Vol. 172, No. 11JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6339-63470021-9193/90/116339-09$02.00/0Copyright © 1990, American Society for Microbiology

Cloning, Expression, and Nucleotide Sequence of the Lactobacillushelveticus 481 Gene Encoding the Bacteriocin Helveticin Jt

M. C. JOERGER't AND T. R. KLAENHAMMERl 2*

Departments ofFood Science' and Microbiology,2 Southeast Dairy Foods Research Center,North Carolina State University, Raleigh, North Carolina 27695-7624

Received 24 April 1990/Accepted 9 August 1990

Lactobacilus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomalDNA from L. helveticus were prepared in Agtll and probed for phage-producing fusion proteins that couldreact with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologousinserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clonesspecifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not producehelveticin J or strains producing unrelated bacteriocins. HJ1 and H14 lysogens produced 0-galactosidase fusionproteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) wasisolated by screening a library of L. helveticus chromosomal DNA in XEMBL3 with the insert DNA from phage1P4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete openreading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' endof another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putativepromoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticusDNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from thetermination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end ofanother ORF (ORF4) was found. A BglM fragment containing ORF2 and ORF3 was cloned into pGK12, andthe recombinant plasmid, pTRK135, was transformed into LactobaciUus acidophilus via electroporation.Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an activityspectrum that was the same as that of helveticin J.

Bacteriocins are defined as proteinaceous antimicrobialagents that exhibit a bactericidal mode of action. Lactoba-cillus helveticus 481 produces a bacteriocin designated ashelveticin J (16). This bacteriocin was partially purified andcharacterized as a heat sensitive, 37-kDa protein that inhibitsgrowth of closely related Lactobacillus species. Bacteriocingenes are typically plasmid borne (43), but the geneticdeterminants encoding helveticin J appear to reside on thechromosome (16). Evidence for chromosomally located bac-teriocin genes has also been reported for lactacin B producedby Lactobacillus acidophilus N2 (2) and lactocin 27 pro-duced by L. helveticus LS18 (44).Many of the bacteriocins (colicins) produced by Esche-

richia coli have been genetically characterized (for a review,see reference 26). A common feature of colicin plasmids isthe contiguous arrangement of genes whose products deter-mine bacteriocin synthesis, immunity, and release via lysis.DNA sequence analysis and transcriptional studies haveshown the presence of an SOS-inducible promoter, whichquite often precedes an operon of colicin, immunity, andlysis genes that are transcribed in the same direction (as inthe case of CloDF13 genes). Colicin E2 and E3 operons havebeen studied in which the immunity gene is transcribed froma secondary promoter located in the terminus of the bacte-riocin structural gene. The colicin El plasmid has beenshown to have yet a different genetic organization, in which

* Corresponding author.t Paper 12544 of the Journal Series of North Carolina Agricultural

Research Service, Raleigh, N.C.t Present address: E.I. Du Pont de Nemours and Co., Agricul-

tural Products, Newark, DE 19714-6101.

the immunity gene is transcribed in the opposite directionfrom its own promoter.

In lactic acid bacteria, the cloning and expression ofbacteriocin genetic determinants have been limited to thelactococci (4, 19, 39, 45). Bacteriocin genes of Lactococcuslactis subsp. diacetylactis WM4 were localized to an 18.4-kbXhoI fragment originating from the 131.3-kb plasmid pNP2by analyzing clones for bacteriocin production and usingrestriction mapping techniques (39). Van Belkum et al. (45)were able to identify two regions within the L. lactis subsp.cremoris 9B4 plasmid p9B4-6, each encoding a uniquebacteriocin along with the corresponding immunity geneticdeterminants. In addition, nisin genes from L. lactis havebeen cloned and sequenced but not expressed (4, 19).To date, the bacteriocin genes of lactobacilli have not been

isolated or characterized. Here, we report on the cloning andsequencing of DNA containing the helveticin J (hlv) gene(s)from L. helveticus 481.

(This work was submitted in partial fulfillment of thePh.D. requirements of M. C. Joerger at North Carolina StateUniversity.)

MATERIALS AND METHODSBacterial strains, phage, and plasmids. The bacterial

strains, phage, and plasmids used in this study are listed inTable 1. Lactobacillus strains were propagated at 37°C inMRS broth (Difco Laboratories, Detroit, Mich.). E. colistrains were propagated at 32, 37, or 42°C in LB mediumwith shaking. Agar media contained 1.5% agar; overlaymedium contained 0.75% (MRS) or 0.80% (LB) agar. Forphage propagation, 10 mM MgCl2 was added to LB medium.The following concentrations of antibiotics were used: ampi-cillin at 100 ,ug/ml, chloramphenicol at 10 ,ug/ml, erythromy-

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TABLE 1. Bacteria, phage, and plasmids

Bacterium, phage, or plasmid Relevant characteristics Source or reference

Lactobacillus helveticus103 NCK229481 NCK228481-C NCK2461846 NCK230

Bac+aHlv+ Hlvr, pMJ1008Hlv+ HlvF, plasmid curedHlvS LaP

NCDOb, ATCC 8001NCDO (16)16NCDO

Lactobacillus bulgaricus1373 NCK2321489 NCK231

Lactobacillus acidophilus88 NCK8888-C NCK6489 NCK89NCK247NCK248NCK249NCK25011694 NCK121ADH NCK100

Lactobacillus jugurti1244 NCK233

Lactobacillus fermentum1750 NCK127

Lactobacillus lactis970 NCK234NCK252

HlvS LafPHlv5 LaP

LafF Laf!Laf LafrLaf-LaPHlv+/Hlv-Hlv-Hlv+, pTRK135LaC Lafr, pGK12LaP Hlv'Bac+ Bacr, pTRK15

NCDONCDO, ATCC 11842

173232This studyThis studyThis studyThis study20MS02 (22)

NCKC collectionHlvs LaP

LafP Hlv'

Hlvs LafPSpontaneous Cm' isolate of NCK234

NCDO (2)

NCDOThis study

Lactobacillus leichmannii4797 NCK235

Escherichia coliY1089(r-) NCK74Y1089-HJ1 NCK75Y1089-HJ4 NCK78Y1090(r-) NCK79

LE392 NCK57

XL1 Blue NCK80

NCK81NCK82NCK83NCK84K12 71-18 NCK238GM1829 NCK239

NCK241

PhageLambdaHJ1 nck79.gtll-HJ1HJ4 nck79.gtll-HJ4Hlv3 nck57.EMBL3-Hlv3

M13

mpl8mpl9

AlacUl69 pro' Alon araDl39 strA hflA [chr::TnlO](pMC9)Y1089::Xgtll-HJ1Y1089::Xgtll-HJ4AlacUl69 proA+ Alon araDl39 strA supF [trpC22::TnJO] hsdRhsdM+ (PMC9)

F hsdRSl4(rMC m-) supE44 supF58 lacY) or (lacIZY)6 galK2galT22 metBi trpR55 lambda-

endA1 hsdRJ7 (rK- mKE) supE44 thi-1 lambda- recAl gyrA96relAl (Lac-)[F' proAB lacPZM15 TnJO (Tet9)]

