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Development and road-testing of an inducible hypermutator strain of Pseudomonas aeruginosa 10.17863/CAM.89061 On, Yue Yuan Welch, Martin Hypermutation occurs when the mismatch repair (MMR) system becomes defective due to e.g., mutation. In Pseudomonas aeruginosa (PA), the mutS gene is commonly mutated in hypermutator strains. MutS mutants display an elevated mutation rate (>100-fold, compared with the wild-type (WT)) and are often associated with the acquisition of antibiotic resistance in clinical PA isolates. In the current work, I construct a strain (PAYY01) of PA in which the expression of mutS (and therefore, the mutation frequency of the strain) can be modulated through the addition of an inert chemical inducer, rhamnose. In PAYY01, the mutation rate can be set to any desired value – either higher or lower than the WT, by adding an appropriate concentration of rhamnose. Indeed, and to my knowledge, this is the first time that a state of controlled hypomutability in PA has been reported. I therefore have a tool that allows me to either “step on the evolutionary accelerator” or “slam on the evolutionary brakes”, and this has opened up a wealth of research avenues to explore. For example, I used whole genome sequencing to investigate the impact of hypermutation during growth of PAYY01 in artificial sputum medium (ASM) and Luria Broth (LB). Very low levels of mutation were observed when mutS expression was “on”, whereas very high levels of mutation were observed when mutS expression was “off”. I also examined how induced hypermutability affects the interaction of PA with Staphylococcus aureus (SA) during co-culture. Somewhat surprisingly, the hypermutator was less invasive than the WT in batch culture during both inter- and intra-species competitions. By contrast, the hypermutator demonstrated a better ability to survive antibiotic (colistin) challenge than the WT. Overall, my data indicate that controlled mutability is an important tool in our efforts to better understand the evolution of drug resistance, inter-species interactions, and pathoadaptation. In addition, I also explore the architecture of the gene cluster surrounding mutS, and find that mutS forms an operon with the adjacent fdxA gene. This, in turn, stimulated an investigation of the possible participation of FdxA in mismatch detection/repair. University of Cambridge 2022-06-26 Thesis Doctoral Doctor of Philosophy (PhD) eng Government of Malaysia https://www.repository.cam.ac.uk/handle/1810/341635 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/28640b73-b119-4610-9f54-726408cb4610/download 208bfa12b38bc784d34d0bb2579cf901 https://www.rioxx.net/licenses/all-rights-reserved/ Pseudomonas aeruginosa inducible hypermutator mutS inter-species interaction intra-species interaction mismatch repair mechanism (MMR) controllable mutation