Isolation, cloning, mapping, and nucleotide sequencing of the gene encoding flavodoxin in Escherichia coli

Abstract

The flavodoxins constitute a highly conserved family of small, acidic electron transfer proteins with flavin mononucleotide prosthetic groups. They are found in prokaryotes and in red and green algae, where they provide electrons at low potentials for the reduction of nitrogen by nitrogenase, for the light-dependent reduction of NADP+ in photosynthesis, and for the reduction of sulfite. Proteins with the physical characteristics of flavodoxins have been implicated in the reductive activation of pyruvate formate-lyase and cobalamin-dependent methionine synthase in Escherichia coli. We have purified flavodoxin to homogeneity from E. coli, determined its N-terminal amino acid sequence, and used this sequence to construct a 64-fold degenerate oligonucleotide probe for the flavodoxin gene. Because the phenotype of a flavodoxin mutant is not known, we used this degenerate probe to screen the phages of the Kohara library and identified two phages, with inserts mapping at approximately 16 min, that hybridized to the probe. The flavodoxin gene, designated fldA, was subcloned from the DNA in the overlap region of these two clones. The deduced amino acid sequence, determined by nucleotide sequencing of the flavodoxin gene, shows strong homology with flavodoxins from nitrogen-fixing bacteria and cyanobacteria. The fldA gene maps at 15.9 min on the E. coli chromosome and is transcribed in a counterclockwise direction.