XL1 Blue (pTRK131)XL1 Blue (pTRK132)XL1 Blue (pTRK133)XL1 Blue (pTRK134)A(lac pro)F' LacPqZM15 pro' supEF- recA441 sulA thr-1 leu-6 his4 argE3 ilv(Ts) galK2 rpsL31 Str'lacUl69 dinDl::Mu d(Ap lac) dam4

GM1829(pTRK135)

M13 cloning vector

M13 cloning vector

51This studyThis study51

33

5

This studyThis studyThis studyThis study308

This study

This studyThis studyThis study

5050

Continued on following page

LafP Hlvr ATCC 4797

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LACTOBACILLUS BACTERIOCIN GENE 6341

TABLE 1-Continued

Bacterium, phage, or plasmid Relevant characteristics Source or reference

PlasmidsBluescript M13 KS(+) lacZ, Apr, 3.0 kb Stratagene, LaJolla,

Calif.pTRK131 Bluescript M13 KS(+)::HJ1, Apr, 3.35 kb This studypTRK132 Bluescript M13 KS(+)::HJ2, Apr, 3.9 kb This studypTRK133 Bluescript M13 KS(+)::HJ3, Apr, 3.75 kb This studypTRK134 Bluescript M13 KS(+)::HJ4, Apr, 3.6 kb This studypGK12 Cmr Emr, 4.4 kb 23pTRK135 pGK12::Hlv-BglII, Cmr Ems, 8.4 kb This study

a Bac, Bacteriocin; Hlv, helveticin J; Laf, lactacin F; (+), producer; (-), nonproducer; s, sensitive; r, resistant.b NCDO, National Collection of Dairy Organisms.c NCK, Culture collection of T. R. Klaenhaemmer at North Carolina State University.

cin at 400 ,ug/ml, and tetracycline at 10 pug/ml for E. coli;chloramphenicol at 7.5 ,ug/ml and a combination of erythro-mycin at 3 ,ug/ml and chloramphenicol at 3 ,ug/ml forLactobacillus strains.

Bacteriocin detection. Lactobacillus strains were examinedfor bacteriocin production by overlaying colonies with alawn of a sensitive indicator microorganism by deferredmethods (2, 20). An adaptation of the critical-dilution assay(29) was used for titration of bacteriocin activity (16).Antibody methods. Helveticin J was partially purified as

previously described (16). After preparatory sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (24) ofpartially purified helveticin J, proteins were electrophoreti-cally transferred (6) to nitrocellulose (BA85, 0.45-,m poresize; Schleicher & Schuell, Keene, N.H.), and the bandcorresponding to the 37,000-Da bacteriocin was excisedfrom the membrane. The nitrocellulose strip with immobi-lized helveticin J was dissolved in dimethyl sulfoxide. Ap-proximately 10 ,ug of bacteriocin was emulsified with Freundcomplete or incomplete adjuvant (Sigma Chemical Co., St.Louis, Mo.) and administered to New Zealand White rabbitsat 2-week intervals. Immune serum was obtained after 11weeks. The immunoglobulin G (IgG) fraction was purifiedwith a protein A-Sepharose (Sigma) column (34). A cellextract was employed to remove E. coli-reactive IgG fromthe antiserum before screening the Agtll library.

P-Galactosidase fusion protein was purified with a Pro-tosorb P-galactosidase immunoaffinity column (PromegaCorp., Madison, Wis.). Purified fusion protein (0.5 to 1.0 mgper injection) was emulsified with adjuvant and injectedevery 2 weeks. After 6 weeks, immune serum was collectedand the IgG fraction was isolated as described above. IgGthat was reactive to the P-galactosidase portion of the fusionprotein was removed by using a P-galactosidase columnprepared with an ImmunoPure Ag/Ab immobilization kit(Pierce, Rockford, Ill.).

Protoblot immunoscreening and Western immunoblot al-kaline phosphatase systems (Promega), for the detection ofantibody bound to proteins immobilized on nitrocellulosemembranes, were used as described by the suppliers withthe following modifications. Calf serum (20% in Tris-buff-ered saline) was employed as a blocking agent. Tween 20was omitted from the wash solutions for filters that wereprobed with helveticin J IgG because of the low affinity ofthe antibody. 1-Galactosidase monoclonal antibody wasobtained from Promega.For antibody neutralization or precipitation experiments,

samples of partially purified helveticin J were mixed withdilutions of IgG and held overnight at 4°C in phosphate-buffered saline with gentle agitation. After centrifugation at

16,000 x g for 10 min in a microcentrifuge, the supernatantswere titered against the helveticin J-sensitive indicator L.bulgaricus 1489.

Antibodies that were reactive with helveticin J epitopesencoded by Xgtll recombinants were affinity purified fromthe polyvalent rabbit IgG as described by Snyder and Davis(41). The antigen-selected antibodies were used to probeimmunoblots (6) of partially purified helveticin J.DNA manipulations and isolation. Total genomic DNA and

plasmid DNA from Lactobacillus strains were isolated andpurified as previously described (1, 16, 21). Extraction ofplasmid DNA from E. coli and purification of X phage DNAwere as described previously (3, 27, 28, 36). All restrictionand DNA-modifying enzymes were employed as prescribedby the manufacturers. Southern blots (42) and plaque lifthybridizations were performed with [35S]dCTP-labeledprobes prepared by using a Multiprime DNA labeling system(Amersham Corp., Arlington Heights, Ill.) or with nonradio-active probes labeled with digoxigenin-dUTP by using aDNA labeling and detection kit (Boehringer MannheimBiochemicals, Indianapolis, Ind.). DNA was immobilized onMagna graph nylon membrane (0.45-,um pore size; MSI,Westboro, Mass.). Hybridizations employing [35S]dCTP-labeled probes were conducted at 42°C in the presence of 6xSSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)and 50% formamide. Digoxigenin-dUTP-labeled probeswere hybridized with DNA at 68°C in the presence of SxSSC.

Construction of genomic libraries and subclones. TotalDNA from L. helveticus 481-C was sonicated to obtain DNAfragments 2 to 6 kb in size. A Xgtll library was constructedas described by Huynh et al. (15) by using sonicated andend-repaired L. helveticus DNA fragments. L. helveticus481-C total DNA was partially cleaved with Sau3A togenerate fragments of 15 to 23 kb. These sized fragmentswere ligated into the BamHI-cleaved vector XEMBL3 (28).DNA fragments from insert DNA in XEMBL3 or Agtllclones were ligated into Bluescript M13 KS(+) DNA (Strat-agene, La Jolla, Calif.) or into the shuttle vector pGK12 (22).

Preparation and analysis of recombinant lysogens. E. coliY1089 was infected with Agtll clones, and lysogens wereisolated as described previously (15). Crude lysates from theXgtll recombinant lysogens were prepared (15) and sub-jected to SDS-polyacrylamide gel electrophoresis (24). P-Ga-lactosidase fusion proteins were purified via affinity columnchromatography as outlined above. The crude lysates andpurified fusion proteins were subjected to Western blotanalysis (6) with antibodies reactive to helveticin J and theHJ4 fusion protein.

