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Osmotic regulation of the Streptomyces lividans thiostrepton-inducible promoter, ptipA

Molecular Biology Research Group, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK¹

Author for correspondence: Paul Dyson. Tel: +44 1792 295667. Fax: +44 1792 295447. e-mail: p.j.dyson{at}swansea.ac.uk

Transcriptional activation of the thiostrepton-inducible promoter, ptipA, in Streptomyces lividans is mediated by TipAL. This transcriptional activator belongs to the MerR/SoxR family that characteristically binds an operator sequence located between the -10 and -35 hexamers normally occupied by RNA polymerase. As for the Escherichia coli merT promoter, the ptipA hexamers are separated by a long 19 bp spacer and hence a topological transition of the DNA is likely to be a requisite for alignment with RNA polymerase. Growth conditions that could facilitate this conformational change were investigated using transcriptional fusions of ptipA with reporter genes. Adjustment of growth medium osmolarity led to increased and prolonged TipAL-dependent expression, both with and without the inducer, thiostrepton. These effects correlated with increases in negative DNA supercoiling. Moreover, an inability to induce the promoter with thiostrepton in strain TK64 was corrected by increasing the concentration of osmolyte, compensating for an apparent reduced level of negative DNA supercoiling in the strain. Prolonging the time of activation of tipA in the wild-type by manipulating growth conditions revealed that mycelial autolysis could be induced by thiostrepton in 4-d-old cultures.

Keywords: Streptomyces, gene expression, morphological development, DNA supercoiling

Abbreviations: NE, nutrient extract; RNAP, RNA polymerase; TPE, Tris-phosphate-EDTA buffer

^a Present address: Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, UK.

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