FolM, a new chromosomally encoded dihydrofolate reductase in Escherichia coli

Abstract

Escherichia coli (thyA DeltafolA) mutants are viable and can grow in minimal medium when supplemented with thymidine alone. Here we present evidence from in vivo and in vitro studies that the ydgB gene determines an alternative dihydrofolate reductase that is related to the trypanosomatid pteridine reductases. We propose to rename this gene folM.

FIG. 1.

Alignment of the Leishmania major PTR1 protein with the E. coli ydgB gene product. The arrows indicate the conserved amino acid residues that are involved in the catalytic activity of PTR1 (5).

FIG. 2.

PCR analyses for the deletions of folA (A) and ydgB (B) genes. (A) PCR was performed with the primers eD-short-up and eD-short-down (Table 2). Genomic DNA of MM612 (lane 1) and MM512 (lane 2) served as templates for the PCR. (B) PCR was performed with the primers ydgB short up and ydgB short down (Table 1). Genomic DNA of MM777 (lane 1) and MG1655 (lane 2) served as templates. DNA size markers are shown (MW).

FIG. 3.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of E. coli FolM. Expression and purification of FolM was carried out as described in the text. Protein extract of induced cells (lane 1) and the purified FolM after elution from the QIAGEN Ni-NTA agarose column (lane 2) are shown.