Linking phenotype to genotype in pseudonas aeruginosa

Correia, Annapaula (2016) Linking phenotype to genotype in pseudonas aeruginosa. Doctoral thesis, University of East Anglia.

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Abstract

The global transcriptional regulator mexT, is a mutational hotspot; the
sequence variants commonly seen to co-exist within the P. aeruginosa
population are: drug susceptible (e.g. PAO1) and chloramphenicol and
norfloxacin non-susceptible (nfxC mutant). The nfxC phenotype, selected for on chloramphenicol agar is characterised by reduced virulence. The conversion between PAO1 and nfxC phenotypes is associated with an 8-bp repeat sequence in mexT. To investigate the effects of the 8-bp repeat on the adaptive mode of survival of P. aeruginosa, isogenic mutants were generated: PA (8-bp, two copies) and PAdel (8-bp, one copy). The mutants were characterised using phenotypic microarrays (PM), motility, antibiotic susceptibility, Galleria virulence models and RNA-seq in defined media. PM revealed differences in central metabolism indicating that PAdel/PAnfxC were associated with a biological metabolic cost. Strains with the single copy of the 8-bp sequence showed reduced motility and virulence. Transcriptome analysis revealed that mexT, in PA, consists of two regulatory elements defined by an intact helix-turn-helix motif (across the repeat region) which is capable of regulating the downstream LysR region via repressor and autoregulative mechanisms. Whole genome sequencing identified regions of compensatory mutations that were associated with differences in phenotype between PAdel (genetically modified) and PAnfxC (selected). To link phenotype and genotype and to understand the metabolic effects of this mutation, a genome wide metabolic reconstruction was performed. This revealed differences in key metabolic pathways such as glycolysis, gluconeogenesis and oxidative phosphorylation. This study has shown that an 8-bp repeat in mexT is a driver of genetic diversity. Regulatory elements linked to the effect of the 8-bp sequence on antibiotic resistance, central metabolism, chemotaxis, motility and virulence have also been identified. These methods can be used to define phenotype in any pair of isogenic mutants, at the genome level, and to investigate the clinical risk of strains.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Brian Watkins
Date Deposited: 08 Nov 2017 10:37
Last Modified: 08 Nov 2017 10:37
URI: https://ueaeprints.uea.ac.uk/id/eprint/65369
DOI:

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