Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transposon insertion sequencing (Tn-Seq) to identify fitness determinants during nitrogen starvation, carbon starvation, and transitions between them in Pseudomonas aeruginosa

ABSTRACT: We performed Tn-Seq to identify genetic fitness determinants for starvation survival in Pseudomonas aeruginosa. A pooled mutant library of approximately 180,000 randomly inserted transposon mutants was grown into stationary phase in rich medium (lysogeny broth) and an aliquot of this mutant pool was taken for gDNA extraction and high throughput sequencing of the transposon-genome junctions to determine and count the sites of transposon insertion. This culture was then split and starved for either nitrogen or carbon for 48 hours. Additional aliquots were taken and allowed to grow out in LB medium for 2-3 generations before harvesting for gDNA extraction. The remaining cultures were then split and transitioned again into either carbon starvation or nitrogen starvation for 24 more hours, after which final aliquots were taken, allowed 3-4 generations of outgrowth in LB, and harvested for extracting gDNA. All time points were sampled in biological triplicate in two separate experiments, for a total of 6 replicates. Transposon insertion counts per gene were tallied from high-throughput sequencing data and used to calculate changes from one condition to the next, in order to infer mutation impacts on fitness. Genes whose transpsoson insertion counts decreased in a starvation condition were inferred to benefit survival in that starvation condition, while genes whose transposon insertion counts increased were inferred to perform functions that limited fitness in that condition.

INSTRUMENT(S): NA, QSonica Q800R, NextSeq 2000

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Megan Bergkessel

PROVIDER: E-MTAB-15727 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

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.

Dataset Information

Transposon insertion sequencing (Tn-Seq) to identify fitness determinants during nitrogen starvation, carbon starvation, and transitions between them in Pseudomonas aeruginosa

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets