The <i>In Vivo</i> Effect of Osmolytes on Folate Metabolism

Previous studies have found that addition of osmolytes weakens the binding of dihydrofolate (DHF) to R67 dihydrofolate reductase (DHFR), chromosomal DHFR from E. coli and a pteridine reductase. These results support the preferential interaction of DHF with osmolytes compared to water. Thus, a working model where interaction of DHF with osmolytes shifts the binding away from the protein-DHF complex towards the free species was proposed. As tetrahydrofolate and other folate redox states have similar structures to DHF, we predict osmotic stress will lower the catalytic efficiencies of other folate pathway enzymes. In this thesis, we explore the in vivo effects of increasing osmolality on the activity of folate pathway enzymes. Essential folate enzymes were selected and the genes cloned into a tunable plasmid (pKTS) with a tetracycline promoter (P_tet) and a SsrA degradation tag. The appropriate clone was transformed into a knockout strain of E. coli followed by optimization of the in vivo protein concentration with tetracycline dependent cell growth. Then, the intracellular osmolality of the knockout E. coli strain was increased by adding sorbitol to the growth media. Finally, the effects of increasing osmolality on the function of the clone were determined by comparing the cell growth between the control and test plates.

Our in vivo assays showed R67 DHFR rescued DH5a E. coli from trimethoprim pressure as well as E. coli LH18 (delta fol::kan) from folate end product auxotrophy. Growth of test cells on minimal media was blocked at a lower osmolality compared to growth of a positive control on supplemented media. These results demonstrate a proof of concept that our assay conditions evaluated the in vivo activity of R67 DHFR and found it to be sensitive to osmotic stress.

The genes for two other folate pathway enzymes, methylene tetrahydrofolate reductase and serine hydroxymethyl transferase, were cloned into the pKTS vector. Their ability to respond to in vivo osmotic pressure can now be performed.

Finally, the ability of a strain carrying a mutant folylpolyglutamate synthase gene to withstand osmotic stress was explored. The results were limited by the strain’s lower sensitivity to osmolality, thus further experiments need to be performed.