In Vivo Titration of Folate Pathway Enzymes

Applied and Environmental Microbiology
Deepika NambiarE E Howell

Abstract

How enzymes behave in cells is likely different from how they behave in the test tube. Previous in vitro 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 in vivo, osmotic stress titrations were performed with Escherichia coli 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 ΔfolA strain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetF strain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyA strain. Additionally, an R67 dihydrofolate reductase clone allowed E. coli ...Continue Reading

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Citations

Jun 26, 2020·The Journal of Physical Chemistry. B·Deepika NambiarElizabeth E Howell
Apr 27, 2021·ChemCatChem·Pratul K AgarwalNicolas Doucet

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