Vol. 172, No. 9

Isolation and Characterization of a 3-Lactamase-Inhibitory Proteinfrom Streptomyces clavuligerus and Cloning and

Analysis of the Corresponding GeneJAMES L. DORAN,t BRENDA K. LESKIW,t SVEN AIPPERSBACH, AND SUSAN E. JENSEN*

Department of Microbiology, University of Alberta, Edmonton, Alberta, T6G 2E9 Canada

Received 22 February 1990/Accepted 13 June 1990

Culture filtrates of Streptomyces clavuligerus contain a proteinaceous I-lactamase inhibitor (BLIP) inaddition to a variety of ,-lactam compounds. BLIP was first detected by its ability to inhibit Bactopenase, apenicillinase derived from Bacillus cereus, but it has also been shown to inhibit the plasmid pUC- andchromosomally mediated ,-lactamases of Escherichia coli. BLIP showed no inhibitory effect against Entero-bacter cloacae I(-lactamase, and it also showed no activity against an alternative source of B. cereuspenicillinase. BLIP was purffied to homogeneity, and sodium dodecyl sulfate-polyacrylamide gel electropho-resis gave a size estimate for BLIP of 16,900 to 18,000. The interaction between purified BLIP and the E.coli(pUC) ,-lactamase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis anddetermined to be noncovalent, with an estimated 1:1 molar stoichiometry. The BLIP gene was isolated on a13.5-kilobase fragment of S. clavuligerus chromosomal DNA which did not overlap a 40-kilobase region ofDNAknown to contain genes for I8-lactam antibiotic biosynthesis. The gene encoded a mature protein with a deducedamino acid sequence of 165 residues (calculated molecular weight of 17,523) and also encoded a 36-amino-acidsignal sequence. No significant sequence similarity to BLIP was found by pairwise comparisons using variousprotein and nucleotide sequence data banks or by hybridization experiments, and no BLIP activity wasdetected in the culture supernatants of other Streptomyces spp.

,B-Lactam compounds account for more than 60% ofworldwide consumption of antibiotics as a result of the highselectivity, low toxicity, and versatility of these compounds.However, the chemotherapeutic application of ,-lactamantibiotics has been continually threatened by the develop-ment of bacterial resistance. The most common mechanismof resistance among both gram-positive and gram-negativebacteria involves the production of 3-lactamases, enzymeswhich cleave the p-lactam ring. The fact that some ,B-lactamases are encoded on transferable plasmids or trans-posable elements has contributed substantially to the inci-dence of clinically significant P-lactam resistance (12). Whilechemical modification has helped to improve the resistanceof the naturally occurring P-lactam antibiotics to cleavage,the need for further improvement resulted in an effort to finduseful ,-lactamase inhibitors. Several effective, mechanism-based, low-molecular-weight P-lactamase inhibitors havebeen developed, but only clavulanic acid, a clavam-typeP-lactam compound, has been used clinically (33). Clavu-lanic acid is one of several P-lactam compounds produced byStreptomyces clavuligerus, a filamentous gram-positive bac-terium which also produces penicillin N, desacetoxyceph-alosporin C, and cephamycin C (28). The significance of theproduction of ,-lactamase inhibitors to S. clavuligerus isunclear, but this is also true of the production of ,-lactamantibiotics. No consensus exists as to how antibiotic-produc-ing organisms benefit from the production of antibiotics (1,14). Although the majority of Streptomyces species produce,-lactamases (43), S. clavuligerus produces no detectable

* Corresponding author.t Present address: Microtek Research and Development Ltd.,

Sidney, British Columbia, V8L 3Y3 Canada.t Present address: John Innes Institute, Norwich, NR4 7UH

United Kingdom.

levels of this enzyme (45). However, ,-lactamase inhibitorscould function to protect the P-lactam antibiotics that S.clavuligerus produces from attack by P-lactamases fromother organisms.Other ,-lactamase-inhibitory compounds have been de-

scribed elsewhere (33). In general, like clavulanic acid, theyare P-lactam compounds which act as suicide inhibitors of,-lactamase. Few other types of active-site-directed P-lac-tamase inhibitors are known, but a class of boronic acid-typeinhibitors which reversibly inhibit P-lactamases has beendescribed elsewhere (13). These compounds are not relatedto ,-lactam compounds in chemical structure. Rather, theyare known as inhibitors of serine type proteases (32) andinhibit P-lactamases because of the similarities between theactive sites of these two classes of enzymes.Few peptide or proteinaceous inhibitors of ,-lactamase

have been described. One preliminary description of aproteinaceous material isolated from S. gedanensis whichinhibits the ,3-lactamase from Staphylococcus aureus hasbeen reported (20), but no further studies were conducted toclarify the nature of the P-lactamase-inhibitory agent. Thepresent study reports the discovery and partial characteri-zation of a P-lactamase-inhibitory protein (BLIP) producedby S. clavuligerus and the cloning and sequencing of thecorresponding gene.

(Preliminary results of this study were presented at theAmerican Society for Microbiology Conference on the Ge-netics and Molecular Biology of Industrially Important Mi-croorganisms, Bloomington, Ind., 2 to 7 October 1988.)

MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophage. S. cla-

vuligerus NRRL 3585, S. jumonjinensis NRRL 5741, S.lipmanii NRRL 3584, and S. cattleya NRRL 3841 wereobtained from the Northern Regional Research Laborato-

4909

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ries, Peoria, Ill. S. lividans 1326 was provided by D. A.Hopwood (John Innes Institute, Norwich, United King-dom). S. griseofuscus NRRL B-5429 and S. parvulus NRRLB-1628 were obtained from L. C. Vining (Department ofMicrobiology, Dalhousie University, Halifax, Nova Scotia,Canada). S. fradiae NCIB 8233, S. griseus NCIB 8237, S.phaeochromogenes NCIB 8505, S. rimosus NCIB 8229, andS. violaceoruber SANK 95570 were obtained from ourdepartmental culture collection. All strains were stored aslyophilized spore stocks or as spore suspensions in 20%glycerol. Streptomyces spp. were grown in Trypticase soybroth containing 1% (wt/vol) soluble starch (Difco Labora-tories, Detroit, Mich.) by incubation at 28°C on a rotaryshaker at 280 rpm.

Escherichia coli MV1183 (55) harbored pUC118 andpUC119 and derivative recombinant plasmids. E. coli DH5a(Bethesda Research Laboratories, Inc., Gaithersburg, Md.)was used as a host for recombinant M13 bacteriophage. E.coli VCS257 (Stratagene Corp., San Diego, Calif.) was usedfor the propagation of recombinant cosmids. All E. colistrains were grown in LB medium (38) which containedampicillin (50 ,ug/ml) or tetracycline (12.5 jig/ml) whenrequired for the maintenance of plasmid or cosmid DNA,respectively. pLAFR3, a broad-host-range cosmid vector(53), was obtained from J. M. Foght (Department of Micro-biology, University of Alberta, Edmonton, Alberta, Cana-da). The cloning vectors pUC118 and pUC119 (55) wereprovided by J. Vieira (Waksman Institute of Microbiology,Rutgers University, Piscataway, N.J.). The replicativeforms of the M13 bacteriophage derivatives mpl8 and mpl9(57) and X c1857 DNA were purchased from Pharmacia Inc.(Uppsala, Sweden).

I8-Lactamase inhibition assays. (i) Agar diffusion bioassay.Clavulanic acid content of culture supernatants was esti-mated by the indirect agar diffusion assay procedure de-scribed previously (30).

(ii) Spectrophotometric ,-lactamase inhibition assay. -Lactamase inhibition assays were conducted as described byGutman et al. (19) using Bactopenase concentrate as thesource of 3-lactamase and penicillin G as the substrateunless otherwise indicated. One unit of BLIP was defined asthat amount of material which would give 50% inhibition ofthe 2 x 104 U of Bactopenase used in the spectrophotometric3-lactamase inhibition assay.In studies in which crude ,B-lactamases from E. coli strains

were used in place of Bactopenase or in which assaymixtures were supplemented with bovine serum albumin,high background absorbances were encountered. In thosecases, the colored cephalosporin {7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethylaminophenylazo)-pyridinium methyl]-3-cephem-4 carboxylic acid} (PADAC) (51) was used as sub-strate at a concentration of S jiM in place of penicillin G, and,B-lactamase activity was monitored as a decline in A572. Allother assay conditions remained unchanged.

Protease assays. Protease activity was monitored withazocasein as substrate as previously described (29). Fibrino-lytic activity was measured with the specific plasmin sub-strate D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochlo-ride as described by Jeffries and Buckley (27).

Protein assays. Protein contents of BLIP and ,3-lactamasepreparations were measured by the dye-binding assay ofBradford (7) with bovine gamma globulin as standard.

Purification of BLIP from S. clavuligerus. Liquid culturesof S. clavuligerus were grown in Trypticase soy brothcontaining 1% (wt/vol) soluble starch on a rotary shaker at28°C and 280 rpm. Culture filtrate from a 64-h culture was

collected by filtration through no. 604 filter paper (Schleicher& Schuell, Inc., Keene, N.H.).

(i) Ammonium sulfate precipitation. Ammonium sulfate(44.9 g) was gradually added with stirring to 87 ml of culturefiltrate at 4°C to give 80% saturation. The precipitatedprotein was collected by centrifugation for 15 min at 27,000x g and dissolved in 20 ml of 0.1 M sodium phosphate buffer(pH 7.0) (phosphate buffer). The protein solution was con-centrated by ultrafiltration with a YM5 filter to a final volumeof 4.0 ml.

(ii) Gel filtration chromatography. The protein concentratewas applied to a Sephadex G-75 superfine column (2.5 by 30cm) which was equilibrated and eluted with phosphatebuffer. Fractions (2.5 ml) were collected and monitored forprotein content (A280) and BLIP by the spectrophotometric,B-lactamase inhibition assay. Fractions containing BLIPwere pooled and concentrated by ultrafiltration to 2.5 ml aspreviously described.

(iii) Ion-exchange chromatography. The BLIP concentrateobtained by gel filtration was applied to a DEAE-trisacrylcolumn (1.6 by 25 cm) which was equilibrated and elutedisocratically with phosphate buffer. Fractions (2.5 ml) werecollected and monitored for protein (A280) and BLIP by thespectrophotometric 1-lactamase inhibition assay. Fractionscontaining BLIP were pooled and stored at -20°C.

Preparation of 1-lactamases from E. coli. Crude 1-lacta-mase was prepared from a 16-h culture of E. coli MV1183grown in Trypticase soy broth on a rotary shaker at 37°C and250 rpm. The cells were harvested by centrifugation at10,000 x g for 10 min. The cells were washed once withphosphate buffer, suspended to 1/20 of the original culturevolume in phosphate buffer, and disrupted by sonication(twice for 15 s each time at power level 7; Branson Sonifier,0.75-in [ca. 1.9-cm] probe). Cell debris was removed bycentrifugation for 15 min at 27,000 x g, and the supernatantwas used as a source of crude 13-lactamase. This 3-lactamasepreparation showed cephalosporinase activity predomi-nantly and is presumed to be the product of the chromo-somal ampC gene (26).

Partially purified 1-lactamase was prepared from culturesof E. coli MV1183 carrying the plasmid pUC119. Cultureswere grown on Trypticase soy broth containing ampicillin at100 ,ug/ml for 16 h on a rotary shaker at 37°C and 250 rpm.The cells were subjected to osmotic shock treatment (42),and 1-lactamase was purified from the shock fluid. Shockfluid was concentrated by ultrafiltration with a PM-10 filter toa final volume of 1.5 ml. The concentrate was applied in0.5-ml amounts to a MonoQ HR 5/5 column which had beenequilibrated with 0.03 M Tris hydrochloride (pH 8.0). Thecolumn was eluted at a flow rate of 0.5 ml/min with a lineargradient of NaCl in phosphate buffer varying from 0 to 0.3 Mover 30 min. ,-Lactamase activity was located in the eluantfractions by the spectrophotometric ,B-lactamase inhibitionassay described above but with 25-,ul amounts of columnfractions replacing Bactopenase as a source of ,B-lactamase.The resulting ,B-lactamase preparation showed penicillinaseactivity predominantly and is presumed to be the product ofthe bla gene of pUC119 (9).

Interaction of BLIP with E. coli penicillinase. Purified BLIP(0.1 mg of protein, 5.6 nmol) was mixed with partiallypurified penicillinase from E. coli (0.2 mg of protein, 5.6nmol [assuming 80% purity]) in a final volume of 1.8 ml ofphosphate buffer. After 5 min of incubation at 25°C, themixture was applied to a DEAE-trisacryl column (1.6 by 20cm) which was equilibrated and eluted with phosphatebuffer. Fractions (1.9 ml) were collected and monitored for

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protein (A280) and penicillinase activity as described above.Purified BLIP (0.1 mg of protein, 5.6 nmol) and partiallypurified penicillinase (0.2 mg of protein, 5.6 nmol [assuming80% purity]), each at a final volume of 1.8 ml in phosphatebuffer, were also chromatographed separately.SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) was carried out with 10%polyacrylamide gels as described by Blackshear (6). Proteinswere localized by staining with Coomassie blue.Recombinant DNA technology. The purification of Strepto-

myces spp. chromosomal DNA on CsCl gradients wasconducted as described previously (22). The large-scalepurification of pLAFR3 was achieved by using a modifiedcleared-lysate procedure (38). Established techniques wereused for the small-scale preparation of plasmid DNA by analkaline lysis procedure and for the construction of recom-binant cosmids, plasmids and bacteriophage (38). E. colistrains were made competent and transformed by plasmid orbacteriophage recombinant DNA by the procedure of Mor-rison (40).A library of S. clavuligerus DNA was constructed in the

cosmid vector pLAFR3. Fragments of chromosomal DNAprepared by partial digestion with Sau3AI were size frac-tionated by sucrose gradient centrifugation (38), and 15- to22-kilobase (kb) fragments were ligated into BamHI-di-gested, alkaline phosphatase-treated pLAFR3. The ligatedDNA was packaged in bacteriophage lambda particles byusing the Giga Pack Plus kit (Stratagene) and was introducedinto E. coli VCS257 by transduction.A collection of 1,800 isolated E. coli colonies containing

recombinant cosmids was screened by hybridization underthe stringent conditions previously described (36). The hy-bridization probe was constructed by end labeling (38) abiased mixture of 44-mer oligonucleotide probes by using T4polynucleotide kinase (Pharmacia). Physical mapping of theselected recombinant cosmid was conducted by an analysisof the products of single- and double-restriction enzymedigestions and consideration of the results of correspondinghybridization experiments (38, 48).DNA sequence determination and analysis. Appropriate

DNA fragments identified by Southern hybridization (48) tooligonucleotide probes or to recombinant M13 hybridizationprobes (24) were used for DNA sequencing. Prior to beingsubcloned into M13mpl8 or M13mpl9 vectors, a DNAfragment of 488 base pairs (bp) generated by Sall digestionwas purified from a 5% polyacrylamide gel by electroelutionusing an Elu-trap apparatus (Schleicher & Schuell). DNAsequencing of an overlapping 840-bp KpnI-ClaI fragmentwas conducted using single-stranded DNA cloned inM13mpl8 or M13mpl9 and using double-stranded DNAcloned in pUC118 or pUC119 (56) and propagated in E. coliDHSa.DNA sequencing was conducted by various modifications

of the enzymatic (dideoxy) method of Sanger et al. (50),including the use of the Klenow fragment of E. coli DNApolymerase I (50), Sequenase (United States BiochemicalCorp., Cleveland, Ohio) (54), or Taq DNA polymerase (47;Promega Corp., Madison, Wis.) and the commercially avail-able 17-mer universal sequencing primer (57) or one of threeother 17-mers (dCAGCAGCTTCTCCTGGC, dCTGCACCACCTGGAGTG, or dGTCCTCGACATAGCCGG) pre-pared by using an Applied Biosystems Inc. (Mississauga,Ontario, Canada) model 381A DNA synthesizer. In order torelieve compressions in the banding pattern on DNA se-quencing gels, some sequencing reactions conducted withthe Klenow fragment of DNA polymerase I or Taq DNA

polymerase utilized 7-deaza-GTP in place of dGTP (39). Forthe same reason, some reactions conducted with Sequenaseincluded dITP in place of dGTP. The DNA sequence of eachfragment was determined repeatedly with several dideoxy-nucleotide-sequencing techniques in order to provide un-equivocal results in the regions of high G+C content whichare characteristic of Streptomyces DNA and difficult tosequence.The nucleotide sequence data were analyzed for the

presence of restriction sites, regions of dyad symmetry,RNA secondary structure, and codon usage by using thePC-Gene programs (Intelligenetics Corp., Mountan View,Calif.). The corresponding protein sequence was examinedby using the FASTP protein sequence analysis programs (46)and their accompanying protein sequence data banks. Boththe nucleotide and amino acid sequences were analyzed byusing the programs and data banks available on PC-Gene.The G+C content as a function of codon position wasdetermined by using the algorithm of Bibb et al. (5).

