Construction and analysis of β-lactamase-inhibitory protein (BLIP) non-producer mutants of Streptomyces clavuligerus The GenBank accession number for the sequence determined in this work is M34538.

Abstract

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 β-lactamase-inhibitory activity, indicating that only a single β-lactamase-inhibitory protein exists in S. clavuligerus. The lack of any proteinaceous β-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 β-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 wildtype 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 pathwayspecific 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.

Binding Hot Spots in the TEM1–BLIP Interface in Light of its Modular Architecture

“…Therefore, it is not surprising that until now no biological connection was proven between these two proteins. 31,32 The scenario of optimizing binding to a set of other proteins is very common in the biological setup, and stems from the flexibility of the network characteristics of the proteom.…”

The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

“…In previous studies (2), it was reported that production of both 5S clavam and clavulanic acid was lost in a ccaR mutant, whereas in our hands, mutation of ccaR had no effect on 5S clavam production. This inconsistency may be attributed to differences in the methodologies and growth media used for culture propagation in the two studies or to the extensive variability that has been observed in 5S clavam production profiles within this species (43).…”

Insights into Positive and Negative Requirements for Protein–Protein Interactions by Crystallographic Analysis of the β-Lactamase Inhibitory Proteins BLIP, BLIP-I, and BLP

“…52 Approximately 10 7 viable protoplasts of each mutant type were combined, sedimented by centrifugation at 3000g for 7 min, and washed once with 5 ml of P-buffer ( 52 The sedimented protoplasts were resuspended gently in 0.8 ml of a 1:1 mixture of P-buffer and molten polyethylene glycol (PEG) 1000 (cooled to 21°C after mixing). After 2 min at 21°C, the protoplast mixture was diluted in P-buffer, and aliquots were plated on R5B agar plates.…”

“…After 2 min at 21°C, the protoplast mixture was diluted in P-buffer, and aliquots were plated on R5B agar plates. 52 After 40 h at 28°C, the regenerating protoplasts were overlaid with thiostrepton (thio) and apr at final concentrations of 5 μg/ml and 25 μg/ml, respectively, to counterselect the parental strains. After an additional 8 days of incubation at 28°C, developing fusants were patched onto MYM 52 plates and MYM + apr + thio plates.…”