Abstract
The Universal Stress Proteins (USPs) are an ancient and widespread group of proteins involved in stress response. The USPs protect against an extensive array of biotic and abiotic stresses, but no biochemical explanation exists for this behaviour. Many USP proteins have been observed to bind nucleotides, but the significance of this behaviour is similarly unknown. An improved mechanistic understanding of this behaviour and a more intense examination of existing phenotypes may reveal their role and how they achieve a seemingly universal role in stress.
This thesis focuses on the biochemical and microbiological characterisation of UspN, a USP from Pseudomonas aeruginosa, and a heptamutant of UspN and six other USPs. UspN/PA4352 is involved in starvation and persistence in anoxic conditions, and UspN mutants are also susceptible to the action of respiratory uncouplers. UspN expression is also a component of the stringent stress response.
In an extension of previous work on bacterial USPs, this thesis demonstrates that uspN, and other USP mutants show aberrant growth phenotypes. Extensive metabolite screening reveals that the effect of USP mutation is greatest in conditions containing central carbon metabolites and amino acids, especially proline. Biochemically, UspN was able to be expressed and purified through recombinant means. UspN was also shown to bind ATP and other adenine-containing nucleotides selectively through circular dichroism spectroscopy. UspN is also denatured at physiologically relevant temperatures and forms high-molecular-weight complexes.
This work combines biochemical and microbiological approaches to support the emerging role of USPs as ATP-binding proteins that control central carbon metabolism and growth. P. aeruginosa is a major human pathogen, infecting the mucosal fluid of patients with incurable respiratory illnesses such as cystic fibrosis, and requires ongoing discovery to overcome antibiotic resistance. The understanding of USPs, and UspN in particular, may have important implications for targeting and designing drugs for UspN and the treatment of P. aeruginosa.