Materials. Penicillinase type I from Bacillus cereus, peni-cillinase type III from Enterobacter cloacae, penicillin G,D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, andazocasein were purchased from Sigma Chemical Co. (St.Louis, Mo.). Bactopenase concentrate was from Difco.PADAC was from Calbiochem-Behring (San Diego, Calif.).Restriction endonucleases and other DNA-modifying en-zymes were purchased from Bethesda Research Laborato-ries, Boehringer-Mannheim (Penzberg, Federal Republic ofGermany), or Pharmacia and were used under the conditionsrecommended by the suppliers. The biased mixture of 44-mer oligonucleotides was purchased from the Alberta Heri-tage Foundation for Medical Research DNA Synthesis Fa-cility, University of Calgary, Calgary, Alberta, Canada.Trypticase soy broth was from BBL Microbiology Systems,(Cockeysville, Md.). Sephadex G-75 superfine and MonoQHR 5/5 columns were from Pharmacia. DEAE-trisacryl wasfrom Reactifs IBF (Villeneuve-la-Garenne, France). Ultrafil-tration membranes were from Amicon Corp. (Danvers,Mass.). Clavulanic acid was a gift from B. Morgan, BeechamPharmaceuticals (Betchworth, Surrey, England). All otherfine chemicals were reagent grade and were purchased fromSigma or BDH (Poole, United Kingdom).

Nucleotide sequence accession number. Sequencing data forthe BLIP gene have been submitted to GenBank and as-signed the accession number M34538.

RESULTS

Demonstration ofBLIP in culture filtrates of S. clavuligerus.S. clavuligerus produces the ,-lactamase inhibitor clavu-lanic acid. Several assay procedures have been developed toquantitate clavulanic acid, including indirect agar diffusionbioassays (30) and direct spectrophotometric P-lactamaseinhibition assays (19). When the clavulanic acid content ofculture filtrates of S. clavuligerus was estimated by indirectagar diffusion bioassay and by spectrophotometric 1-lacta-mase inhibition assay, major discrepancies were noted.Estimates of clavulanic acid content as determined by thespectrophotometric P-lactamase inhibition assay (472 jig/ml)were much higher than those based on the bioassay (3.7,ug/ml). This suggested that culture filtrates contain someinhibitory material in addition to clavulanic acid, whichaffects the spectrophotometric P-lactamase inhibition assayonly.When samples of S. clavuligerus culture filtrate were

mixed with an equal volume of methanol or were heated at

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0 20 40 60Incubation time , hours

200

-100

IIIII

-I

im

20 -

10 -

0E

a-

co

80 1 00

FIG. 1. Production of BLIP and protease activity throughout the growth cycle of S. clavuligerus. Liquid cultures of S. clavuligerusgrowing in Trypticase soy broth containing 1% soluble starch were sampled at 16-h intervals throughout the growth cycle. Growth wasestimated by measuring optical density at 600 nm (OD6w) on appropriately diluted samples. Protease activity and BLIP activity of culturefiltrates were measured by following azocasein digestion and Bactopenase inhibition activities, respectively. BLIP activity measurementswere corrected for clavulanic acid by subtracting the activity seen in methanol-treated culture filtrates.

98°C for 2 min, treatments which should denature proteins,the ability of the culture filtrate to inhibit Bactopenase wasreduced. Untreated culture filtrate caused an 85.4% inhibi-tion of Bactopenase activity, while either methanol or heattreatment reduced the inhibition to 6.8%. Authentic clavu-lanic acid was not affected by either of these treatments.This suggested that the additional material contributing tothe ,-lactamase inhibition was a protein.

S. clavuligerus produces exocellular proteases (8, 27). Anexocellular protease could give the appearance of 3-lacta-mase inhibition in an enzyme-based assay by causing pro-teolytic degradation of the P-lactamase. When P-lactamaseinhibition assay mixtures were supplemented with bovineserum albumin at a 10-fold excess over the amount of,-lactamase protein, no diminution of the ,-lactamase-inhib-itory effect of culture filtrate was seen. Thus, the addition ofan excess of unrelated protein did not protect the Bactope-nase from inhibition, as would be expected if the inhibitionwas actually due to a protease.When culture filtrates were collected throughout the

growth cycle of S. clavuligerus and assayed for proteaseactivity (azocasein digestion), activity was low in all samples(Fig. 1). In contrast, the production of the BLIP was lowearly in the growth cycle, rose to a maximum by 64 h, andthen dropped slightly. The possibility that BLIP is a proteasewith a very restricted substrate specificity still remained. S.clavuligerus is reported to produce a serine-type proteasewith fibrinolytic activity (8). The specificity of this proteasemight prevent it from showing activity against azocasein.When culture filtrates were tested for fibrinolytic activitywith the specific plasmin substrate D-valyl-L-leucyl-D-lysine-p-nitroanilide dihydrochloride, no activity was seen (datanot shown). These results suggest that BLIP is proteina-ceous in nature but is not a protease.