Electroporation. E. coli and lactobacilli were transformed

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with recombinant DNA via electroporation (10, 25) with aGene Pulser apparatus (Bio-Rad Laboratories, Richmond,Calif.). Electroporation of L. acidophilus NCK64, NCK88,and NCK89 was conducted as described previously (25) withthe following modifications. Cells (200 ml) were harvestedafter they reached an optical density at 600 nm of 0.78 to0.80, and the pellet was washed two times with 150 ml ofelectroporation buffer (1 M sucrose, 2.5 mM CaCl2 [pH 7.0]).After the second wash, the cells were suspended in 2.6 ml ofthe same buffer and held on ice. Cells (400 RI) and DNA weremixed and pipetted into a chilled 0.2-cm cuvette. An elec-trical pulse was delivered to the cuvette with the Gene Pulserapparatus set at 25 pLF, 2.1 kV, and infinite ohms.DNA sequencing and analysis. Contiguous 2.8-kb EcoRI

and 5.5-kb HindlIl fragments, containing the putative hlvgenes, were subcloned from the XEMBL3 recombinant Hlv3into M13 mpl8 and mpl9. These two fragments were alsocleaved with AluI, RsaI, Sau3A, TaqI, HindIII-SphI, andSau3A-SphI, and the resulting subfragments were clonedinto M13 mpl8 and mpl9 (50). HJ1 and HJ4 insert DNA wasalso ligated with EcoRI-cleaved M13 mpl8 and mpl9. DNAfor sequencing was prepared from the phage by a procedurepublished by International Biotechnologies, Inc. (New Ha-ven, Conn.). Sequencing of both DNA strands was carriedout by the method of Sanger et al. (38) with [35S]dATP (NENResearch Products, Boston, Mass.) and the Sequenase DNAsequencing kit (United States Biochemical Corp., Cleve-land, Ohio). A number of site-specific primers were synthe-sized with a Pharmacia Gene Assembler oligonucleotidesynthesizer (Pharmacia, Piscataway, N.J.) to sequence re-gions for which no suitable subfragments were available.

Individual sequences were analyzed for overlaps andorganized into a contiguous sequence (18). Restriction sitesand predicted amino acid sequences were determined byusing programs written by Mount and Conrad (7, 31). TheDNA sequence was subjected to a base-preference analysiswith the UWGCG computer program Testcode (9, 11).Molecular weights and pls were calculated with the programPeptideSort (UWGCG). The GenBank and the NBRF pro-tein sequence data bank were searched for similar DNA oramino acid sequences by using the program of Wilbur andLipman (48).

Nucleotide sequence accession number. These data havebeen submitted to GenBank under accession no. M30121.

RESULTS

Characterization of Xgtll clones reactive with polyclonalantibody to helveticin J. Bacteriocin-reactive IgG, isolatedfrom antiserum made against helveticin J, was employed toscreen 350,000 clones representing several Xgtll libraries oftotal DNA isolated from L. helveticus 481-C. Two phages,designated HJ1 and HJ4, produced positive signals. Theinserts from these two clones hybridized to each other and tototal genomic DNA isolated from L. helveticus 481, theproducer of helveticin J. The clones did not hybridize toDNA from L. helveticus 103 or 1846 or L. jugurti 1244,which either produce a bacteriocin different from helveticinJ or do not produce a bacteriocin.

E. coli Y1089 containing HJ1 or HJ4 lysogens producedlarge amounts of ,-galactosidase fusion proteins. The HJ4fusion protein was slightly larger than the HJ1 fusion pro-tein. This would be expected, since HJ4 has a 600-bp insert,whereas HJ1 has a 350-bp insert. Helveticin J activity wasnot detected when the titers of the lysates were determinedagainst the sensitive indicator L. bulgaricus 1489.

Both ,B-galactosidase monoclonal antibody and helveticinJ antibody bound to the fusion protein bands present inlysates obtained from isopropyl-,-D-thiogalactopyranoside-induced E. coli Y1089 carrying the HJ1 or HJ4 lysogens.Signals produced by the bacteriocin IgG binding to thefusion proteins were faint in comparison with the signalgenerated by the antibody binding to helveticin J. The signalwas also faint on immunoblots of fusion proteins purified viaa P-galactosidase immunoaffinity column. This phenomenonis most likely due to the fusion proteins displaying fewerantigenic epitopes as compared with those displayed byhelveticin J, and hence fewer antibody molecules bound tothe hybrid proteins.

Affinity purification of antibodies recognizing recombinantprotein. One strategy for testing whether a Agtll recombi-nant encodes the particular protein is to use the phage clonesto affinity purify antibody from the screening IgG (41). Theclone-purified antibody is then used to probe immunoblots ofthe protein in question. HJ1 and HJ4 were employed toaffinity purify a fraction of the screening IgG that boundantigenic epitopes of the fusion proteins produced by eachrespective clone. The wild-type Xgtll was employed as anegative control in the affinity purification of helveticin Jantibody. Very faint signals were obtained when wild-typeXgtll-purified IgG, representing P-galactosidase-reactive an-tibodies, was probed against immunoblots of HJ1 and HJ4fusion proteins. No signal was detected against membrane-immobilized helveticin J. HJ1- or HJ4-purified IgG producedsignals with both HJ1 and HJ4 fusion proteins. HJ4-purifiedIgG produced a faint signal with helveticin J, and an evenweaker signal was produced by the HJ1-purified IgG. Thedifference in signal intensity is logical, since the HJ4 clone islarger than the HJ1 clone. Therefore, the HJ1 and HJ4 fusionproteins shared some of the same antigenic epitopes as thosedisplayed on helveticin J.

Fusion protein antibody studies. The ,-galactosidase fusionprotein produced by the lysogen E. coli Y1089-HJ4 waspurified via a ,B-galactosidase immunoaffinity column. Thepurified fusion protein was injected into rabbits to producepolyclonal antibody specific for epitopes expected to bepresent on the HJ4-encoded portion of the fusion protein.P-Galactosidase-specific antibody was removed from thetotal IgG fraction with an agarose column containing immo-bilized P-galactosidase. A high-affinity antibody was ob-tained that gave strong signals when bound to membrane-immobilized helveticin J.To aid in clone verification, an immunoblot of partially

purified helveticin J, P-galactosidase, and the HJ1 and HJ4fusion proteins was probed with the HJ4-specific antibodylacking the P-galactosidase-reactive portion of IgG. A dupli-cate protein blot was stained to detect protein bands. Theantibody bound and produced signals with the HJ1 and HJ4fusion proteins and the 37,000-Da protein band correspond-ing to helveticin J (Fig. 1). The antibody also producedsignals with a triplet of bands of approximately 43,000 Da inthe partially purified bacteriocin preparation. These bandswere not detected previously with the antibody that wasmade against denatured helveticin J. Possibly, the secondantibody made against the fusion protein detected anotherform of the bacteriocin. No signals were detected in theP-galactosidase lane (Fig. 1), indicating that all of the n-ga-lactosidase-reactive IgG had been successfully removedfrom the screening antiserum. These results unequivocallydemonstrated that the portion of the fusion proteins encodedby HJ1 and HJ4 shared similar antigenic epitopes withhelveticin J.

