain, which interacts with DnaK resulting in stabilizing σ32 and subsequently up-regulating heat shock proteins. Our findings indicate the expression of BnTR1 confers thermoprotective effects on E. coli cells, and it may provide useful clues to engineer thermophilic bacterial strains.
Project description:Distribution of R-loops on genomic sites was studied for exponentially growing Escherichia coli in different conditions using strand-specific DRIP-Seq with S9.6 antibodies.
Project description:We examined the chromosomal distribution of GFP-tagged Tus (Tus-GFP) in exponentially growing E. coli (KRX) using chromosome immunoprecipitation (ChIP)-Seq. Our ChIP-seq allowed delineation of a refined minimal replication fork trap within E. coli comprising two clusters of three Ter sites
Project description:Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading organism with respect to global bacterial protein-protein networks. However, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. In presenting use cases for this resource to be exploited, we show that its stable association with 30S ribosomes gives the seemingly noncoding RNA RyeG of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study complements existing interactome resources for the primary model bacterium E. coli with valuable RNA/protein complexome information and should facilitate functional discovery in this and many related species.
Project description:Two E. coli genotypes (type_II and type_III) that showing a mutation either within rpoH or upstream of groESL were isolated in the thermal adaptative evolution. Transcriptome analyses of the two genotypes exponentially growing at both regular and high temperatures were performed.