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A novel cosmid (pABC6.5) whose DNA insert fromStrep-tomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reportedpCAR11 andpCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identifi-cation of a DNA stretch of 19 kbbetweenard1andard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster | Eur. J. Biochem. 269 5527-5535 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03258.x Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus Irene Saugar Eloisa Sanz Miguel Angel Rubio1 Juan Carlos Espinosa and Antonio Jimenez Centro de Biologia Molecular Universidad Autonoma 28049 Madrid Spain A novel cosmid pABC6.5 whose DNA insert from Strep-tomyces capreolus the A201A antibiotic producer overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ardl respectively. Together these three cosmids have permitted the identification of a DNA stretch of 19 kb between ardl and ard2 which should comprise a large region of a putative A201A biosynthetic ata gene cluster. The sequence of the 7 kb upstream of ardl towards ard2 reveals seven consecutive open reading frames ataP3 ataP5 ataP4 ataP10 ataP7 ata12 and ataPKSl. Except for the last two their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6 N6-dimethyl-3 -amino-3 -deoxyade-nosine moiety of puromycin. Because A201A contains this chemical moiety these ataP genes are most likely implicated in its biosynthesis. Accordingly the ataP4 ataP5 and ataP10 genes complemented specific puromycin nonproducing Dpur4 Dpur5 and DpurlO mutants of S. alboniger respectively. Amino acid sequence comparisons suggest that atal2 and ataPKSl could be implicated in the biosynthesis of the D-rhamnose and a-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ardl has disclosed a region which might contain one end of the ata cluster. Keywords aminonucleosides A201A ata cluster Strepto-myces capreolus pur cluster. Nucleoside .