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A B C D E F G H I charged amino acid (histidine) was present within the amino-terminal region of the signal peptide. However, this regioncarried a net negative charge, a property that would beunusual for signal peptides.ORF2 and ORF3 were separated by a small noncoding

region of 30 bp. A putative ribosomal binding site (13) waslocated 7 bp upstream of the initiation codon of ORF3. The

-- _*1,002-bp ORF3 could encode a hydrophilic protein with a pIof 6.03 and a molecular weight of 37,511, which is very closeto the weight of helveticin J of 37,000 as predicted bySDS-polyacrylamide gel electrophoresis (16). The L. helve-ticus DNA inserts in HJ1 and HJ4 were contained within

_m, ORF3; HJ4 (515 bp) was located at nucleotide positions 2314to 2828 (Fig. 2). The smaller HJ1 insert (338 bp) was internalto HJ4 and resides between nucleotides 2491 and 2828.Searches ofNBRF and GenBank sequence data libraries didnot identify any polypeptides with significant sequence ho-mology to the predicted products of ORF2 or ORF3.A 312-bp noncoding region separated ORF3 and ORF4. A

FIG. 1. Lanes A through E show an SDS-polyacrylamide gel putative ribosomal binding site lay 5 bp upstream of theprotein blot stained with amido black: A, molecular weight markers initiation codon of ORF4. Since L. helveticus 481-C is(phosphorylase b, 94,000; albumin, 67,000; oval,bumin, 43,000; adenine and thymine rich, many areas in the noncodingcarbonic anhydrase, 30,000; from top to bottom); B, partially region could serve as the -10 promoter site. However, apurified helveticin J; C, P-galactosidase; D, HJ1 fusion protein; E, -35 region was not evident within the same stretch of DNAHJ4 fusion protein. Lanes F through I show an SDS-polyacrylamide P tgel protein blot probed with HJ4 fusion protein IgG: F, partiallypurified helveticin J; G, P-galactosidase; H, HJ1 fusion protein; I region, or perhaps ORF4 is a continuation of the ORF2-HJ4 fusion protein. The arrow indicates a 37,000-Da protein band ORF3 putative operon.corresponding to helveticin J. Expression of helveticin J. Attempts to clone the 5.5-kb

HindIII fragment derived from X recombinant Hlv3 in thehigh-copy-number E. coli vector Bluescript M13 were un-

Attempts to demonstrate neutralization or precipitation of successful due to plasmid rearrangements in the transfor-helveticin J with antibodies specific for the bacteriocin or mants. A smaller 4-kb BglII fragment of the same L. helve-fusion protein were unsuccessful. ticus 481-C DNA was ligated into the BclI site of pGK12 and

Isolation of the intact helveticin J gene and DNA sequence successfully transformed into E. coli GM1829. Restrictionanalysis. To isolate a DNA fragment that encoded the entire enzyme analysis of the recombinant plasmid, pTRK135,bacteriocin, a library of L. helveticus 481-C DNA in verified that the 4-kb BglII insert was intact. Helveticin JXEMBL3 was constructed and probed with the insert DNA activity was not detected in broth supernatants or cellfrom HJ4. A number of positive XEMBL3 clones were extracts of E. coli GM1829 harboring pTRK135.detected and purified for further analysis. The HJ4 insert Electroporation was employed to introduce pTRK135 intohybridized to a 2.8-kb EcoRI fragment and a 5.5-kb HindIII L. acidophilus NCK88, NCK64, NCK89, and ADH and L.fragment from the XEMBL3 recombinant Hlv3. Both of fermentum 1750. Plasmid DNA analysis of Cmr coloniesthese fragments were cloned into phage M13 in both orien- revealed three classes of transformants. Class I transfor-tations. Smaller subfragments of the EcoRI and HindIII mants, obtained with strains NCK88, NCK64, ADH, andfragments, which could readily be sequenced, were gener- 1750, were visually devoid of pTRK135 but exhibited a Cmrated with restriction enzymes and subcloned into M13. Also, phenotype. Deleted forms of pTRK135 were present in thethe HJ1 and HJ4 inserts were subcloned into M13. class II transformants acquired with strains NCK88,Both strands of a contiguous 3,364-bp region were se- NCK64, and NCK89. Several L. acidophilus NCK64 trans-

quenced (Fig. 2). The gene organization of the sequenced formants in class III appeared to have the intact plasmid.region is shown in Fig. 3. Open reading frame (ORF) 2 and After repeated transfers of the L. acidophilus NCK64 classORF3 have been completely sequenced and are possibly III transformants through MRS broth containing chloram-cotranscribed. Only the 3' end of ORF1 and the 5' end of phenicol, pTRK135 remained intact.ORF4 were present in the region where the nucleotide The class III transformant, L. acidophilus NCK247sequence has been determined. DNA sequence analysis by (pTRK135), was plated on MRS agar containing chloram-the Testcode program (11) predicted that a 1,400-bp region phenicol and overlaid with an indicator lawn of L. lactiscontaining ORF2 and ORF3 has a high probability of being a NCK252 to detect bacteriocin-producing colonies. The ma-coding region. jority of colonies exhibited zones of growth inhibition in theORF2 consisted of 315 bp and possibly encodes a protein indicator lawn. A colony producing a zone of inhibition

of 11,808 Da with a pl of 6.81. Upstream of ORF2, putative (NCK249) and a colony lacking a similar zone (NCK248)-10 and -35 promoter regions (37) could be identified (Fig. were isolated and examined for bacteriocin-producing abil-2). The putative -35 region was identical to corresponding ity. The isolate NC}K249 gave rise to a homogeneous popu-regions in promoters from E. coli (37). A possible ribosomal lation of colonies with inhibition zones (Fig. 4). The "no-binding site (13) was located 5 bp upstream of the initiation zone" phenotype of NCK248 was also stable. Plasmid DNAcodon for ORF2. The putative protein encoded by ORF2 examination ofNCK249 and NCK248 revealed that NCK249contained an amino terminus with characteristics of a signal carried the intact pTRK135, whereas NCK248 had incurredpeptide (46, 47), because it contained a stretch of nonpolar a deletion in the recombinant plasmid. Total plasmid DNAamino acids that preceded a glycine residue. A positively was isolated from NCK249 and used to transform E. coli

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ORF 1 ->1 G AAT TCC AAA GAT GCA GAT CCT ATT TAT GTA GGA AAA AAC AAC 43

