Inducible transposon mutagenesis for genome-scale forward genetics

Area of Science:

• Microbiology
• Bacterial Genetics
• Infectious Disease

Background:

• Transposon insertion sequencing (Tn-seq) is vital for bacterial functional genetics.
• Traditional Tn-seq often suffers from limited mutant diversity due to inefficient delivery or population bottlenecks.
• This limits the detection of subtle gene fitness defects and accurate essentiality classification.

Purpose of the Study:

• To introduce InducTn-seq, a novel method for inducible transposition with temporal control.
• To enhance mutant diversity for sensitive fitness defect detection across all genes.
• To enable quantitative fitness measurements and overcome host bottlenecks in bacterial screens.

Main Methods:

• Developed InducTn-seq leveraging inducible mutagenesis for controlled transposition.
• Generated millions of transposon mutants from a single bacterial colony.
• Applied InducTn-seq in a mouse model of infectious colitis to assess bacterial pathogenesis.

Main Results:

• InducTn-seq generates highly diverse mutant populations, even after a restrictive host bottleneck.
• The method enables sensitive detection of subtle fitness defects in both essential and non-essential genes.
• Revealed the role of oxygen-related metabolic plasticity in bacterial pathogenesis during infection.

Conclusions:

• InducTn-seq overcomes limitations of traditional Tn-seq, enhancing genome-scale forward genetic screens.
• Provides a quantitative measure of gene fitness, transforming binary essentiality classifications.
• Unlocks new possibilities for studying bacterial adaptation and virulence in complex environments.