Titration of Folate Pathway Enzymes.

How enzymes behave in cells is likely different from how they behave in the test tube. Previous studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs , osmotic stress titrations were performed with Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a Δ strain, for a methylenetetrahydrofolate reductase clone rescuing a Δ strain, and for a serine hydroxymethyltransferase clone rescuing a Δ strain. Additionally, an R67 dihydrofolate reductase clone allowed DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol. can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactors , and/or viscosity effects.