THE 2 BETA-LACTAMASE GENES OF STREPTOMYCES-CACAOI, BLAL AND BLAU, AREUNDER THE CONTROL OF THE SAME REGULATORY SYSTEM

The production of beta-lactamase in Streptomyces cacaoi, which contain s two beta-lactamase-encoding genes, blaL. and blaU, is inducible by b eta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone di d not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but tran scribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to e xamine the role of BlaA in regulation, we here report on over-expressi on of a GST-BlaA fusion protein in Escherichia coli and its use for an tibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA interg enic region. The BlaA DNA binding-site was further restricted to a 30- bp sequence containing a T-N-11-A motif, a characteristic of LysR-type promoters. Another T-N-11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N-11-A motifs in Bla A binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confe r on an S. lividans host the capacity to produce inducible beta-lactam ase. It can thus be concluded that the S. cacaoi blaL and blaU genes a re controlled by the same regulatory system.