Cloning, nucleotide sequence and expression of the pyrBI operon of Salmonella typhimurium LT2

Abstract

The pyrB-pyrI region of the Salmonella typhimurium LT2 chromosome has been cloned and sequenced. The two genes were found to constitute an operon, with pyrI being the distal gene and separated from pyrB by a 15-bp intercistronic region. Sequence analysis revealed the presence of two potential promoters; transcription initiated from the promoter proximal to pyrB would produce a transcript which could direct the synthesis of a 33-amino-acid leader peptide. The leader sequence possesses the requisite features of a rho-independent transcriptional terminator (attenuator) which is positioned 22 bp upstream from the pyrB structural gene. A regulatory mutation imparting a 30-fold elevated expression of pyrBI was identified as a two-base-pair deletion in the track of A X T base pairs located on the 3' side of the region of dyad symmetry of the attenuator. The leader sequence also has an additional region of dyad symmetry (putative transcriptional pause site) located 33 nucleotides upstream from the start of the proposed attenuator. The intervening sequence between the putative pause site and the indicated attenuator is characterized by encoding a high content of uracil residues in the transcript. Construction and analysis of transcriptional and translational fusions provided evidence that the leader region has the necessary features to mediate polypeptide synthesis in vivo, the removal of the region corresponding to the pause site and attenuator results in constitutive expression and the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from Escherichia coli K-12 but notable differences are observed.