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Transcriptional regulation of the novobiocin biosynthetic gene cluster

¹ Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen;
² Institute for Medical Microbiology and Hygiene, Universit&aumltsklinikum Tübingen;
³ Institute for Medical Microbiology and Hygiene, Universitätsklinikum Tübingen;
⁴ Institute de Génétique et Microbiologie, Université Paris-Sud

The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and comprises 20 coding sequences, among them the two regulatory genes novE and novG. We now investigated the location of transcriptional promoters within this cluster by insertion of transcriptional terminator cassettes and reverse transcriptase-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 novH, but apparently not to any of the other seven promoters. Quantitative real-time PCR was now 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 novG or insertion of a terminator cassette into the biosynthetic gene novH led to a strong reduction of the number of transcripts of the genes located between novH and novW. This suggested that these sixteen biosynthetic genes form a single operon. Three internal promoters are localized within this operon but appear to be of minor importance, if any, under our experimental conditions. Transcription of novG was found to depend on the presence of NovE, suggesting that the two regulatory genes novE and novG act in a cascade-like mechanism. The resistance gene gyrBR, coding for an aminocoumarin-resistant gyrase B subunit, may initially be co-transcribed with the genes from novH to novW. However, when the gyrase inhibitor novobiocin accumulates in the cultures, gyrBR is transcribed from its own promoter. Previous work suggested that this promoter may be controlled by the superhelical density of chromosomal DNA.

⁵ E-mail: heide{at}uni-tuebingen.de