1887

Abstract

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes and . We investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and RT-PCR analysis of the resulting mutants. The cluster was found to contain eight DNA regions with promoter activity. The regulatory protein NovG binds to a previously identified binding site within the promoter region located upstream of , but apparently not to any of the other seven promoters. Quantitative real-time PCR was used to compare the number of transcripts in a strain carrying an intact novobiocin cluster with strains carrying mutated clusters. Both in-frame deletion of the regulatory gene and insertion of a terminator cassette into the biosynthetic gene led to a strong reduction of the number of transcripts of the genes located between and . This suggested that these 16 biosynthetic genes form a single operon. Three internal promoters are located within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of was found to depend on the presence of NovE, suggesting that the two regulatory genes, and , act in a cascade-like mechanism. The resistance gene , encoding an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from to . However, when the gyrase inhibitor novobiocin accumulates in the cultures, is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA.

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2009-12-01
2020-04-10
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