The 1-kb-repeat-encoded DNA-binding protein as repressor of an alpha-glucosidase operon flanking the amplifiable sequence AUD1 of Streptomyces lividans

High-copy-number amplification of the AUD1 element is frequently associated with the large chromosomal deletions responsible for genetic instability i n Streptomyces lividans TK64, Five ORFs were found in a 7 kb region directl y adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed sim ilarities to ATP-binding cassette (ABC) sugar transporters, the deduced pro tein sequence of ORF4 displayed similarities to alpha-glucosidases whilst n o homology to proteins with known functions was found for ORF5, ORF4 (renam ed aglA) was expressed in Escherichia coli and the protein purified and cha racterized. An alpha-glucosidase activity was detected using the synthetic alpha-glucoside p-nitrophenyl alpha-D-glucopyranoside, Of the many oligosac charides tested, only sucrose was hydrolysed at a measurable rate [specific activity 32.4 units (mg protein)(-1)] but no growth of 5, lividans TK64 on sucrose was observed. A strain in which aglA was disrupted showed the same low alpha-glucosidase activity as strain TK64 and in both strains no stimu lation of activity was seen by sucrose, trehalose or maltose; dextrin incre ased alpha-glucosidase activity about 10-fold. This probably resulted from induction of a second a-glucosidase-encoding gene. The AUD1 element contain s three 1 kb repeats which encode DNA-binding proteins necessary for high-f requency amplification. In strains with a unique 1 kb repeat, disruption of the repeat led to a significant increase in the alpha-glucosidase activity . These results strongly suggest that the 1-kb-repeat-encoded proteins of A UD1 have a dual function: they are the repressors of the agl genes and they promote amplification of AUD1.