N S K D A D P I Y V G K N N

44 TAC AAA TAT GCT TTA ACG CAT TAT GAA ACC TTC AAG GGC AAG 85Y K Y A L T H Y E T F K G K

86 ACA ATT AGT CCA GCT AAG GTT CAA AAC GTT AAA TTT AGA GTT 127T I S P A K V Q N V K F R V

128 GAA AAG ATA GTC AGA TTC CAC GGC AAA ATT AGT GGT GCT CCA 169E K I V R F H G K I S G A P

170 TTG TAC TTA GTG GCT TCT AAG GAT AAG AAG TAC AGC TGC TGG 211L Y L V A S K D K K Y S C W

212 ACT ACG CAA GCA ATG CTT CAA TAT TAT TAC TTC AAT AGC AAG 253T T Q A M L Q Y Y Y F N S K

254 GGG ATG CGC GGA GTA GTA AAT CCA TTG AAG AGA ATT GCT AAT 295G M R G V V N P L K R I A N

296 AGA AGT GCT GAT AAG AAT ATT ATT AGT CTA AAG AAT AAA CAA 337R S A D K N I I S L K N K Q

338 AAT AAA CGT GAC TTT AAT GCA GCA ATG AAG GCT GCT AAT AAG 379N K R D F N A A M K A A N K

380 CTT AAG GGC AGT CAG AAG AAG TTT GTT GTA AAT TCT TTG AAG 421L K G S 0 K K F V V N S L K

422 CAA CTT AAG AAA GAT AAC AAT ATT GGC GTT GAA GGC GAC AAC 463Q L K K D N N I G V E G D N

464 TTG CTT TTG TTT GGT TTT TAA AGTGAAAAAKTACGTTAAAAGAACA 510L L L F G F *

511 TAGAAAAACTGATGCATCTAAGCTATGAAGCTAGGAGCATCAGTTTTTTGTTA 563

564 CATTAGATCGTCATTGTCTTTAAAAAGTTGCTTGGTTGCAGCGGTCATTCTTT 616

617 CGGGGATGCCATAGTTCTGCATAAAGGTTCCTGGTTCTTGAATGTAGTCACCA 669

670 TTTTTGCAGGAATAGTCGTAAATAATCTTAAGAGAAAATAGATCTGCTTCCTC 722

723 TTCTTCCGAGTTTTGTCTTGAAAAGCTAGGCCAATAGGCGATACCTGAATCAC 775

776 CTAACATTAAGTGACCAATTTCGTGACCAATGATAAAAGGCAATTCATTAGGA 828

829 TTATGCCAATTAGTATTGATTACCATTTTATGAGCATTCTTAAATGAAAGTGC 881

882 TGGGTCGTATGGCTCTCCTTTAACCAAGATGTATGAAAGACCGTGCTTAAAAG 934

935 CATAATTGAGTAAGTACTCAATTAACTCTTCTTTACCTAAGTTTCTCATTTCT 987

988 TGTCTGCACGACCTTCCTTAATGTCATCTTCCATTAGTCCTCGAATCATATTGA 1041

1042 GATATCTCTCAGGAACGTTGTAACCATGATATGAATAAGGCTTTTCTTCGTCTA 1095

1096 GTGGAATAGAAGTTGGTTGTCCTTTTGCAGGTATAGGGGTAGGATCCATCAGTT 1149

1150 TTACCTTTTAGGTAATCGATTGAAGTATTTAGTACTTCGGCAACGGTTCTAAGG 1203

1204 CATCGCCACCAGGTCTTTTATTCTTCCAATTATAGATAGAATTAGTACCTAACC 1257

1258 CAGCCTTATCATTCACTTCACGTAAGTTCATTTTATATTTTTTCGCTAATTCTT 1311

1312 TAGTACGCTCTAATTCAATCATGGCAAGCATTCTCCGAATATTTTAAATTATTA 1365

1366 AATAAGTATTTTTACTAAAAATGTATfGACAATATTAGTCTTTGGACGTA&TAT 1419

1420 AAGAACTGTCAACGAGATATGAAAGAAATAAGAAAAAGCCTAGAAACACGTGTG 1473

1474 TAAGTAAGAAAGGAATCATGTGCTTAAGGTTATTTCTTATACCATTATATTAGT 1527

1528 ATAAATACTAATATAAGTCAACGAATATTAGTATCTAGTTGATAAATTTTATTCA 1582

1583 AAGAGGTGGTGCCAATGACTGTGCAATAAGGAAAAAGAGAGGTiAAGIA&AAATC 1637ORF 2 ->

1638 ATG GAT ATT CAT GAT TAC GTT GAA TTG ATA GCT TTA GCG TTT 1679M D I H D Y V E L I A L A F

1680 TGG GTT ATT AGT GTT GTA AGT GTT GGT ATC TTG AGT CAT GTT 1721W V I S V V S V G I L S H V

1722 CAT TTT AAG AAT AAG AGG CTG GAA CAG TTT CGT ATT ACT GCT 1763H F K N K R L E Q F R I T A

1764 GAT GAT TTG ATG AAA AAC TAC GTT GGT TTG TAC AAC AAA GAA 1805D D L M K N Y V G L Y N K E

1848 GTA GTA GAC GGA CTA GAA GCT AAA GGT TTT AAA GTG GAA GAC 1889V V D G L E A K G F K V E D

1890 CAA GAT GTA AAG GAT ATT TTT GCA AAG GTC GCA AAA ATT ATT 1931Q D V K D I F A K V A K I I

1932 AAT GAA AAT TCT TCT AAG TAG GAAGATAGAGATTTTTTC2AGGTTTT 1979N E N S S K *

ORF 3 ->1980 ATT ATG AAG CAT TTA AAT GAA ACA ACT AAT GTT AGA ATT TTA 2021

M K H L N E T T N V R I L

2022 AGT CAA TTT GAT ATG GAT ACT GGC TAT CAA GCA GTA GTT CAA 2063S 0 F D M D T G Y Q A V V 0

2064 AAA GGC AAT GTA GGT TCA AAA TAT GTA TAT GGA TTA CAA CTT 2105K G N V G S K Y V Y G L Q L

2106 CGC AAA GGT GCT ACT ACT ATC TTG CGT GGT TAC CGT GGA AGT 2147R K G A T T I L R G Y R G S

2148 AAA ATT AAT AAC CCT ATT CTT GAA TTA TCT GGT CAA GCA GGT 2189K I N N P I L E L S G Q A G

2190 GGT CAC ACA CAG ACA TGG GAA TTT GCT GGT GAT CGT AAA GAC 2231

G H T 0 T W E F A G D R K D

2232 ATT AAT GGT GAA GAA AGA GCA GGT CAA TGG TTT ATA GGT GTT 2273

I N G E E R A G 0 W F I G V

HJ4->2274 AAA CCA TCG AAA ATT GAA GGA AGC AAA ATT ATT TGG GCA AAG 2315

K P S K I E G S K I I W A K

2316 CAA ATT GCA AGA GTT GAT CTT AGA AAT CAA ATG GGA CCT CAT 2357

0 I A R V D L R N 0 M G P H

2358 TAT TCA AAT ACT GAC TTT CCT CGA TTA TCC TAC TTG AAT CGC 2399

Y S N T D F P R L S Y L N R

2400 GCC GGT TCT AAT CCA TTT GCT GGT AAT AAG ATG ACG CAT GCC 2441A G S N P F A G N K M T H A

2442 GAA GCC GCA GTA TCA CCT GAT TAT ACT AAG TTT TTA ATT GCT 2483

E A A V S P D Y T K F L I A

HJ1->2484 ACT GTT GMA AAT AAC TGT ATT GGT CAT TTT ACT ATA TAC AAT 2525

T V E N N C I G H F T I Y N

2526 TTA GAT ACA ATT AAT GAA AAA CTT GAT GAA AAG GGA AAT AGT 2567L D T I N E K L D E K G N S

2568 GAA GAT GTT AAT CTC GAA ACT GTT AAA TAC GAA GAT AGT TTT 2609E D V N L E T V K Y E D S F

2610 ATC ATT GAT AAT TTA TAT GGT GAT GAT AAT AAT TCT ATT GTA 2651I I D N L Y G D D N N S I V

2652 AAT TCA ATT CAA GGG TAT GAT TTG GAT AAT GAT GGA AAT ATT 2693N S I G Y D L D N D G N I

2694 TAT ATT TCC AGT CAA AAA GCG CCA GAT TTT GAT GGC TCT TAT 2735Y I S S 0 K A P D F D G S Y

