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

Department of Biological Sciences, CW-405 Biological Sciences Building, University of Alberta, Edmonton, Alberta, CanadaT6G 2E9 1 Author for correspondence: Susan E. Jensen. Tel: +1 780 492 0672. Fax: +1 780 492 2216. e-mail: susan.jensen{at}ualberta.ca The gene encoding BLIP, a b eta- l actamase- i nhibitory p rotein, 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 ß-lactamase-inhibitory activity, indicating that only a single ß-lactamase-inhibitory protein exists in S. clavuligerus . The lack of any proteinaceous ß-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 ß-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. Keywords: clavulanic acid, clavams, antibiotic resistance, exocellular protein Abbreviations: ABC, ATP-binding cassette; BLIP, ß-lactamase-inhibitory protein; BLP, BLIP-like protein; MSD, membrane-spanning domain; MYM, maltose/yeast extract/malt extract medium; SSPE, sodium chloride/sodium phosphate/EDTA; TSB, trypticase soy broth; TSBS, TSB-starch; TSBM, TSB-maltose; YEME, yeast extract/malt extract; YEMEM, YEME-maltose The GenBank accession number for the sequence determined in this work is M34538 . a Present address: Diversa Corporation, 10665 Sorrento Valley Road, San Diego, CA 92121, USA.