BiochemicalPathwaysUnderlyingMulti-metalResistanceinPseudomonasfluorescens.docVIP

Biochemical Pathways Underlying Multi-metal Resistance in Pseudomonas fluorescens By Jacob Fabio Costanzi An Undergraduate Thesis submitted to the Department of Chemistry and Biochemistry at Laurentian University In partial fulfilment for an Honours Bachelor of Science Degree in Biochemistry. Approved by: Supervisor: Date: _____________________ Second Reader: Date: _____________________ Abstract The goal of this research project was to bestow a multiple metal stress consisting of five metals (Al, Fe, Zn, Ca, Ga) on a model biological system, in this case the Pseudomonas fluorescens (ATCC 13525 strain) bacterial strain. There is of great interest in attempting to observe how bacteria are able to thrive under stress conditions. By making use of various techniques such as high pressure liquid chromatography (HPLC) and blue-native polyacrylamide gel electrophoresis (BN), a full metabolic and enzymatic investigation were conducted. Using previous knowledge of how the microbe can effectively eradicate single metal stressors, the goal was to uncover how the cells are able to reconfigure their internal biochemical processes in order to effectively stave off stress. Enzyme activity analysis revealed the up-regulation of several key enzymes most importantly isocitrate lyase (ICL) and acylating glyoxyalate dehydrogenase (AGODH). These enzymes were a part of a specific pathway within the TCA cycle which saw a large production of oxalate in the stress spent fluid samples, thus hinting at the possibility that oxalate serves as a means for scavenging metal stress. The electron transport chain (ETC) was also shut down evidenced by a down-regulation of both complex I and complex IV indicating the cells are applying new measures to produce ATP. Acknowledgements This research project was the culmination of four ye