2736 TAT GCA CAT CAT AAG CAG ATT GTT AAG ATT CCA TAT TAT GCT 2777Y A H H K 0 I V K I P Y Y A

2778 CGG TCT AAA GAA AGC GAA GAC CAA TGG AGA GCT GTA AAT TTA 2819R S K E S E D Q N R A V N L

<- HJ1, HJ42820 AGC GAA TTQ GGT GGC TTG GAT ATT CCA GGT AAA CAT AGT GAA 2861

S E F G G L D I P G K H S E

2862 GTT GAA AGC ATC CAA ATT ATT GGT GAG AAT CAT TGT TAC TTA 2903V E S I 0 I I G E N H C Y L

2904 ACT GTT GCA TAT CAT TCT AAA AAT AAA GCG GGT GAA AAT AAA 2945T V A Y H S K N K A G E N K

2946 ACT ACT TTG AAT GAG ATT TAT GAA TTA TCT TGG AAT TAG ATT 2987T T L N E I Y E L S N N *

2988 CTTGTTAGTGGTCTCGATTTAGATATAAACTAACAAAAGCGGATGAAATATTCAT 3042

3043 TATTGAAATTCATCGCTTTTTATTTTTAATTAAATTATTGGATATACTTATAATA 3097

3098 TATATTGCTGGATATATTGCTGGGATAAGAGTAAAATAATTATAGGCATTATTTC 3152

3153 TAAATTAAAAGGACAATTATTATGATAAAAAACAAGATTATATCAGCTTCAATTG 3207

3208 CAGCATTAATGGCTGTAAGTCCAGTGCTTCCACTTAGCTCACAGGCTCATACGGT 3262ORF 4 ->

3263 TCAAGCTGCAGATAATTCTGTCAOAMMCAGTT ATG CAT AAT TCA ATT 3311M H N S I

3312 GCT TAT GAT AAA GAT GGC AAT TCA ACA GGT CAA AAG TAT TAC 3353

A Y D K D G N S T G 0 K Y Y

1806 AGT TTA GCC AGC GAT CAA AAA ATC AAT CGG ATT GTC AAT GCA 1847 3354 GCT TAC GGA TC 3364

S L A S D Q K I N R I V N A A Y G

FIG. 2. Nucleotide sequence containing the helveticin J structural gene (ORF3) and amino acid sequences of the translation products of

the ORFs. The putative promoter regions, potential ribosomal binding sites, and the location of the Agtll clones HJ1 and HJ4 are underlined.The termination codons are indicated by asterisks.

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LACTOBACILLUS BACTERIOCIN GENE 6345

ORFI ORF2 ORF3 ORF4(C-term.) -_ (N-term.)

I~

R a a S R B I

---------_ --------- .

I I I I --------- .---------I I

0 1 2 3 4 5 6 ]cbFIG. 3. Location of ORFs and restriction sites in the sequenced ( ) and contiguous (---- ) L. helveticus 481-C DNA. The entire

sequenced fragment contains 3,364 bases. Restriction sites: EcoRI (R), 1 bp, 2,823 bp; HindIII (H), 377 bp, -5,877 bp; BgIII (B), 709 bp,-4,709 bp; SphI (S), 2,435 bp. The upper arrow indicates a putative promoter region.

GM1829. Cmr transformants carrying pTRK135 were ob-tained. EcoRI and SphI digests of the recombinant plasmidverified that the pTRK135 insert consisted of fragmentsidentical in size to those of the BgIII fragment cloned from L.helveticus 481-C genomic DNA (Fig. 3). When NCK249 wascured of pTRK135, the bacteriocin-producing phenotypedisappeared. Plasmid pGK12 in L. acidophilus NCK64 failedto confer bacteriocin production on host cells, verifying that

FIG. 4. (a) Colonies of L. acidophilus NCK249 overlaid with an

L. lactis NCK252 indicator lawn. (b) effect of heat treatment on

helveticin J, lactacin F, and the bacteriocin produced by the putativeHlv+ clone, NCK249(pTRK135). A, NCK249(pTRK135) superna-tant before heat; B, NCK249(pTRK135) supematant after heat(100°C, 15 min); C, 481-C supernatant before heat; D, 481-Csupernatant after heat; E, NCK88 supernatant before heat; F,NCK88 supernatant after heat. The indicator was L. lactis NCK252.

the insert DNA was responsible for the antimicrobial phe-notype.

Since L. acidophilus NCK64 is a Laf Laf' derivative ofL. acidophilus NCK88 (Laf' Laf') (32), we considered thepossibility that latent lactacin F genes might be activatedupon introduction of pTRK135. Therefore, experimentswere conducted to confirm that helveticin J was the bacte-riocinogenic substance responsible for the formation of theinhibition zones. Indicator strains were screened via directantagonism assays for sensitivity to NCK249(pTRK135).Inhibition of growth was detected only among indicatorstrains that were sensitive to helveticin J (16). Sensitivestrains included L. helveticus 1846, Lactobacillus jugurti1244, L. bulgaricus 1373 and 1489, and L. lactis 970.Insensitive strains included L. leichmannii 4797, L. acido-philus 11694 and NCK89, and L. fermentum 1750. Theselatter Lactobacillus strains are sensitive indicators for lac-tacin F (32).

Lactacin F is heat resistant (32) as compared with helveti-cin J, which is heat sensitive (16). Titers of supernatants ofMRS broth cultures of L. acidophilus NCK88 and NCK249and L. helveticus 481-C were determined before and after a15-min, 100°C heat treatment. The NCK88 supernatantretained full activity (400 AU/ml before and after heating),whereas the NCK249 and 481-C supernatants (NCK249,6,400 AU/ml; 481-C, 3,200 AU/ml) had no detectable inhib-itory activity after the heat treatment (Fig. 4b). The narrowactivity spectrum and heat sensitivity of the bacteriocin, ascompared to corresponding lactacin F characteristics,proved that L. acidophilus NCK249 harboring pTRK135produced helveticin J.

