Gallagher, Maurice

Blakely, Garry

Ferguson, Gail

Woodall, Katy Anna

Biotechnology and Biological Sciences Research Council (BBSRC)

2011-11-22T15:57:49Z

2011-11-22T15:57:49Z

2011-11-24

Salmonella enterica sv. typhimurium is a major food-borne pathogen, in part because of its ability to persist and multiply at low temperatures. Adaptation to refrigerated temperatures involves induction of a multigenic cold shock response (CSR); where gene expression is co-ordinately modified, to express cold shock proteins (CSPs). Characterisation of CspA, the major cold shock protein, instigated the identification of other CspA paralogues; which are highly conserved and widespread across species. Six CspA paralogues have previously been identified in S. typhimurium and a csp null strain, lacking all CspA paralogues made. This strain is unable to grow following cold shock, demonstrating that the CspA paralogues play an essential role during low temperature adaptation. The individual CspA paralogues exhibit distinct expression profiles; including expression of CspC and CspE at optimal temperature and CspA and CspB following cold shock. This work investigates the transcriptional changes of S. typhimurium during cold shock and the role of the CspA paralogues under both optimal and cold shock conditions. Using a bacteriophage Mu transposon library (Francis and Gallagher, 1993) this study identifies 7 novel cold induced targets and analyses their native expression levels in SL1344 and the csp null strain during cold shock. This revealed that the regulation of 5 discrete loci including tRNApro2, cpxP and 3 uncharacterised ORFS are mediated by CspA paralogues. In addition, the transcriptional profiles of a highly conserved and essential set of genes encoding known cold shock proteins, NusA, IF2, RbfA, PNPase and CsdA have been characterised. Comparative Northern analysis of SL1344 and the csp null strain has identified a role for CspA paralogues in mediating low temperature induction of three of these genes, through transcription anti-termination. Taken together these results demonstrate that during adaptation to low temperature CspA paralogues regulate expression of genes involved in the translational machinery and metabolic biosynthetic pathways: possibly through a number of transcriptional and post transcriptional processing events. Furthermore this study provides in vivo evidence of the RNA binding activity of the S. typhimurium CspA paralogues. Using fusion proteins, the RNA targets of CspE at 37°C and CspA at 10°C were isolated and analysed. This work identifies 17 direct binding targets for CspE and these indicate that CspE performs a role at optimal growth temperature in regulating components of metabolic (coaA and plsX), translational (EF-Tu, EF-G and IF3) and virulence associated (hha) pathways. Functional redundancy between CspE and CspA was suggested as both paralogues bound 16s rRNA. In light of these findings, the functions of CspA & CspE at optimal and low temperature are discussed. Overall this study has revealed novel information about low temperature adaptation of S. typhimurium, expanding our knowledge of the complexity and importance of the CSR in bacterial pathogens. In addition this work enhances our comprehension of the roles of the CspA paralogues at both optimal and low temperature.

http://hdl.handle.net/1842/5652

en

The University of Edinburgh

salmonella

cold shock

CspA paralogues

Cold shock response of Salmonella enterica serovar typhimurium; the involvement of the CspA paralogues

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy