Identification of genes regulated by cold temperature - a study of mdtJI in E. coli O157:H7
Urlich, S. (2009). Identification of genes regulated by cold temperature - a study of mdtJI in E. coli O157:H7 (Thesis, Master of Science (MSc)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/3595
Permanent Research Commons link: https://hdl.handle.net/10289/3595
E. coli O157:H7, a serious food-borne pathogen, is capable of adapting to distinctly different environments, ranging from the ruminant, to soil and water. Critical to the success of this pathogen, is the ability to adapt rapidly to changes in the environment. These changes rarely occur in isolation and bacteria, through regulatory networks, can respond to multiple challenges simultaneously, often through master regulators. Understanding the adaptive process of E. coli O157:H7, particularly in response to cold temperatures, is vital for elucidating the pathogens ability to persist during food processing. Of major concern to the meat industry, is the ability of E. coli O157:H7 to survive multiple hurdle intervention strategies that include both chilling and freezing. The aim of this project was to identify genes involved in the cold shock response of E. coli O157:H7 when exposed to refrigeration temperatures (4, 0, -1.5 C). We hypothesized that E. coli O157:H7 is able to withstand chill temperatures by up-regulating genes that allow survival in unfavorable conditions, for example, when the cell is expelled from the ruminant host, into soil or water environments. It is likely that E. coli O157:H7, utilizing similar adaptive mechanisms can withstand prolonged periods at refrigeration temperatures. Furthermore, we speculated that quorum sensing (QS) has overtime become integrated into these adaptive pathways, potentially forming an integrated component of the E. coli O157:H7 adaptive stress response, including the cold shock response. A number of genes were identified as being up-regulated in E. coli O157:H7 during incubation at 4 C on meat. Of these, four were of particular interest, as they had been previously linked to cell survival processes: slp (carbon starvation lipoprotein), hslJ (heat inducible protein), mdtI (multidrug efflux pump) and mdtJ (multidrug efflux pump). RT-PCR data showed that slp and the mdtJI complex are expressed more at refrigeration temperatures than at 37 C while hslJ expression was greatest at 37 C. mdtJI was selected for further analysis, because mdtJI was the only gene that was not expressed at 37 C when grown on BHI media, plus these genes had, at the initiation of this project, not been annotated or assigned function. Using a luxCDABE promoter reporter, real time analysis of the effect of temperature downshift on mdtJI expression was confirmed. Furthermore, expression was demonstrated to increase at high cell density at 37 C, suggesting a regulatory connection to quorum sensing. This coupled with the finding that 400nt upstream of the mdtJI promoter was a gene encoding a transporter of AI-2, a QS autoinducer, suggested a link to the LuxS/AI-2 QS regulatory network. Data presented here was unable to confirm a regulatory link to AI-2 itself but it did reveal a link to LuxS. In conclusion, data presented in this thesis has confirmed that mdtJI is involved in the adaptive response, specifically adaptation to cold temperatures in E. coli O157:H7, and possibly, to growth cessation. The influence of LuxS on mdtJI expression in E. coli O157:H7 is most likely to be through metabolic activity, rather than via a QS mechanism.
The University of Waikato
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- Masters Degree Theses