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