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

Abstract

The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did 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 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 over-expression 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 beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.

β-Lactamase Gene Expression in a Penicillin-Resistant Bacillus anthracis Strain

“…Regulation of ␤-lactamase synthesis has been studied in certain other gram-positive bacteria (11,36). In Streptomyces cacaoi, the expression of ␤-lactamase genes is controlled by a regulatory protein, BlaB (22), and mutations in blaB can lead to silencing or constitutive expression of ␤-lactamase genes (30). Homologs of bla regulators are present in the B. anthracis genome, but they are not linked to either bla gene.…”

The diversity, structure and regulation of β-lactamases

“…In S. cacaoi, blaU is also inducible by i-lactams, and its promoter region is very similar to that of blaL. The control of blaU also relies on the blaA and blaB genes [19].…”

“…They are also located upstream of and diverge from the controlled structural gene, blaL. BlaA is a transcriptional activator, belonging to the LysR family, and binds to the intergenic region, blaA-blaL [19] at the level of a T-N 11 -A motif, as described in Goethals et al [20]. The function of the product of blaB remains unknown.…”

BlaB, a protein involved in the regulation of Streptomyces cacaoi ?-lactamases, is a penicillin-binding protein

“…It binds to DNA through a helix-turn-helix motif and recognizes a T-N 11 -A sequence including a double overlapping palindromic sequence [17]. This motif, characteristic of the LysR-type promoters, is present in the blaA-blaL intergenic region and upstream of blaU [14,16]. To date, no function has been attributed to BlaB.…”

“…S. cacaoi possesses two different unlinked b-lactamase structural genes called blaL and blaU [9]. The two genes encode two extracellular active-site serine class A b-lactamases sharing 50 % strict identities [9 -13] and they are under the control of the same regulatory system [14]. Sequence analysis and genetic study of the blaL regulatory region identified two genes, blaA and blaB, located just upstream of blaL but transcribed divergently and required for the induction and for the basal expression of BlaL and BlaU [8,15,16].…”