Characterisation of the S. Typhimurium σE regulon

Abstract

The extracytoplasmic stress response (ESR) of the Enterobacteriacea consists mainly of two partially overlapping pathways, one regulated by the alternative sigma factor, σE encoded for by rpoE and the other by the two component regulator cpxRA. This thesis mainly focuses on the regulated arm of the ESR but does discuss potential crosstalk between these two systems. Characterisation of the rpoE regulon in E. coli has been performed using a number of methods, each of which identified different genes (Dartigalongue, Missiakas, and Raina, 2001;Rezuchova et al., 2003). RpoE has been described as essential for survival of E. coli laboratory strains (De Las, Connolly, and Gross, 1997a), but deletion of the rpoE structural gene in S. Typhimurium is not lethal (Humphreys et al, 1999). However the S. Typhimurium rpoE mutation is critical for survival in vivo, as it appears to be for a number of pathogenic bacteria (Humphreys et al, 1999;Hild et al., 2000;Craig, Nobbs, and High, 2002;Heungens, Cowles, and Goodrich-Blair, 2002;Kovacikova and Skorupski, 2002;Testerman et al, 2002;Heusipp, Schmidt, and Miller, 2003). Considering the different locations and microenvironments encountered by these different pathogens it is likely that each of the rpoE regulons may contain different genes. To establish which are important for S. Typhimurium our collaborators and I have utilised a number of techniques to identify RpoE regulated genes. In collaboration with the group of Dr. Jan Kormanec, Slovakia, a two plasmid system has been utilised. We have also been given access to the S. Typhimurium microarrays at the Institute of Food Research, and used a promoter consensus search derived from known σE regulated genes to search the S. Typhimurium genome for putatively σE regulated genes. With a combination of these techniques we have identified a number of putatively σE regulated genes, and have selected a portion of these to be mutated with lambda red mutagenesis and characterised both in vitro and in vivo