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 in Streptomyces lividans TK64. Five ORFs were found in a 7 kb region directly adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed similarities to ATP-binding cassette (ABC) sugar transporters, the deduced protein sequence of ORF4 displayed similarities to alpha-glucosidases whilst no homology to proteins with known functions was found for ORF5. ORF4 (renamed aglA) was expressed in Escherichia coli and the protein purified and characterized. An alpha-glucosidase activity was detected using the synthetic alpha-glucoside p-nitrophenyl alpha-D-glucopyranoside. Of the many oligosaccharides tested, only sucrose was hydrolysed at a measurable rate [specific activity 32.4 units (mg protein)(-1)] but no growth of S. 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 stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased alpha-glucosidase activity about 10-fold. This probably resulted from induction of a second alpha-glucosidase-encoding gene. The AUD1 element contains three 1 kb repeats which encode DNA-binding proteins necessary for high-frequency 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 AUD1 have a dual function: they are the repressors of the agl genes and they promote amplification of AUD1.
Volff
Jean-Nicolas
JN
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany1.
Altenbuchner
Josef
J
Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany1.
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005959:Glucosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics; D013302:Streptomyces / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D005959:Glucosidases