JOURNAL

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Vol. 172, No. 11

BACTERIOLOGY, Nov. 1990, p. 6339-6347

0021-9193/90/116339-09$02.00/0 Copyright © 1990, American Society for Microbiology

Cloning, Expression, and Nucleotide Sequence of the Lactobacillus helveticus 481 Gene Encoding the Bacteriocin Helveticin Jt M. C. JOERGER't AND T. R. KLAENHAMMERl 2* Departments of Food 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 chromosomal DNA from L. helveticus were prepared in Agtll and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and H14 lysogens produced 0-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in XEMBL3 with the insert DNA from phage 1P4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A BglM fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into LactobaciUus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an activity spectrum that was the same as that of helveticin J. the immunity gene is transcribed in the opposite direction from its own promoter. In lactic acid bacteria, the cloning and expression of bacteriocin genetic determinants have been limited to the lactococci (4, 19, 39, 45). Bacteriocin genes of Lactococcus lactis subsp. diacetylactis WM4 were localized to an 18.4-kb XhoI fragment originating from the 131.3-kb plasmid pNP2 by analyzing clones for bacteriocin production and using restriction 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 unique bacteriocin along with the corresponding immunity genetic determinants. In addition, nisin genes from L. lactis have been 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 and sequencing of DNA containing the helveticin J (hlv) gene(s) from L. helveticus 481. (This work was submitted in partial fulfillment of the Ph.D. requirements of M. C. Joerger at North Carolina State University.)

Bacteriocins are defined as proteinaceous antimicrobial agents that exhibit a bactericidal mode of action. Lactobacillus helveticus 481 produces a bacteriocin designated as helveticin J (16). This bacteriocin was partially purified and characterized as a heat sensitive, 37-kDa protein that inhibits growth of closely related Lactobacillus species. Bacteriocin genes are typically plasmid borne (43), but the genetic determinants encoding helveticin J appear to reside on the chromosome (16). Evidence for chromosomally located bacteriocin genes has also been reported for lactacin B produced by Lactobacillus acidophilus N2 (2) and lactocin 27 produced by L. helveticus LS18 (44). Many of the bacteriocins (colicins) produced by Escherichia coli have been genetically characterized (for a review, see reference 26). A common feature of colicin plasmids is the contiguous arrangement of genes whose products determine bacteriocin synthesis, immunity, and release via lysis. DNA sequence analysis and transcriptional studies have shown the presence of an SOS-inducible promoter, which quite often precedes an operon of colicin, immunity, and lysis genes that are transcribed in the same direction (as in the case of CloDF13 genes). Colicin E2 and E3 operons have been studied in which the immunity gene is transcribed from a secondary promoter located in the terminus of the bacteriocin structural gene. The colicin El plasmid has been shown to have yet a different genetic organization, in which

MATERIALS AND METHODS Bacterial strains, phage, and plasmids. The bacterial strains, phage, and plasmids used in this study are listed in Table 1. Lactobacillus strains were propagated at 37°C in MRS broth (Difco Laboratories, Detroit, Mich.). E. coli strains were propagated at 32, 37, or 42°C in LB medium with shaking. Agar media contained 1.5% agar; overlay medium contained 0.75% (MRS) or 0.80% (LB) agar. For phage propagation, 10 mM MgCl2 was added to LB medium. The following concentrations of antibiotics were used: ampicillin at 100 ,ug/ml, chloramphenicol at 10 ,ug/ml, erythromy-

* 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., Agricultural Products, Newark, DE 19714-6101.

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TABLE 1. Bacteria, phage, and plasmids Relevant characteristics

Bacterium, phage, or plasmid

Lactobacillus helveticus 103 NCK229 481 NCK228 481-C NCK246 1846 NCK230

Bac+a

Source or reference

Hlv+ Hlvr, pMJ1008 Hlv+ HlvF, plasmid cured HlvS LaP

NCDOb, ATCC 8001 NCDO (16) 16 NCDO

Lactobacillus bulgaricus 1373 NCK232 1489 NCK231

HlvS LafP Hlv5 LaP

NCDO NCDO, ATCC 11842

Lactobacillus acidophilus 88 NCK88 88-C NCK64 89 NCK89 NCK247 NCK248 NCK249 NCK250 11694 NCK121 ADH NCK100

