The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argil laceus, a mithramycin producer, revealed the presence of two open reading f rames (ORFs), The first one (orfA) codes for a protein that resembles sever al transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII -orf4, CcaR, and Redo) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently nonc oding region. Replacement of the mtmR region by an antibiotic resistance ca ssette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in m ithramycin production. The mtmR gene restored actinorhodin production in St reptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activa tor ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmX was found to be repeated approximately 50 kb downstream of mtmR at the oth er end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is p roposed.