Transposon and exponential mutagenesis approaches for determining essential genomic functions

Transposon mutagenesis is an increasingly used technique in molecular microbiology, antimicrobial development, and bioproduction of compounds. However limited experimental evidence is available for the reproducibility and intrinsic biases of the technique. The reliance on annotated genomes for determination of essentiality is problematic as annotations can change rendering a gene list inaccurate. Genomic features form networks and functional redundancies can be overlooked using the technique, so methods producing double mutations in a similar fashion would provide better resolution of essential processes within a cell. In this thesis, the reproducibility of transposon library construction was assessed by generating mutant libraries. One library is demonstrated to be reproducible when using the BioTradis pipeline, as long as a sufficient density of mutants with insertions inside coding regions is achieved. Transposon insertion frequency was found to be dependent on factors other than gene essentiality, and these should be taken into account during insertion frequency analysis. Some of these insertion biases can be measured and accounted for and insertion counts normalised to reflect this. Also presented is an annotation-independent approach for determining essentiality using change point analysis that can accept normalised insertion data. This enables identification of essential regions that may or may not code for a known product. Finally, the development of Exponential Mutagenesis, the production of mutants containing multiple transposon insertions is demonstrated, producing preliminary data identifying known genetic interactions in metabolic pathways for folate biosynthesis; these are targeted by sulphonamides and trimethoprim, two therapeutic antimicrobials. A high throughput paired mutant approach will lead to a better understanding of the mechanisms and redundancy involved in bacterial survival and antimicrobial resistance. Investigating the full complement of double mutants should also allow us to understand gene interactions in both metabolic and biosynthetic pathways and highlight essential genomic functions rather than gene products.