DISCUSSIONTo our knowledge, this was the first attempt to clone a

bacteriocin gene by employing the expression vector Xgtll.Adequate protein characterization and purification of hel-veticin J had been accomplished (16) to allow production ofan antibody probe. Also, the suspected chromosomal loca-tion of the helveticin J gene (16) necessitated the screening ofa library representing the entire genome of this organism.Immunological, nucleotide sequence, and genetic data dem-onstrated that the cloning strategy was successful. Thecloned fragment contained an ORF (ORF3) that potentiallyencodes a 37,511-Da protein, whose molecular weight wouldbe close to that of helveticin J (37,000) as estimated fromSDS-polyacrylamide gel electrophoresis. Database searchesfor proteins with similar amino acid sequences did notindicate that the predicted product of ORF3 was similar toother bacteriocins. The ice nucleation protein produced byPseudomonas syringae (14) had the highest homology scoreswhen compared with the predicted product of ORF3. This isdue to imperfect repeats of a consensus octapeptide withinthe ice nucleation protein sequence that lined up with glycine

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6346 JOERGER AND KLAENHAMMER

residues in ORF3. The glycine residues in the putativebacteriocin gene also exhibit some periodicity, and 48% ofthe total glycines in the protein can be found within the first100 amino acids. This finding may be significant, since a

number of colicins also contain a higher percentage ofglycines at the amino-terminal end of the protein (35, 49).This has led to the prediction of ,-turns and ,-sheets at theamino-terminal ends, which are thought to be involved in theuptake of colicin molecules into the target cell. Interestingly,the amino terminus of the putative protein encoded by ORF3contains a pentapeptide similar to a consensus sequencefound among TonB-dependent colicins and outer membraneproteins involved with iron siderophore and vitamin B12transport (40). Uptake of these colicins across the outermembrane is dependent on TonB protein function. Theconsensus pentapeptide begins within the first 23 residues ofthe mature polypeptide and consists of an acidic amino acidfollowed by an almost invariant threonine and then by twouncharged amino acids and an invariant valine. The ORF3pentapeptide Glu-Thr-Thr-Asn-Val starts at the sixth aminoacid and matches the consensus sequence. However, it isnot obvious what function this pentapeptide might have in a

protein produced by a gram-positive bacterium, since theyhave no outer membrane.The apparent location of the helveticin J structural gene

(ORF3) within an operon is reminiscent of the geneticorganization of many colicin genes (26). The function of theORF2-encoded protein product is unknown; however, thefeatures of the amino terminus might permit its secretionfrom the cell. Since helveticin J is hydrophilic in nature, onecan speculate that ORF2 encodes the immunity protein thatbinds to helveticin J and facilitates its export from the cell.Some colicins have been shown to be released as a complexwith their immunity protein (35).

Expression of the helveticin J gene could not be demon-strated in E. coli. Due to the adenine and thymine-richcomposition ofthe bacteriocin gene as compared with E. coliDNA in general, there is a strong preference for codonscomposed of the two bases. Some of these codons are usedinfrequently by E. coli and are recognized by only minortRNA species. Therefore, the expression of adenine- andthymine-rich genes appear to be limited by the availability oftRNA in E. coli. Gamier and Cole (12) reported and dis-cussed a similar scenario, where they unsuccessfully tried toexpress the Clostridium perfringens bacteriocin BCN5 in E.coli.The recombinant plasmid pTRK135 was successfully in-

troduced into L. acidophilus NCK64. The 4-kb BglII insertoriginating from L. helveticus 481-C conferred on L. acido-philus NCK249 the ability to produce helveticin J as judgedby inhibitory spectrum and heat lability.

Future efforts to establish the function of the ORF2-encoded protein product and the completion of the DNAsequence analysis of ORFI and ORF4 should yield moreinformation concerning the regulation of helveticin J synthe-sis, export, and uptake. Such data will facilitate the cloningof related bacteriocin genes and will promote the construc-tion of food-grade cloning vectors carrying bacteriocin phe-notypic markers. An understanding of the genetic organiza-tion and regulation of Lactobacillus bacteriocin genes willexpand our capabilities to engineer novel protein antimicro-bial agents for food and dairy products.

ACKNOWLEDGMENTSSupport of this investigation was provided by the National Dairy

Promotion and Research Board and by the North Carolina DairyFoundation.We thank P. M. Muriana for providing strains and sharing the

electroporation protocol for L. acidophilus NCK64, NCK88, andNCK89; L. Morelli for suggestions on electroporation of L. helve-ticus, M. A. Conkling, H. M. Hassan, and P. M. Foegeding forinsightful discussion and guidance; P. E. Bishop for the use ofinstrumentation to synthesize primers; and R. D. Joerger for assis-tance with VAX sequence analysis programs.

LITERATURE CITED1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid

method for isolating large plasmid DNA from lactic strepto-cocci. Appl. Environ. Microbiol. 46:549-552.

2. Barefoot, S. F., and T. R. Klaenhammer. 1983. Detection andactivity of lactacin B, a bacteriocin produced by Lactobacillusacidophilus. Appl. Environ. Microbiol. 45:1808-1815.

3. Birnboim, H. C., and J. Doly. 1979. A rapid extraction proce-dure for screening recombinant plasmid DNA. Nucleic AcidsRes. 7:1513-1523.

4. Buchman, G. W., S. Banerjee, and J. N. Hansen. 1988. Struc-ture, expression, and evolution of a gene encoding the precursorof nisin, a small protein antibiotic. J. Biol. Chem. 263:16260-16266.

5. Bullock, W. O., J. M. Fernandez, and J. M. Short. 1987.XL1-Blue: a high efficiency plasmid transforming recA Esche-richia coli strain with beta-galactosidase selection. Biotech-niques 5:376-378.

6. Bunette, W. N. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacryla-mide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal. Bio-chem. 112:195-203.

7. Conrad, B., and D. W. Mount. 1982. Microcomputer programsfor DNA sequence analysis. Nucleic Acids Res. 10:31-38.

8. Craig, R. J., J. A. Arraj, and M. G. Marinus. 1984. Induction ofdamage inducible (SOS) repair in dam mutants of Escherichiacoli exposed to 2-aminopurine. Mol. Gen. Genet. 194:539-540.

9. Devreux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-sive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

10. Dower, W. J., J. F. Miller, and C. W. Ragadae. 1988. Highefficiency transformation of E. coli by high voltage electropora-tion. Nucleic Acids Res. 16:61274145.

11. Fickett, J. W. 1982. Recognition of protein coding regions inDNA sequences. Nucleic Acids Res. 10:5303-5318.

12. Gamier, T., and S. T. Cole. 1986. Characterization of a bacte-riocinogenic plasmid from Clostridium perfringens and molecu-lar genetic analysis of the bacteriocin-encoding gene. J. Bacte-riol. lo8:1189-1196.

13. Gold, L., D. Pribnow, T. Schneider, S. Shindling, B. S. Singer,and G. Stormo. 1981. Translational initiation in prokaryotes.Annu. Rev. Microbiol. 35:365-403.

14. Green, R. L., and G. J. Warren. 1985. Physical and functionalrepetition in a bacterial ice nucleation gene. Nature (London)317:645-648.

15. Huynh, T. V., R. A. Young, and R. W. Davis. 1984. Constructingand screening cDNA libraries in X-gtlO and X-gtll, p. 49-78. InD. Glover (ed.), DNA cloning techniques: a practical approach.IRL Press, Oxford.