LafF Laf! Laf Lafr Laf-LaP Hlv+/HlvHlvHlv+, pTRK135 LaC Lafr, pGK12 LaP Hlv' Bac+ Bacr, pTRK15

17 32 32

This study This study This study This study

Lactobacillus jugurti 1244 NCK233

Hlvs LaP

NCKC collection

Lactobacillus fermentum 1750 NCK127

LafP Hlv'

NCDO (2)

Lactobacillus lactis 970 NCK234 NCK252

Hlvs LafP Spontaneous Cm' isolate of NCK234

NCDO This study

Lactobacillus leichmannii 4797 NCK235

LafP Hlvr

ATCC 4797

Escherichia coli Y1089(r-) NCK74 Y1089-HJ1 NCK75 Y1089-HJ4 NCK78 Y1090(r-) NCK79

AlacUl69 pro' Alon araDl39 strA hflA [chr::TnlO](pMC9) Y1089::Xgtll-HJ1 Y1089::Xgtll-HJ4 AlacUl69 proA+ Alon araDl39 strA supF [trpC22::TnJO] hsdR

51 This study This study 51

hsdM+

LE392 NCK57 XL1 Blue NCK80 NCK81 NCK82 NCK83 NCK84 K12 71-18 NCK238 GM1829 NCK239

NCK241

20

MS02 (22)

(PMC9)

hsdRSl4(rMC m-) supE44 supF58 lacY) or (lacIZY)6 galK2 galT22 metBi trpR55 lambdaendA1 hsdRJ7 (rK- mKE) supE44 thi-1 lambda- recAl gyrA96 relAl (Lac-)[F' proAB lacPZM15 TnJO (Tet9)] XL1 Blue (pTRK131) XL1 Blue (pTRK132) XL1 Blue (pTRK133) XL1 Blue (pTRK134) A(lac pro)F' LacPqZM15 pro' supE F- recA441 sulA thr-1 leu-6 his4 argE3 ilv(Ts) galK2 rpsL31 Str' lacUl69 dinDl::Mu d(Ap lac) dam4 F

GM1829(pTRK135)

33 5

This study This study This study This study 30 8 This study

Phage Lambda HJ1 nck79.gtll-HJ1 HJ4 nck79.gtll-HJ4 Hlv3 nck57.EMBL3-Hlv3

This study This study This study

M13

mpl8 mpl9

M13

cloning

vector

M13 cloning vector

50 50

Continued on following page

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TABLE 1-Continued Bacterium, phage, or plasmid

Plasmids Bluescript M13 KS(+)

Relevant characteristics

lacZ, Apr, 3.0 kb

pTRK131 pTRK132 pTRK133 pTRK134 pGK12 pTRK135

Bluescript M13 KS(+)::HJ1, Apr, 3.35 kb Bluescript M13 KS(+)::HJ2, Apr, 3.9 kb Bluescript M13 KS(+)::HJ3, Apr, 3.75 kb Bluescript M13 KS(+)::HJ4, Apr, 3.6 kb Cmr Emr, 4.4 kb pGK12::Hlv-BglII, Cmr Ems, 8.4 kb a Bac, Bacteriocin; Hlv, helveticin J; Laf, lactacin F; (+), producer; (-), nonproducer; s, sensitive; r, resistant.

Source or reference

Stratagene, LaJolla, Calif. This study This study This study This study 23 This study

