Cloning and characterization of the pyrE gene and of PyrE::Mud1 (Ap lac) fusions from Salmonella typhimurium

Abstract

A lambda-specialized transducing phage carrying the pyrE gene from Salmonella typhimurium LT2 was constructed and used as the source of DNA for subcloning the pyrE gene into pBR322. The pyrE gene product was identified as a 23-kDa polypeptide using a minicell system for analysis of plasmid-encoded proteins. Studies utilizing a promoter-cloning vehicle provided evidence for the existence of two promoter regions, one located close to the start of the structural gene and the other positioned more than 300 base pairs upstream. Transcription from the more distal promotor was the only situation in which significant regulation by pyrimidines was observed. Additional studies served to localize sites involved in the regulation of pyrE expression and led to the inference that regulation does not occur at the level of initiation of transcription. A procedure was developed for the construction of plasmids through recombination in vivo, whereby pyrE::Mud1 (Ap lac) fusions were transferred to a recipient pyrE+ plasmid by bacteriophage P22-mediated transduction. This enabled the identification of the integration sites of Mud within pyrE and also verified the deduced orientation of the pyrE gene in the parental plasmid. The nucleotide sequence of the 5' end of the pyrE gene was determined, including 150 nucleotide residues encoding the first 50 N-terminal amino acids of orotate phosphoribosyltransferase, and 400 nucleotides upstream from the start of the coding region. The leader region contains sequences characteristic of a rho-independent transcriptional terminator preceded by a cluster of thymidylate residues. In addition, the leader RNA contains an open reading frame with a UGA stop codon immediately preceding the putative transcriptional terminator. The nucleotide sequence suggests that pyrE expression is regulated by modulated attenuation, as has been proposed to be the case for both pyrB and pyrE expression in Escherichia coli.