STRUCTURE AND EXPRESSION OF A PYRIMIDINE GENE-CLUSTER FROM THE EXTREME THERMOPHILE THERMUS STRAIN ZO5

On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we ident ified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, a nd gene pyrC. The transcription initiation site was mapped at about 11 5 nucleotides upstream of the pyrR translation start codon. The cognat e Thermus pyr promoter also functions in heterologous expression of Th ermus pyr genes in E. coli. In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three- to fourfold by uracil and increased two fold by arginine. Based on the occurrence of several transcription sig nals in the Thermus pyr promoter region and strong amino acid sequence identities (about 60%) between Thermus PyrR and the PyrR attenuation proteins of two Bacillus sp., we propose a regulatory mechanism involv ing transcriptional attenuation to control pyr gene expression in Ther mus. In contrast to pyr attenuation in Bacillus spp., however, central of the Thermus pyr gene cluster would not involve an antiterminator s tructure but would involve a translating ribosome for preventing forma tion of the terminator RNA hairpin. The deduced amino acid sequence of Thermus strain ZO5 aspartate carbamoyltransferase (ATCase; encoded by pyrB) exhibits the highest similarities (about 50% identical amino ac ids) with ATCases from Pseudomonas sp. For Thermus strain ZO5 dihydroo rotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B. caldolyticus and Bac illus subtilis. The enzyme properties of ATCase expressed from truncat ed versions of the Thermus pyr gene cluster in E. coli suggest that Th ermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase comple x.