Gallagher, Maurice

Blakely, Garry

Gwynne, Peter John

Medical Research Council (MRC)

2015-04-16T11:00:53Z

2015-04-16T11:00:53Z

2015-06-29

Despite their name, a number of the cold shock proteins are expressed during normal growth, and not just during cold shock, in several species. The function of these constitutively expressed CspA paralogues is unclear. In Salmonella Typhimurium (a major worldwide cause of gastrointestinal disease) they have been linked to various stress responses and the establishment of virulence. Study of the cold shock proteins as gene regulators is therefore of great interest, and they also have potential as targets for antimicrobial development. CspE in Salmonella Typhimurium is constitutively expressed during normal growth. In order to determine its function, attempts were made to identify the interactions it forms with other cellular proteins. Initially, a proteomic investigation attempted to identify proteins which complex with CspE by in vivo cross-linking and affinity purification followed by mass spectrometry. Although no defined complex was consistently identified, the results suggested a handful of proteins which might interact with CspE in a weak or transient manner. These proteins included many from the nucleoid and ribosomal entry site, hinting at CspE’s cellular localisation. In order to investigate these transient interactions, a bacterial two-hybrid system was employed. Interactions between CspE and HupA, a nucleoid protein identified in the proteomic analysis, were probed, as were interactions between CspE and CsdA, an RNA helicase thought to function co-operatively with CspE. The twohybrid system also allowed investigation of CspE dimerisation, which has been reported in vitro but not investigated in vivo until this study. CspE appears not to interact significantly with either HupA, CsdA, or itself at 37oC. Finally in a further attempt to identify interactions of CspE, a genomic library was created to test CspE interactions by two-hybrid assay with random peptides derived from the whole Salmonella genome. The library was successfully created and screened for evidence of interaction, and revealed an association between CspE and a transcriptional repressor, DeoT. DeoT is a repressor of several genes for catabolic processes, suggesting a role for CspE in the regulation of central metabolism. The findings of this work present a number of novel discoveries and several interesting opportunities for further studies.

http://hdl.handle.net/1842/10048

en

The University of Edinburgh

Salmonella

cold shock

post-transcriptional regulation

RNA chaperone

Protein-protein interactions of the cold shock protein CspE of salmonella typhimurium

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy