The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.The de novo synthesis of pyrimidines is universal. The pathway consists of six enzymatic steps leading to the formation of UMP, which is further converted into UTP, CTP, dCTP, and dTTP. In order to coregulate expression, genes are often found to be organized in operons in procaryotes. The pyrimidine biosynthetic genes (pyr genes) have been found to constitute a single operon in a number of different gram-positive organisms including Bacillus subtilis (37), Bacillus caldolyticus (13), Enterococcus faecalis (23), and Lactobacillus plantarum (9). In addition a pyrimidine biosynthetic operon is found in Mycobacterium tuberculosis (6). The pyrimidine metabolism in Lactococcus lactis has been studied for a number of years. Surprisingly it was found that the pyr genes of L. lactis are scattered on the chromosome in small operons, the pyrKDbF operon (2), the carB gene (35), the pyrEC operon (5), and the pyrDa gene (1). This paper describes the genomic organization of the pyrimidine biosynthetic genes of L. lactis, since we demonstrate the presence of an operon including pyrB, encoding aspartate transcarbamoylase, and carA, encoding the small subunit of the carbamoyl-phosphate (CP) synthetase (CPSase). Moreover, the operon includes pyrR, the regulatory gene controlling expression of the pyr genes, and pyrP, encoding the uracil transporter.Regulation of the expression of the pyr genes of L. lactis has not been subject to a detailed analysis. It has, however, been shown that the transcription of the carB gene is repressed by addition of uracil to the growth medium (35). Moreover, in front of both the carB gene (35) and the pyrKDbF operon (2) a putative attenuator similar to those found in B. subtilis (41) can be identified. These findings suggest that the expression of the pyr genes of L. lactis is regulated by the same attenuator mechanism as that suggested for B. subtilis (41). An RNA binding protein, Pyr...