Purification of BLIP from culture filtrates of S. clavuligerus.BLIP was purified from culture filtrate from a 64-h culture ofS. clavuligerus as described in Materials and Methods.When samples from the various stages of purification wereexamined by SDS-PAGE, the purified BLIP sample ap-

peared homogeneous (Fig. 2). SDS-PAGE analysis of crudesalt-precipitated exocellular protein indicated that BLIP is amajor component (about 10%) of the total exocellular proteinproduced by S. clavuligerus under these growth conditions.Comparison with molecular weight marker proteins gave

1 2 3

<67

445

A25

4 421.5

FIG. 2. Analysis of BLIP throughout the purification procedureby SDS-PAGE. Lane 1, Ammonium sulfate-precipitated culturefiltrate, 20 jig of protein; lane 2, pooled fractions after SephadexG-75 chromatography, 2 jig of protein; lane 3, pooled fractions afterDEAE-trisacryl chromatography, 2 ,ug of protein. Arrows indicatethe locations of appropriate molecular weight markers (in thou-sands).

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TABLE 1. Purification of BLIP

Total Total Sp act Recove Purifi-Sample BLIP protein (U/mg of (%) cation

(Ua) (mg) protein) factor

Culture filtrate 12,015 37.4 321.2 100 1Ammonium sulfate 11,112 29.2 380.2 92.5 1.18

concentrateSephadex G-75 7,802 3.85 2,026 64.9 6.31pooled fractions

DEAE-trisacryl 5,265 1.24 4,246 43.8 13.2pooled fractions

a One unit of BLIP activity is defined as that amount of material which gives50% inhibition of the Bactopenase used in the standard spectrophotometric,-lactamase inhibition assay.

molecular weight estimates ranging from 16,900 to 18,000 forBLIP.

Final recovery of BLIP upon complete purification was43.8%, with a purification factor of 13.2 (Table 1). Thepurified BLIP showed complete stability to storage with noloss of activity noted after 6 months at -20°C with repeatedfreezing and thawing (data not shown).

Interaction between BLIP and P-lactamases. The nature ofthe interaction between BLIP and ,-lactamase was investi-gated by comparing the SDS-PAGE profiles of ,B-lactamasebefore and after exposure to BLIP. However, Bactopenasecould not be used for these studies because SDS-PAGEanalysis of Bactopenase produced a broad smear rather thana discrete band of protein (data not shown). No standardmethods of protein purification, including ultrafiltration, gelfiltration, or anion-exchange chromatography, would resolvethe Bactopenase into a form which would give a discreteprotein band by SDS-PAGE. Therefore, a selection of otherP-lactamases was tested for susceptibility to inhibition bypurified BLIP. Commercially available (Sigma) 1-lactamasesderived from B. cereus (penicillinase) and E. cloacae (ceph-alosporinase) were not inhibited by BLIP. This result wasunexpected in the case of the B. cereus enzyme, sinceBactopenase is also a penicillinase prepared from B. cereus(Technical Services Department, Difco, personal communi-cation) and is strongly inhibited by BLIP. No artifactualexplanation for the difference between the two B. cereuspenicillinases was apparent, since neither dialysis against acommon buffer nor mixing of the two preparations couldeliminate the difference (data not shown). Both chromo-somal (cephalosporinase) and plasmid-encoded (penicillin-ase) P-lactamases of E. coli were inhibited by BLIP.The plasmid-encoded penicillinase of E. coli was purified

by anion-exchange chromatography to about 80% homoge-neity as judged by SDS-PAGE and then combined withpurified BLIP. Based on a molecular weight estimate of17,500 for BLIP (according to total-amino-acid analysis) and28,900 for E. coli penicillinase (9), the two proteins werecombined in an approximate 1:1 molar ratio and incubatedfor 5 min at 25°C. The mixture of penicillinase and BLIPshowed complete inhibition of penicillinase activity. Whenthis mixture was subjected to anion-exchange chromatogra-phy on DEAE-trisacryl, the penicillinase and BLIP coelutedin the unretained fraction. The penicillinase was still fullyinhibited, and no evidence of free BLIP was seen. Whenpenicillinase alone was chromatographed on the DEAE-trisacryl column, it also eluted in the nonretained fraction.When BLIP alone was chromatographed, it was retarded butnot retained by the resin and eluted in a position whichwould not overlap with the penicillinase.

12 3 4

67

A45

FIG. 3. Analysis of the E. ccli penicillinase-BLIP complex bySDS-PAGE. Lane 1, Inactive E. coli penicillinase-BLIP complex aseluted from DEAE-trisacryl, 5.0 [ig of protein; lane 2, partiallypurified E. coli penicillinase as eluted from DEAE-trisacryl, 2.5 jigof protein; lane 3, purified BLIP, 2.5 jig of protein; lane 4, themolecular weight markers serum albumin (67,000), ovalbumin(45,000), chymotrypsinogen (25,000), and trypsin inhibitor (21,500),2.5 jig of each protein.

The penicillinase-BLIP complex which was eluted fromthe DEAE-trisacryl column was analyzed by SDS-PAGE(Fig. 3). The complex was resolved into two separate bandswhich were indistinguishable from the original BLIP andpenicillinase samples. The staining intensity of the penicil-linase band was about twice that of the BLIP band, as wouldbe expected if the two proteins interacted in a 1:21 molarratio.BLIP gene cloning. The sequence of the 29 N-terminal

amino acids of BLIP was determined by analysis of theproducts of Edman degradation to be NH2-Ala-Gly-Val-Met-Thr-Gly-Ala-Lys-Phe-Thr-Gln-Ile-Gln-Phe-Gly-Met-Thr-Arg-Gln-Gln-Val-Leu-Asp-Ile-Ala-Gly-Ala-Glu-Asn. A44-mer, mixed-oligonucleotide probe was designed from thesequence of the N-terminal 15 amino acids since this regionhad the least codon degeneracy if the biased codon usage ofStreptomyces spp. is taken into account (52). The 44-mer,biased, mixed probe had the sequence dGTCAT(C/G)CCGAACTGGATCTG(GIC)GTGAACTT(GIC)GC(GIC)CC(GIC)GTCAT(G/C)AC, where CIG indicates that there was eithera G or a C residue at this position.A library of S. clavuligerus chromosomal DNA fragments

