Construction and analysis of beta-lactamase-inhibitory protein (BLIP) non-producer mutants of Streptomyces clavuligerus

The gene encoding BLIP, a beta-lactamase-inhibitory protein, was disrupted in wild-type Streptomyces clavuligerus and in a clavulanic acid non-produci ng mutant. The resulting BLIP mutant and BLIP/clavulanic acid double mutant showed no residual proteinaceous beta -lactamase-inhibitory activity, indi cating that only a single beta -lactamase-inhibitory protein exists in S, c lavuligerus, The lack of any proteinaceous beta -lactamase-inhibitory activ ity in the bli and bli/claR mutants also indicates that BLP, the BLIP-like protein, encoded by S, clavuligerus does not possess beta -lactamase-inhibi tory activity despite its similarity to BLIP, The bli mutant and the bli/cl aR double mutant did not show any aberrant growth morphology, sporulation d efects, or alterations in cephamycin C production or penicillin C resistanc e when compared to wildtype S. clavuligerus or to the claR single mutant. M utants bearing the bli gene disruption did show an elevated level of produc tion of clavam-2-carboxylate and hydroxymethyl clavam as well as clavulanic acid, This phenomenon was observed in the middle stages of production of t hese clavams but was not detected during maximum production. The production of BLIP was also determined to be down-regulated in a ccaR mutant, lacking the pathway-specific transcriptional regulator required for production of cephamycin C and clavulanic acid. Sequencing of the regions flanking the bl i gene showed the presence of a partial open reading frame that encodes a D NA-binding protein, and several open reading frames apparently involved in the production of an ABC transporter.