Unknown,Transcriptomics,Genomics,Proteomics

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Prior exposure strongly influences mechanisms underpinning survival of heat shock in Escherichia coli

ABSTRACT: In past, heat stress mechanisms have been identified by analysis of RNA-seq or proteomics. Recently, high-throughput transposon mutagenesis coupled with sequencing (TraDIS-Xpress) is another approach proving useful for elucidating the roles of genes involved in the overall cellular response to a particular stress. In this study, we used TraDIS-Xpress to determine the role played by genes following exposure to heat stress at different temperatures. We used two approaches for heat stress, direct heat (heat shock) and stepwise heat increase (gradient heat) stress. Approximately 10^8 cells from the mutant library were inoculated into LB broth at 37C, 44C, 47C, 50C, 44C to 47C and 44C to 50C. Experiments were performed with induction using 0.2mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). All experiments were performed in triplicate.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Muhammad Yasir

PROVIDER: E-MTAB-15219 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A whole genome screen was used to assay every gene of Escherichia coli strain BW25113 to identify genes involved in susceptibility to the monobactam (beta-lactam) antibiotic aztreonam. The methodology has been called TraDIS-Xpress, and is a version of TraDIS or Tn-seq. A transposon mutant library consisting of several hundred thousand mutants was constructed using a Tn5-derived transposon incorporating an inducible outward transcribing promoter. All the mutants were grown in LB broth cultures supplemented with aztreonam at 2 x, 1 x, 0.5 x and 0.25 x MIC with induction of the transposon promoter using 0.2 mM IPTG or 1 mM IPTG or without induction. Following growth, mutants with increased susceptibility show reduced numbers and those with reduced susceptibility show increased numbers. Each condition was performed in duplicate. The methodology enable genes to be assayed by insertional inactivation or by changes in expression. Expression changes result from altered transcription from upstream transposon insertions transcribing into the gene, or downstream insertions transcribing into the gene in the reverse direction leading to RNA interference through the generation of reverse and complementary RNA. Thus, essential genes into which transposon insertions are not tolerated may be assayed also by changes in numbers of upstream or downstream insertion mutants. Changes to high throughput sequencing protocols permit the generation of nucleotide sequence reads from the known transposon sequences into the surrounding insertion site for all the mutants in the mixture simultaneously. Matching the sequence of the reads to the genome nucleotide sequence of E. coli BW25113 then allows the precise locations of all the transposon insertion sites of all the mutants to be mapped simultaneously. The relative changes in mutants between control (without) and selective condition (with aztreonam) then indicates which genes are involved in susceptibility. The numbers of sequence reads that match is reflected by the number of mutants, and so the degree of susceptibility can also be estimated.

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Prior exposure strongly influences mechanisms underpinning survival of heat shock in Escherichia coli

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