in pLAFR3, constructed as described in Materials andMethods, was screened by hybridization to the pool of44-mer oligonucleotides. A physical map of the 13.5-kb S.clavuligerus chromosomal-DNA fragment isolated from thesingle strongly hybridizing recombinant cosmid, designatedpBLIP1, is presented in Fig. 4. The relative size, position,and orientation of the BLIP gene was determined by hybrid-ization and DNA sequencing. The 13.5-kb fragment does notcontain recognition sites for the restriction endonucleasesBamHl, EcoR3 , HindIII, Pstl, Ssti, Xbai, and Xhol.

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B/S C K KI I I

K BgB/SI II

BLIP

a I a I I I I I I I I I I 130 2 4 6 8 10 12 13.5kb

FIG. 4. Restriction map of S. clavuligerus DNA fragment con-taining the BLIP gene. The BLIP gene was isolated on a 13.5-kbpartial Sau3AI digest fragment which was ligated into the BamHIsite of pLAFR3 to create the plasmid pBLIP1. The position, size,and orientation of the BLIP gene is indicated by the underlying boldarrowhead. Abbreviations: B/S, BamHI-Sau3AI hybrid site; Bg,BgII; C, ClaI; K, KpnI.

DNA and protein sequence analyses. The 840-bp KpnI-ClaIfragment shown in Fig. 4 hybridized to the mixed 44-merprobe and was analyzed to obtain the complete sequence ofthe BLIP gene (Fig. 5). The BLIP open reading frame (ORF)was initially recognized by the presence of a DNA sequencewhich corresponded precisely to the N-terminal 29-amino-acid sequence of the exocellular protein. Analysis of theG+C content of the entire sequenced region as a function ofcodon position, using the algorithm of Bibb et al. (5),confirmed the presence and position of a typical Streptomy-ces ORF (data not shown).The BLIP ORF was assumed to begin at a methionine

codon located 4 bp downstream of the sequence GGAAGGGGacGG. This sequence is complementary to the 3' end ofthe 16S rRNA of S. lividans (4) and therefore may representa Shine-Dalgamo-type ribosome-binding site (Fig. 5). Whilethis represents the most likely ribosome-binding site andstart codon, the GUG codon which exists just a few basepairs downstream from the AUG codon must also be con-sidered as a possible start codon. However, the potentialribosome-binding site preceding the GUG start codon is lesstypical than that preceding the AUG start codon. The ORFterminated at a UAA translation stop codon adjacent to aninverted repeat sequence (Fig. 5) which would encode astable RNA stem-loop structure (AG = -42.4 kcal [ca.-177.4 kJ]/mol; 58) of the type considered to serve as atranscription terminator (52). Assuming that the ORF startsat the AUG codon, the BLIP gene is 606 bp in length. Acomparison of this ORF with the N-terminal sequence of themature protein revealed that the structural gene corre-sponded to a protein precursor which included 36 additionalN-terminal amino acid residues (Fig. 5). This signal se-quence contained four positively charged arginine residuesin the N-terminal region and an adjacent hydrophobic regionwhich included a series of 14 hydrophobic residues (Table 2).The deduced amino acid composition of the mature proteincompared favorably with the results of total amino acidanalysis (Table 3). The calculated molecular weight of themature form of BLIP is 17,523. BLIP is composed of 165

AMT (xT GOC NAG GIf G =CIS(X CitM W0 GM0 TIGGr C GM AM GMB

GGMG Mt000 GG CGGGAM COG0 G G COG COG0GA GIGGM T- fMtet Arg Thr Val Gly Ile Gly Ala Gly Val Ag Arg Leu Gly Arg Ala Val Val

AIG GOG GOGJC GIG 0GGT G CiG GIG CitC GM OG GOG G COT TOG AMC GOXJ(X GOGMt Ala Ala Ala Val Gly Gly Lu Val Leu Gly Ser Ala Gly Ala Ser Asn Ala Ala Gly

GIG AMG ACC (M GM ANG TtC N)G CAG M73CA tCOG AMG ACA COG C3G CPlGiC CiCVal Met lTr Gly Ala Iqs Phe Thr Gln Ile Gln Phe Gly Met hr Arg Gln Gln Val Le

GA: ATAXGC T GOG GK AA 1 GAG A. COC COG TOG TIGC GG0 CA( C ACCMt TOCAsp Ile Ala Gly Ala Glu Asn Cys Glu Thr Gly Gly Ser Phe Gly Asp Ser Ile His Cys

OGG COG 7 (GOG CGC GOG GAC TC T GOC TAC GC AYC TIC GOC TIC C AGC GOC00CArg Gly His Ala Ala Gly Asp Tyr Tyr Ala Tyr Ala Thr Phe Gly Phe Thr Ser Ala Ala

0C GCOG AG GIG QC TMAAAK C C1G GC; AG CIG CTG GOCOG COO COG ANAla Asp Ala Iys Val Asp Ser Lys Ser Gln Glu bI Lu Lou Ala Pro Ser Ala Pro Ttr

Cit ACC Ci COO AG TlC A: CG GIt KC GIG MG ANC AGG GOCC GI7 CiGGMGLeu lTr Lou Ala IeV Phe An Gln Val Thr Val Gly Mt hr Arg Ala Gln Val Lou Ala

ACC GITC OG CG TOG OO NACC AE TOG AGr GWG TZ TC COG COC TAT COG TOG ANGThr Val Gly Gln Gly Ser Cys Thr Thr Tip Ser Glu Tyr Tyr Pro Ala Tyr Pro Ser Thr

GOC MG GTG NE Cit AE C TOOUC lW ltGT GI G C CGT TC TOG TOG EG COG T7CAla Gly Val Thr Lou Ser Lou Ser Cys Phe Asp Val Asp Gly Tyr Ser Ser Thr Gly Pt.

