The aminonucleoside antibiotic A201A is inactivated by a phosphotransferase activity from Streptomyces capreolus NRRL 3817, the producing organism. Isolation and molecular characterization of the relevant encoding gene and its DNA flanking regions

Abstract

A novel resistance determinant (ard2) to the aminonucleoside antibiotic A201A was cloned from Streptomyces capreolus NRRL 3817, the producing organism, and expressed in Streptomyces lividans. Sequencing and subcloning experiments of a 3-kb fragment localized ard2 to an ORF of 591 nucleotides. Cell-free extracts from both S. capreolus and S. lividans (ard2) were shown to phosphorylate A201A in an ATP-dependent reaction. The resulting product (P-A201A) was purified and shown to lack any detectable biological activity against a gram-positive indicator organism. Phosphorylation by Ard2 takes place on the hydroxyl group at C2 of the unsaturated hexofuranose moiety of A201A, as shown by 1H-NMR analysis of purified P-A201A. The expression of ard2 appears to be developmentally controlled. In addition to ard2, the sequenced DNA fragment contained two incomplete ORFs (2 and 5) and one complete ORF (4), which appear to determine enzymes of the A201A biosynthetic pathway. Whereas the deduced product of ORF2 did not show any similarity to proteins in data banks, those of ORF5 and ORF4 encode putative glycosyltransferase and ketoreductase activities, respectively. ard2 and these three ORFs seem to be transcribed in a single polycistronic transcript, which supports the notion that they are a part of an A201A biosynthetic gene cluster.