Construction and analysis of ss-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-producing mutant. The resulting BLIP mutant and BLIP/clavulanic acid double mutant showed no residual proteinaceous beta-lactamase-inhibitory activity, indicating that only a single beta-lactamase-inhibitory protein exists in S. clavuligerus. The lack of any proteinaceous beta-lactamase-inhibitory activity in the bli and bli/claR mutants also indicates that BLP, the BLIP-like protein, encoded by S. clavuligerus does not possess beta-lactamase-inhibitory activity despite its similarity to BLIP. The bli mutant and the bli/claR double mutant did not show any aberrant growth morphology, sporulation defects, or alterations in cephamycin C production or penicillin G resistance when compared to wild-type S. clavuligerus or to the claR single mutant. Mutants bearing the bli gene disruption did show an elevated level of production of clavam-2-carboxylate and hydroxymethyl clavam as well as clavulanic acid. This phenomenon was observed in the middle stages of production of these 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 bli gene showed the presence of a partial open reading frame that encodes a DNA-binding protein, and several open reading frames apparently involved in the production of an ABC transporter.