TPAC O C TOG GM (a CiC TOG TIC ANG G CG GIG Cr Ca M AMCXG TOG GTyr Arg Gly Ser Ala His Lou Tip Pie Thr Asp Gly Val Iu Gln Gly Lye Ag Gln Tip

GK CrT GTA TM CG COUCGTT= Ar GIG COG GCTGC OC( GOr GT COG OO COC

Asp Leu Val Non *

AM 00 ATG AAC C GM GT COG AA CAT CA TNC GIt AG MT CIr OGT COOlCG AM

60

120

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FIG. 5. DNA sequence of region encompassing S. clavuligerus BLIP gene. The deduced amino acid sequence of the BLIP proteinprecursor is presented. The presumed site of N-terminal processing of the precursor is indicated by a vertical arrowhead. The nucleotidesconstituting the putative BLIP Shine-Dalgarno motif are indicated with bold underlining. The region of dyad symmetry presumably encodinga transcription terminator is indicated by underlying horizontal arrows.

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4916 DORAN ET AL.

TABLE 3. Comparison of deduced and measured amino acidcompositions of BLIP

Amino acid residue(s) No. measureda No. deducedb

Asp + Asn 12 9 + 2Thr 16 17Ser 13 14Glu + Gln 15 4 + 10Pro 4 4Gly 20 19Ala 21 20Cys 4 4Val 13 11Met 4 3Ile 3 3Leu 11 11Tyr 8 8Phe 9 9His 3 3Lys 6 6Arg 5 5Trp ND 3

a Determined by total amino acid analysis. ND, Not determined.b Deduced from DNA sequence presented in Fig. 5.

amino acids, of which 7.9% are acidic, 8.5% are basic, and48% are hydrophobic.The nucleotide sequence corresponding to the mature

protein had an overall G+C base composition of 66%, whichis within the range of 62 to 74% G+C characteristic ofStreptomyces genes (52). The pattern of codon usage in theBLIP gene encoding the mature protein was typical ofStreptomyces spp. genes and consistent with the asymmetricG+C distribution.

In an effort to recognize any similarity between BLIP andother proteins for which sequence data are available, theBLIP nucleotide and amino acid sequences were comparedwith sequences contained in GenBank and National Biomed-ical Research Foundation data banks. No significant se-

quence similarities were identified.Analysis of other Streptomyces spp. In order to determine

whether a gene product similar to BLIP might be encoded byother Streptomyces spp., a probe consisting of a 488-bp SalIfragment internal to the BLIP gene (nucleotide positions 45to 539, Fig. 5) cloned in M13mpl8 was hybridized to BglIldigests of the chromosomal DNA of S. cattleya, S. fradiae,S. griseus, S. lipmanii, S. lividans, S. phaeochromogenes,S. rimosus, and S. violaceoruber. No hybridization was

detected (data not shown). Similarly, assays of the superna-tants obtained from cultures of S. cattleya, S. griseofuscus,S. jumonjinensis, S. lipmanii, S. lividans, and S. parvulus forP-lactamase-inhibitory activity also yielded negative results(data not shown). The Streptomyces spp. used in thesestudies were chosen to include a range of ,B-lactam-produc-ing and non-p-lactam-producing species and included S.jumonjinensis, one of the few Streptomyces spp. other thanS. clavuligerus which produces clavulanic acid.

DISCUSSIONS. clavuligerus produces a variety of 1-lactam com-

pounds, including the antibiotics penicillin N and cephamy-cin C and the ,B-lactamase inhibitor clavulanic acid. Theclavulanic acid content of culture filtrates can be quantitatedby several means, including spectrophotometric assaysbased on the inhibition of ,-lactamases. The present studyshows that caution must be used when employing such

assays because S. clavuligerus also produces a proteina-ceous P-lactamase inhibitor, BLIP, which is structurallyunrelated to clavulanic acid but can interfere with clavulanicacid estimates.BLIP is a major protein product in the culture filtrate of S.

clavuligerus, constituting approximately 10% of the totalexocellular protein. Isolation of the gene encoding BLIPconfirmed that it is a protein produced by S. clavuligerus,rather than some natural or modified component of thegrowth medium. No proteolytic activity was detected toexplain the P-lactamase inhibitory activity of BLIP, andSDS-PAGE analysis of the completely inhibited P-lactamase-BLIP complex showed that no alteration or deg-radation of the ,-lactamase had occurred. The ability ofSDS-PAGE to resolve the inhibited P-lactamase-BLIP com-plex into two component proteins indistinguishable from thestarting materials indicated that BLIP interacts with -lactamases in a noncovalent manner.The range of P-lactamases inhibited by BLIP has yet to be

determined, but it is evident that several types of 13-lactam-ases are not inhibited by BLIP and that the inhibitory rangeof BLIP is more restricted than that of clavulanic acid. Sincethe closely related P-lactamases Bactopenase and B. cereuspenicillinase (Sigma) show differences in sensitivity to BLIP,it appears that the pattern of inhibition will not correspond tothose of any of the already described classifications of,-lactamases (9).The BLIP gene encodes a precursor protein of 201 amino

