PYRIMIDINE BIOSYNTHESIS OPERON OF THE THERMOPHILE BACILLUS-CALDOLYTICUS INCLUDES GENES FOR URACIL PHOSPHORIBOSYLTRANSFERASE AND URACIL PERMEASE

A 3-kb DNA segment of the Bacillus caldolyticus genome including the 5 ' end of the pyr cluster has been cloned and sequenced. The sequence r evealed the presence of two open reading frames, pyrR and pyrP, locate d immediately upstream of the previously sequenced pyrB gene encoding the pyrimidine biosynthesis enzyme aspartate transcarbamoylase. The py rR and pyrP genes encoded polypeptides with calculated molecular masse s of 19.9 and 45.2 kDa, respectively. Expression of these ORFs was con firmed by analysis of plasmid-encoded polypeptides in minicells. Seque nce alignment and complementation analyses identified the pyrR gene pr oduct as a uracil phosphoribosyltransferase and the pyrP gene product as a membrane-bound uracil permease. By using promoter expression vect ors, a 650-bp EcoRI-HincII fragment, including the 5' end of pyrR and its upstream region, was found to contain the pyr operon promoter. The transcriptional start point was located by primer extension at a posi tion 153 bp upstream of the pyrR translation initiation codon, 7 bp 3' of a sequence resembling a o(A)-dependent Bacillus subtilis promoter. This established the following organization of the ten cistrons withi n the pyr operon: rR-pyrP-pyrB-pyrC-pyrAa-pyrAb-orf2-pyrD-pyrF-pyrE. T he nucleotide sequences of the region upstream of pyrR and of the pyrR -pyrP and pyrP-pyrB intercistronic regions indicated that the transcri pt may form two mutually exclusive secondary structures within each of these regions. One of these structures resembled a rho-independent tr anscriptional terminator. The possible implication of these structures for pyrimidine regulation of the operon is discussed.