Exploring Folate-Small Molecule Interactions in Bacterial Cells

Abstract

Folate (Vitamin B9) is involved in one carbon transfer reactions required for the synthesis of DNA and amino acids. Our current understanding of the folate pathway is mostly based on in vitro studies, which are very different from the crowded environment in the cell. E. coli produces osmoprotectants during times of osmotic stress. This leads to perturbation of water activity inside the cell, and an increase in macromolecular crowding. We have shown earlier that, in vitro, osmolytes weaken the binding of dihydrofolate to dihydrofolate reductase in the folate pathway. We hypothesize that an increased osmolyte concentration in the cell will also prevent the functioning of other folate pathway enzymes by interaction of osmolytes with the various folate redox states. In this study, we studied the effect of osmotic stress on the folate synthesizing and metabolizing enzymes such as dihydropteroate synthase (folP), dihydrofolate reductase (folA), methylenetetrahydrofolate reductase (metF) and serine hydroxymethyltransferase (glyA) in vivo. Studies were done with knockout and rescued strains. Protein expression in rescued strains was limited to very low levels using a Ptet promoter and a protein degradation tag. We can titrate the enzyme activity in the rescued strains by osmotic stress. Osmotic stress studies have indicated that the rescued strain was unable to grow in higher osmolality conditions when compared to knockout strains. We predict this is due to an increase in osmolyte concentration in vivo which leads to interaction of osmolytes with folate intermediates in the pathway. This is turn decreases the efficiency of the folate pathway enzyme. Finally, we have varied the predominant osmolyte in the cell from trehalose to glycine betaine to study if our model holds for these different conditions.