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 pyrl 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-aminoacid 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 . 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 (a) the leader region has the necessary features to mediate polypeptide synthesis in vivo, (b) the removal of the region corresponding to the pause site and attenuator results in constitutive expression and (c) the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from E . s c~~r i c~i a coli K-12 but notable differences are observed.The pyrimidine biosynthetic enzyme, aspartate transcarbamoylase from Escherichia coli and Sulnionella typhimurium is composed of six catalytic (c) and six regulatory (r) polypeptides, which are organized as two trimeric catalytic (C) subunits and three dimeric regulatory (R) subunits in the native enzyme (reviewed in [l]). The structural genes for the c and r polypeptides have been designated pyrB and pyrl, respectively, and for E. coli the two genes have been shown to comprise an operon, with pyrB being promoter-proximal Previous studies have shown that expression of pyrBI is regulated over a several-hundredfold range [3 -51 and that regulation of expression occurs at the level of transcription [6]. Sequencing studies on the promoter-regulatory region of pyrBI from E. coli led to the identification of a putative rhoindependent terminator (attenuator) located approximately 20 bp upstream of the pyrB gene [7, 81. Also identified was a transcriptional pause site positioned roughly 20 bp before the start of the attenuator region [8]. The region at the pause site encodes a uridine-rich sequence in the transcript. Transcription initiating from a promoter about 160 bp upstream of pyrB, and proceeding without termination at the attenuator, will produce a transcript which can encode a 44-amino-acid peptide, with the termin...

The technique developed by Rosenfeld and Brenchley [J Bacteriol 144, 848-851 (1980)] has been used to introduce Mu d1 (Apr lac) into Salmonella typhimurium for purposes of constructing pyr::lac fusion strains. A stable pyrB::lac fusion mutant was subsequently derived and used for the genetic characterization of the pyrB gene. The direction of transcription of pyrB was determined to be counterclockwise on the S. typhimurium linkage map and argI was shown to be located clockwise of pyrB. Mutants altered in the regulation of expression of pyrB were isolated and two of the isolates chosen for further study were tentatively categorized as promoter or operator mutants.