2:00 pm - 3:00 pm
A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Tongbo Zhu. This seminar is open to the general public to attend.
Thesis Topic: Protein homeostasis in survival and persistence of Escherichia coli, Salmonella Typhimurium and Cronobacter sakazakii at alkaline pH and after desiccation
MSc with Drs. Michael Gaenzle and Tracy Raivio.
Bacteria have evolved a protein homeostasis network to maintain the integrity of their proteome. Chaperones and proteases of the core genome can be complemented by accessory genes encoding additional elements of the protein homeostasis network. The transmissible locus of stress tolerance (tLST) confers exceptional tolerance to high temperature, chlorine, oxidative chemicals and high hydrostatic pressure by reducing protein aggregation. Highly expressed tLST proteins include intracellular small heat shock proteins sHsp20 and sHspGI, disaggregase ClpKGI, and periplasmic stress chaperones PscA and PscB. The tLST also encodes KefBGI, a Na+/H+ antiporter. This study aimed to determine the effects of improved protein homeostasis that is mediated by the tLST on bacterial tolerance to alkaline pH and desiccation.
The expression of the Cpx, a two-component regulatory system in Escherichia coli which responds to envelope stress, was not altered by the presence of the tLST. The tolerance or resistance of E. coli to alkaline pH in the pH range of 6.9 to 9.2 and at pH 11, respectively, were also not changed by the presence of the tLST. The presence of the tLST improved, however, survival at pH 11 in presence of chlorine stress; this effect was attributed to KefBGI rather than protein homeostasis.
The impact of protein homeostasis on desiccation tolerance was characterized in Salmonella Typhimurium, Cronobacter sakazakii and E. coli. The cloning of the shsp20, shspGI and clpKGI decreased desiccation tolerance in S. Typhimurium and C. sakazakii but not in E. coli. Protein aggregates were visualized in vivo using an IbpA-Yfp fusion protein, demonstrating that cloning of shsp20, shspGI and clpKGI reduced intracellular protein aggregates in S. Typhimurium and C. sakazakii but not in E. coli. The presence of resource intensive elements including high copy plasmids as well as cloning of highly expressed proteins had a detrimental effect on desiccation tolerance irrespective of the function of the expressed proteins. Abolishing the ATP-hydrolysis function of ClpKGI by substitution of two amino acids (E383A/E723A) increased cell counts of S. Typhimurium after desiccation more than 100-fold, demonstrating a direct contribution of protein aggregation to desiccation tolerance. Dry storage of S. Typhimurium and C. sakazakii for 112 days revealed that cloning of the tLST or of its protein homeostasis module decreased survival during desiccated storage in infant formula. In conclusion, additional protein quality control provided by tLST does not aid in alkaline resistance or tolerance in E. coli. The desiccation tolerance of S. Typhimurium and C. sakazakii positively correlates with the formation of protein aggregates but are negatively impacted by the presence of resource intensive elements. Taken together, this study improves the understanding of how protein homeostasis affects bacterial stress tolerance by increasing tolerance to some stressors while reducing tolerance to others.