16. Joerger, M. C., and T. R. Klaenhammer. 1986. Characterizationand purification of helveticin J and evidence for a chromoso-mally determined bacteriocin produced by Lactobacillus helve-ticus 481. J. Bacteriol. 167:439-446.

17. Johnson, J. L., C. F. Phelps, C. S. Cummins, J. London, and F.Gasser. 1980. Taxonomy of the Lactobacillus acidophilusgroup. Int. J. Syst. Bacteriol. 30:5348.

18. Johnston, R. E., J. M. Mackenzie, Jr., and W. G. Dougherty.1986. Assembly of overlapping DNA sequences by a programwritten in BASIC for 64K CP/M and MS-DOS IBM-compatiblemicrocomputers. Nucleic Acids Res. 14:517-527.

J. BACTERIOL.

on May 3, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

LACTOBACILLUS BACTERIOCIN GENE 6347

19. Kaletta, C., and K. D. Entian. 1989. Nisin, a peptide antibiotic:cloning and sequencing of the nisA gene and posttranslationalprocessing of its peptide product. J. Bacteriol. 171:1597-1601.

20. Kekessy, D. A., and J. D. Piguet. 1970. New method fordetecting bacteriocin production. Appl. Microbiol. 20:282-283.

21. Klaenhammer, T. R., L. L. McKay, and K. A. Baldwin. 1978.Improved lysis of group N streptococci for isolation and rapidcharacterization of plasmid deoxyribonucleic acid. Appl. Envi-ron. Microbiol. 35:592-600.

22. Kleeman, E. G., and T. R. Klaenhammer. 1982. Adherence ofLactobacillus species to human fetal intestinal cells. J. DairySci. 65:2063-2069.

23. Kok, J., J. M. B. M. Van Der Vossen, and G. Venema. 1984.Construction of plasmid cloning vectors for lactic streptococciwhich also replicate in Bacillus subtilis and Escherichia coli.Appl. Environ. Microbiol. 48:726-731.

24. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

25. Luchansky, J. B., P. M. Muriana, and T. R. Klaenhammer.1988. Application of electroporation for transfer of plasmidDNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria,Pediococcus, Bacillus, Staphylococcus, Enterococcus, andPropionibacterium. Mol. Microbiol. 2:637-646.

26. Luria, S. E., and J. L. Suit. 1986. Colicins and col plasmids, p.1615-1624. In F. C. Neidhartd, J. L. Ingraham, K. B. Low, B.Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esche-richia coli and Salmonella typhimurium: cellular and molecularbiology. American Society for Microbiology, Washington, D.C.

27. Manfioletti, G., and C. Schneider. 1988. A new and fast methodfor preparing high quality A DNA suitable for sequencing.Nucleic Acids Res. 7:2873-2884.

28. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

29. Mayr-Harting, A., A. J. Hedges, and R. C. W. Berkeley. 1972.Methods for studying bacteriocins. Methods Microbiol. 7A:315-422.

30. Messing, J., B. Gronenborn, B. Mueller-Hill, and P. H. Hof-schneider. 1977. Filamentous coliphage M13 as a cloning vehi-cle: insertion of a HindIII fragment of the lac regulatory regionin M13 replicative form in vitro. Proc. Natl. Acad. Sci. USA74:3642-3646.

31. Mount, D. W., and B. Conrad. 1984. Microcomputer programsfor graphic analysis of nucleic acid and protein sequences.Nucleic Acids Res. 12:811-817.

32. Muriana, P. M., and T. R. Klaenhammer. 1987. Conjugaltransfer of plasmid-encoded determinants for bacteriocin pro-duction and immunity in Lactobacillus acidophilus 88. Appl.Environ. Microbiol. 53:553-560.

33. Murray, N. E., W. J. Brammer, and K. Murray. 1977. Lamb-doid phages that simplify the recovery of in vitro recombinants.Mol. Gen. Genet. 150:53.

34. Oppenheimer, C. L., A. E. Eckhardt, and R. L. Hill. 1981. Thenonidentity of porcine N-acetylglucosaminyltransferases I andII. J. Biol. Chem. 256:11477-11482.

35. Pugsley, A. P. 1984. The ins and outs of colicins. I. Production,and translocation across membranes. Microbiol. Sci. 1:168-175.

36. Rodriguez, R. L., and R. C. Tait. 1983. Recombinant DNAtechniques: an introduction, p. 48-49. Addison-Wesley Publish-ing Co., Reading, Mass.

37. Rosenberg, M., and D. Court. 1979. Regulatory sequencesinvolved in the promotion and termination of RNA transcrip-tion. Annu. Rev. Genet. 13:319-353.

38. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain elongation inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

39. Scherwitz-Harmon, K., and L. L. McKay. 1987. Restrictionenzyme analysis of lactose and bacteriocin plasmids from Strep-tococcus lactis subsp. diacetylactis WM4 and cloning of BclIfragments coding for bacteriocin production. Appl. Environ.Microbiol. 53:1171-1174.

40. Schramm, E., J. Mende, V. Braun, and R. M. Kamp. 1987.Nucleotide sequence of the colicin B activity gene cba: consen-sus pentapeptide among tonB-dependent colicins and receptors.J. Bacteriol. 169:3350-3357.

41. Snyder, M., and R. W. Davis. 1985. Screening X-gtll expressionlibraries with antibody probes, p. 397-406. In T. A. Springer(ed.), Hybridoma technology in the biosciences and medicine.Plenum Publishing Corp., New York.

42. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

43. Tagg, J. R., A. S. Dajani, and L. W. Wannamaker. 1976.Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40:722-756.

44. Upreti, G. C., and R. D. Hinsdill. 1975. Production and mode ofaction of lactocin 27: bacteriocin from a homofermentativeLactobacillus. Antimicrob. Agents Chemother. 7:139-145.

45. Van Belkum, M. J., B. J. Hayema, A. Geis, J. Kok, and G.Venema. 1989. Cloning of two bacteriocin genes from a lacto-coccal bacteriocin plasmid. Appl. Environ. Microbiol. 55:1187-1191.

46. Von Heine, G. 1985. Signal sequences, the limits of variation. J.Mol. Biol. 184:99-105.

47. Watson, M. E. E. 1984. Compilation of published signal se-quences. Nucleic Acids Res. 12:5145-5164.

48. Wilbur, W. J., and D. J. Lipman. 1983. Rapid similaritysearches of nucleic acid and protein data banks. Proc. Natl.Acad. Sci. USA 80:726-730.

49. Yamada, M., Y. Ebina, T. Miyata, T. Nakazawa, and A.Nakazawa. 1982. Nucleotide sequence of the structural gene forcolicin El and predicted structure of the protein. Proc. Natl.Acad. Sci. USA 79:2827-2831.

50. Yanisch-Perron, C., J. Viera, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide se-quences of M13 mpl8 and pUC19 vectors. Gene 33:103-110.

51. Young, R. A., and R. W. Davis. 1985. Immunoscreening X-gtllrecombinant DNA expression libraries, p. 29-41. In J. K.Setlow and A. Hollaender (ed.), Genetic engineering, vol. 7.Plenum Publishing Corp., New York.

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