acids which contains an N-terminal 36-amino-acid sequencepresumably important for translocation of this exocellularprotein. The length and distribution of the positively chargedand hydrophobic residues of the N-terminal region unique tothe precursor resembled the signal sequences of severalStreptomyces secreted proteins. The molecular weight ofBLIP calculated from the gene sequence is 17,523, which isin good agreement with the molecular weights determined byamino acid analysis (17,500) and SDS-PAGE (16,900 to18,000). Similarly, the amino acid composition of the ma-ture, exocellular form of BLIP deduced from the gene wasconsistent with the measured values, indicating that theentire gene had been characterized. Although BLIP is nativeto an aqueous environment, hydrophobic residues ac-counted for almost half the protein composition and therewere relatively small proportions of acidic or basic residues.BLIP contained four cysteine residues at positions 30, 42,109, and 131 of the deduced mature protein, which is unusualfor a secreted protein. The putative ribosome-binding site ofthe BLIP gene included a Shine-Dalgarno motif, GGAAGGGG--GG, located 4 bp upstream of an AUG codon. Theclose proximity of these two elements and the presence of afavorable initiation codon may facilitate translation of thisprominent exocellular protein (18).

Streptomyces genes related to antibiotic production andresistance are often physically linked (11, 52). Therefore, itwas of interest to determine whether the BLIP gene isincluded in a cluster of genes involved in ,B-lactam antibioticproduction. A comparison of the restriction map for the13.5-kb fragment containing the BLIP gene (Fig. 4) with themaps describing the approximately 40-kb region of DNAcontaining four enzymes responsible for P-lactam antibioticbiosynthesis in S. clavuligerus (35, 36; J. L. Doran, B. K.Leskiw, A. K. Petrich, D. W. S. Westlake, and S. E. Jensen,J. Ind. Microbiol., in press; M. K. R. Burnham, A. J. Earl,J. H. Bull, D. J. Smith, and G. Turner, European patentpublication 0 320 272, 1989; M. K. R. Burnham, J. E.Hodgson, and I. D. Normansell, European patent publica-

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S. CLAVULIGERUS P-LACTAMASE-INHIBITORY PROTEIN

tion 0 233 715, 1987) did not reveal any overlapping regions.Genomic hybridization experiments confirmed that the BLIPgene and the gene for isopenicillin N synthase (36) are notclosely linked (A. K. Petrich, J. L. Doran, and S. E. Jensen,unpublished data).The biological significance of BLIP remains uncertain.

Streptomyces spp. produce a number of different types ofextracellular enzyme inhibitors. However, no other protein-aceous ,3-lactamase inhibitor from Streptomyces spp. or anyother source has been characterized. Comparison of theBLIP amino acid and nucleotide sequences with all se-quences currently available in the GenBank and NationalBiomedical Research Foundation data banks failed to pro-vide an indication of any evolutionary or structural relation-ship which might reveal the potential mechanism of BLIPactivity. Therefore, BLIP appears to be an unusual proteinwhich may have a unique mode of action. Although it may bedifficult to foresee a clinical application of a proteinaceous,-lactamase inhibitor, the eventual understanding of theinhibitory activity of BLIP may lead to the development ofnew strategies for coping with the clinical occurrence of1-lactam antibiotic resistance.

It is tempting to speculate that the production of BLIP byS. clavuligerus is part of a response to the production of1-lactamases by other organisms in the surrounding soilmicroenvironment which may threaten the antimicrobialactivity of the P-lactam antibiotics produced by S. cla-vuligerus. Inherent in this notion is the hypothesis thatP-lactam antibiotics are produced to challenge competitivebacteria. S. clavuligerus has long been known to produce the,B-lactamase inhibitor clavulanic acid, but since clavulanicacid is structurally related to the other classical ,-lactamcompounds that S. clavuligerus produces, the ,3-lactamase-inhibitory properties of clavulanic acid could be coincidentaland of no particular significance to the organism. Thisdiscovery of BLIP, a second structurally unrelated P-lacta-mase inhibitor, suggests that the inhibitory property of theseproducts is of value to S. clavuligerus.An alternative hypothesis for the significance of BLIP is

that it functions as a regulator of cell wall growth ormorphogenesis. Certain Streptomyces species produce cellwall biosynthetic enzymes known as penicillin-binding pro-teins (PBP), in a soluble form in the culture medium ratherthan in the more usual membrane-bound location (17).Studies on the PBP of S. clavuligerus have shown that incomparison with other Streptomyces species, this organismhas relatively few membrane-bound PBP and produces morekinds of PBP in the culture supernatant and the solublefraction inside the cells than in the membrane (44). Since ithas been suggested that ,-lactamases and PBP have acommon evolutionary origin (31), the characteristics ofBLIP which allow it to interact with and inhibit 1-lactamasesmay also allow it to function as an inhibitor or regulator ofPBP. Studies exploring this possibility are in progress.

ACKNOWLEDGMENTS

We thank Pam Banser for excellent technical assistance. We aregrateful to Sandy Kielland (Protein Microsequencing Laboratory,Department of Biochemistry and Microbiology, University of Vic-toria, Victoria, British Columbia, Canada) for conducting the totalamino acid analysis, N-terminal amino acid sequencing, and proteinsequence data analysis and comparisons using the FASTP pro-grams. We also thank Kenneth L. Roy (Department of Microbiol-ogy, University of Alberta, Edmonton, Alberta, Canada) for thepreparation of oligonucleotide sequencing primers.

This research was supported by grants from the Natural Sciences

and Engineering Research Council of Canada and the AlbertaHeritage Foundation for Medical Research.

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