Cloning and characterization of the pyrF operon of Salmonella typhimurium

Abstract

The pyrF gene of Salmonella typhimurium encoding the sixth enzyme of pyrimidine nucleotide biosynthesis, OMP decarboxylase, was isolated from a pyrF-complementing R' factor. A 2.0-kbp DNA fragment, generated by PvuI cleavage, was subsequently subcloned into the multicopy vector pBR322 and shown to contain the intact pyrF gene. Bacterial strains harbouring the resulting plasmid contain 15-20-fold elevated levels of OMP decarboxylase, and these levels increase 4-5-fold during uracil starvation. Experiments utilizing minicells identified the gene product as a polypeptide with a molecular mass of approximately 27 kDa. Furthermore, it was found that the pyrF gene is expressed as the first gene of a bicistronic operon, wherein the second gene encodes an 11-kDa polypeptide of unknown functions. The complete nucleotide sequence of the pyrF operon was determined. An open reading frame, encoding a polypeptide with a calculated molecular mass of 26213 Da, was deduced to be the coding region for pyrF. Another open reading frame, with a translational start codon which overlaps the translational stop codons of the pyrF gene, encodes a polypeptide of 11513 Da. This open reading frame represents the coding region for the second gene of the operon, orfF. S1-nuclease mapping indicated that pyrF transcription is initiated 54 bases upstream of the translational start. The leader region does not show any features resembling the attenuators found preceding the pyrBI operon and the pyrE gene.