The two β-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system

Abstract

The production of β-lactamase in Streptomyces cacaoi, which contains two β-lactamase-encoding genes, blaL and blaU, is inducible by β-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a β-lactamase-negative species. The blaU clone did not respond to the presence of β-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 transcribed 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 examine the role of BlaA in regulation, we here report on overexpression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N₁₁-A motif, a characteristic of LysR-type promoters. Another T-N₁₁-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N₁₁-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible β-lactamase. It can thus be concluded that the S. cacaoiblaL and blaU genes are controlled by the same regulatory system.