Downloaded from www.microbiologyresearch.org by IP: 54.146.133.247 On: Mon, 18 Jul 2016 15:04:10 Transcriptional regulation of the novobiocin biosynthetic gene cluster Volker Dangel, 1 Johannes Ha ¨ rle, 1 3 Christiane Goerke, 2 Christiane Wolz, 2 Bertolt Gust, 1 Jean-Luc Pernodet 3 and Lutz Heide 1 Correspondence Lutz Heide heide@uni-tuebingen.de 1 Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universita ¨t Tu ¨ bingen, Auf der Morgenstelle 8, 72076 Tu ¨ bingen, Germany 2 Institute for Medical Microbiology and Hygiene, Universita ¨ tsklinikum Tu ¨ bingen, Elfriede-Aulhorn- Strasse 6, 72076 Tu ¨ bingen, Germany 3 Univ. Paris-Sud 11, CNRS, UMR 8621, Institut de Ge ´ ne ´ tique et Microbiologie, 91405 Orsay Cedex, France Received 12 July 2009 Revised 10 September 2009 Accepted 10 September 2009 The aminocoumarin antibiotic novobiocin is a gyrase inhibitor formed by a Streptomyces strain. The biosynthetic gene cluster of novobiocin spans 23.4 kb and contains 20 coding sequences, among them the two regulatory genes novE and novG. 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 novH, 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 novG and 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 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 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 gyrB R , encoding 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, gyrB R is transcribed from its own promoter. Previous work has suggested that this promoter is controlled by the superhelical density of chromosomal DNA. INTRODUCTION Genome mining, i.e. the sequencing of bacterial genomes in search of novel biosynthetic gene clusters, is a promising new strategy for the discovery of bioactive substances, most importantly antibiotics (Wilkinson & Micklefield, 2007; Zerikly & Challis, 2009). The principal bottleneck for the success of this strategy is the step from sequence data, which can be readily obtained by highly automated procedures, to the production of the encoded secondary metabolites in sufficient quantities for pharmacological screening. This can be achieved, e.g. by heterologous expression of the gene clusters. For this purpose, an understanding of the regulation of the expression of bacterial secondary metabolic gene clusters is of great practical importance for future drug discovery. The aminocoumarin antibiotic novobiocin is produced by Streptomyces caeruleus (syn. Streptomyces spheroides) and is a potent inhibitor of bacterial gyrase (Maxwell & Lawson, 2003). It has been licensed for the treatment of infections with Gram-positive pathogens in humans (Albamycin, Pfizer). The closely related aminocoumarins clorobiocin and coumermycin A 1 , produced by different Streptomyces strains, are even more potent antibacterials (Maxwell & Abbreviations: EMSA, electrophoretic mobility shift assay; 4HPP, 4- hydroxyphenylpyruvate; qRT-PCR, quantitative RT-PCR. 3Present address: Pharmaceutical Biology and Biotechnology, Albert- Ludwigs-Universita ¨ t Freiburg, Stefan-Meier-Straße 19, 79104 Freiburg, Germany. A set of supplementary methods, describing electrophoretic mobility shift assays, a supplementary figure, showing quantitative real-time analysis of Streptomyces coelicolor strains, and two supplementary tables, listing bacterial strains, plasmids and cosmids used in this study, and primers used in this study, with references, are available with the online version of this paper. Microbiology (2009), 155, 4025–4035 DOI 10.1099/mic.0.032649-0 032649 G 2009 SGM Printed in Great Britain 4025