MOLECULAR CHARACTERIZATION OF PYRIMIDINE BIOSYNTHESIS GENES FROM THE THERMOPHILE BACILLUS-CALDOLYTICUS

The genes encoding the six pyrimidine biosynthesis enzymes from the th ermophile Bacillus caldolyticus were characterized by cloning and comp lementation in Escherichia coli, and by nucleotide sequence analysis. Nine cistrons are clustered within an 11 kb region of the chromosome. the gene order being: orf1-pyrB-pyrC-pyrAa-pyrAb-orf2-pyrD-pyrF-pyrE. This organization of the cluster is very similar to that of the pyr op eron of Bacillus subtilis. Different parts of the B. caldolyticus clus ter were cloned in two orientations in the expression shuttle vector p HPS9. Complementation studies in B. subtilis established that expressi on of the pyr genes was dependent on the vector-borne promoter, sugges ting that they are part of an operon, and that the native promoter of the operon had not been cloned. The deduced amino acid sequence of the individual cistrons showed 49 to 78% identity with the corresponding B. subtilis cistrons. Measurements of the aspartate transcarbamylase ( pyrB), orotidine monophosphate decarboxylase (pyrF) and orotate phosph oribosyl transferase (pyrE) levels in cells grown under different cond itions indicated that expression of the operon is repressed 7-9-fold b y addition of uracil to the growth medium. Based on the nucleotide seq uence in the intercistronic region between orf1 and pyrB a regulatory mechanism involving transcriptional termination and antitermination is proposed to control expression of the operon.