J Bacteriol. 1988 Jul;170(7):3040-5. doi: 10.1128/jb.170.7.3040-3045.1988.

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

Journal of bacteriology

E E Howell, P G Foster, L M Foster

PMID: 2838456 PMCID: PMC211246 DOI: 10.1128/jb.170.7.3040-3045.1988
Free PMC Article

Abstract

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.

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Substances

• DNA, Bacterial
• Tetrahydrofolate Dehydrogenase
• Thymidylate Synthase
• DNA Restriction Enzymes

MeSH terms

• Alleles
• Chromosome Deletion
• Cloning, Molecular
• DNA Restriction Enzymes
• DNA, Bacterial / analysis
• Escherichia coli / enzymology
• Escherichia coli / genetics
• Escherichia coli / growth & development
• Gene Expression Regulation
• Genetic Vectors
• Mutation
• Nucleic Acid Hybridization
• Plasmids
• Tetrahydrofolate Dehydrogenase / genetics
• Thymidylate Synthase / genetics
• Thymidylate Synthase / metabolism
• Transduction, Genetic
• Transformation, Bacterial

Publication Types

• Journal Article
• Research Support, U.S. Gov't, P.H.S.

Grant support

• GM35308/GM/NIGMS NIH HHS/United States

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