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

Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany¹

Author for correspondence: Josef Altenbuchner. Tel: +49 711 685 7591. Fax: +49 711 685 6973. e-mail: Josef.Altenbuchner{at}po.uni-stuttgart.de

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 -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 -glucosidase activity was detected using the synthetic -glucoside p-nitrophenyl -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 -glucosidase activity as strain TK64 and in both strains no stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased -glucosidase activity about 10-fold. This probably resulted from induction of a second -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 -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.

Keywords: Streptomyces, -glucosidase, ABC transporter, repressor, amplification

Abbreviations: ABC, ATP-binding cassette; AUD, amplifiable unit of DNA; LDR, left direct repeat; LIP, left imperfect palindrome; MDR, middle direct repeat; PBS, potential binding sequence; p-NPG, p-nitrophenyl -D-glucopyranoside; RDR, right direct repeat; RIP, right imperfect palindrome

The GenBank accession number for the sequence reported in this paper is U22894.

^a Present address: Biozentrum/Physiologische Chemie I, Am Hubland, 97074 Würzburg, Germany.

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