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

Abstract

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

Expression of blaA Underlies Unexpected Ampicillin-Induced Cell Lysis of Shewanella oneidensis

“…It has been proposed that PBP5 of E. coli is utilized to form a complex with ampicillin, thereby protecting essential PBPs [8]. In Streptomyces cacaoi , the production of β-lactamase, BlaL, is controlled by two regulators, a LysR-type activator and a PBP protein, BlaB [40]. In P. aeruginosa , inactivation of a nonessential PBP leads to overproduction of the chromosomal β-lactamase, AmpC, and the activation of the CreBC two-component system, a major regulator involved in β-lactam resistance [7].…”

From Dormant to Germinating Spores of Streptomyces coelicolor A3(2): New Perspectives from the crp Null Mutant

“…The 687 bp PCR product was first cloned in pGEM-T Easy for DNA sequencing and then digested with BglII and PstI and subcloned into the high-copy shuttle-vector Vpro p145.10. 9 The resulting crp constitutive expression plasmid was designated pCIP267. pCIP267 was transferred into S. coelicolor wild-type M145 by protoplast transformation 8 resulting in the SAF2 strain (or crp +++ ).…”

“…For overproduction of Crp in S. coelicolor , the cloning was achieved by PCR with oligonucleotides 5‘-CC AGATCT GTGGACGACGTTCTGCGGCGC-3' ( Bgl II site italicized) and 5‘- CTGCAG TCAGCGGGAGCGCTTGGCCAGTCG-3‘ ( Pst I site italicized). The 687 bp PCR product was first cloned in pGEM-T Easy for DNA sequencing and then digested with Bgl II and Pst I and subcloned into the high-copy shuttle-vector Vpro p145.10 . The resulting crp constitutive expression plasmid was designated pCIP267.…”

Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP

“…The PCR product was cloned into pGEM-T-Easy and sequenced. The resulting plasmid pCIP266 was digested with BglII and PstI, and the isolated 675 bp crp fragment was ligated with the Vpro p145.10 vector [14] digested by the same restriction enzymes, giving rise to pCIP267. Protoplasts of S. lividans TK24 were transformed by pCIP267.…”