On a 4.7-kbp HindIII clone of Thermus strain ZO5 DNA, complementing an aspartate carbamoyltransferase mutation in Escherichia coli, we identified a cluster of four potential open reading frames corresponding to genes pyrR, and pyrB, an unidentified open reading frame named bbc, and gene pyrC. The transcription initiation site was mapped at about 115 nucleotides upstream of the pyrR translation start codon. The cognate Thermus pyr promoter also functions in heterologous expression of Thermus pyr genes in E. coli. In Thermus strain ZO5, pyrB and pyrC gene expression is repressed three-to fourfold by uracil and increased twofold by arginine. Based on the occurrence of several transcription signals 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 involving transcriptional attenuation to control pyr gene expression in Thermus. In contrast to pyr attenuation in Bacillus spp., however, control of the Thermus pyr gene cluster would not involve an antiterminator structure but would involve a translating ribosome for preventing formation 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 acids) with ATCases from Pseudomonas sp. For Thermus strain ZO5 dihydroorotase (DHOase; encoded by pyrC), the highest similarity scores (about 40% identity) were obtained with DHOases from B. caldolyticus and Bacillus subtilis. The enzyme properties of ATCase expressed from truncated versions of the Thermus pyr gene cluster in E. coli suggest that Thermus ATCase is stabilized by DHOase and that the translation product of bbc plays a role in feedback inhibition of the ATCase-DHOase complex.Thermus strain ZO5 is an extremely thermophilic gram-negative carotenoid pigmented bacterium which is closely related to Thermus aquaticus YT-1 and to T. thermophilus HB8 (10). The optimal growth temperature of Thermus strain ZO5 is about 72°C, and the maximum temperature for growth 85°C in rich medium. This microorganism has been studied with respect to antibiotic sensitivity, nutritional pattern, primary metabolism (10), and arginine biosynthesis (57).The metabolism of pyrimidines in extreme thermophiles deserves investigation from both fundamental and applied points of view. Genes of the pyrimidine biosynthetic pathway have been successfully used as genetic markers in mesophilic procaryotes and lower eucaryotes. Recently, the pyrE and pyrF genes from Thermus sp. were cloned and sequenced in order to provide selectable markers for developing thermophilic cloning vectors (61, 64). The T. thermophilus HB8 pyrH gene, encoding UMP kinase, was sequenced recently and shown to be located adjacent to the tsf gene, which codes for elongation factor Ts (4).De novo synthesis of pyrimidines involves utilization of energy-rich labile intermediates such as carbamoyl pho...

Bacterial polyynes are highly active natural products with a broad-spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. Through integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), from the biosynthesis gene cluster (BGC) dominant for the production of antifungal polyynes (massilin A, massilin B, collimonin C, and collimonin D) in Massilia sp. YMA4. Phylogenic and chemotaxonomic analyses characterized the core architecture of bacterial polyyne BGC. The crystallographic analysis of the MasL-collimonin C complex indicated that bacterial polyynes serve as a covalent inhibitor of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited cell viability of Candida albicans by targeting ERG10 (homolog of MasL). Overall, understanding the antifungal mechanism of bacterial polyynes presented herein will be useful for the development of polyynes for fungal infections.