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 erythromycin at 3 ,ug/ml and chloramphenicol at 3 ,ug/ml for Lactobacillus strains. Bacteriocin detection. Lactobacillus strains were examined for bacteriocin production by overlaying colonies with a lawn of a sensitive indicator microorganism by deferred methods (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 dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (24) of partially purified helveticin J, proteins were electrophoretically transferred (6) to nitrocellulose (BA85, 0.45-,m pore size; Schleicher & Schuell, Keene, N.H.), and the band corresponding to the 37,000-Da bacteriocin was excised from the membrane. The nitrocellulose strip with immobilized helveticin J was dissolved in dimethyl sulfoxide. Approximately 10 ,ug of bacteriocin was emulsified with Freund complete or incomplete adjuvant (Sigma Chemical Co., St. Louis, Mo.) and administered to New Zealand White rabbits at 2-week intervals. Immune serum was obtained after 11 weeks. The immunoglobulin G (IgG) fraction was purified with a protein A-Sepharose (Sigma) column (34). A cell extract was employed to remove E. coli-reactive IgG from the antiserum before screening the Agtll library. P-Galactosidase fusion protein was purified with a Protosorb P-galactosidase immunoaffinity column (Promega Corp., Madison, Wis.). Purified fusion protein (0.5 to 1.0 mg per injection) was emulsified with adjuvant and injected every 2 weeks. After 6 weeks, immune serum was collected and the IgG fraction was isolated as described above. IgG that was reactive to the P-galactosidase portion of the fusion protein was removed by using a P-galactosidase column prepared with an ImmunoPure Ag/Ab immobilization kit (Pierce, Rockford, Ill.). Protoblot immunoscreening and Western immunoblot alkaline phosphatase systems (Promega), for the detection of antibody bound to proteins immobilized on nitrocellulose membranes, were used as described by the suppliers with the following modifications. Calf serum (20% in Tris-buffered saline) was employed as a blocking agent. Tween 20 was omitted from the wash solutions for filters that were probed with helveticin J IgG because of the low affinity of the antibody. 1-Galactosidase monoclonal antibody was obtained from Promega. For antibody neutralization or precipitation experiments, samples of partially purified helveticin J were mixed with dilutions of IgG and held overnight at 4°C in phosphatebuffered saline with gentle agitation. After centrifugation at

16,000 x g for 10 min in a microcentrifuge, the supernatants were titered against the helveticin J-sensitive indicator L. bulgaricus 1489. Antibodies that were reactive with helveticin J epitopes encoded by Xgtll recombinants were affinity purified from the polyvalent rabbit IgG as described by Snyder and Davis (41). The antigen-selected antibodies were used to probe immunoblots (6) of partially purified helveticin J. DNA manipulations and isolation. Total genomic DNA and plasmid DNA from Lactobacillus strains were isolated and purified as previously described (1, 16, 21). Extraction of plasmid DNA from E. coli and purification of X phage DNA were as described previously (3, 27, 28, 36). All restriction and DNA-modifying enzymes were employed as prescribed by the manufacturers. Southern blots (42) and plaque lift hybridizations were performed with [35S]dCTP-labeled probes prepared by using a Multiprime DNA labeling system (Amersham Corp., Arlington Heights, Ill.) or with nonradioactive probes labeled with digoxigenin-dUTP by using a DNA labeling and detection kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). DNA was immobilized on Magna graph nylon membrane (0.45-,um pore size; MSI, Westboro, Mass.). Hybridizations employing [35S]dCTPlabeled probes were conducted at 42°C in the presence of 6 x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 50% formamide. Digoxigenin-dUTP-labeled probes were hybridized with DNA at 68°C in the presence of S x SSC. Construction of genomic libraries and subclones. Total DNA from L. helveticus 481-C was sonicated to obtain DNA fragments 2 to 6 kb in size. A Xgtll library was constructed as described by Huynh et al. (15) by using sonicated and end-repaired L. helveticus DNA fragments. L. helveticus 481-C total DNA was partially cleaved with Sau3A to generate fragments of 15 to 23 kb. These sized fragments were ligated into the BamHI-cleaved vector XEMBL3 (28). DNA fragments from insert DNA in XEMBL3 or Agtll clones were ligated into Bluescript M13 KS(+) DNA (Stratagene, La Jolla, Calif.) or into the shuttle vector pGK12 (22). Preparation and analysis of recombinant lysogens. E. coli Y1089 was infected with Agtll clones, and lysogens were isolated as described previously (15). Crude lysates from the Xgtll recombinant lysogens were prepared (15) and subjected to SDS-polyacrylamide gel electrophoresis (24). P-Galactosidase fusion proteins were purified via affinity column chromatography as outlined above. The crude lysates and purified fusion proteins were subjected to Western blot analysis (6) with antibodies reactive to helveticin J and the HJ4 fusion protein. Electroporation. E. coli and lactobacilli were transformed

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with recombinant DNA via electroporation (10, 25) with a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, Calif.). Electroporation of L. acidophilus NCK64, NCK88, and NCK89 was conducted as described previously (25) with the following modifications. Cells (200 ml) were harvested after they reached an optical density at 600 nm of 0.78 to 0.80, and the pellet was washed two times with 150 ml of electroporation buffer (1 M sucrose, 2.5 mM CaCl2 [pH 7.0]). After the second wash, the cells were suspended in 2.6 ml of the same buffer and held on ice. Cells (400 RI) and DNA were mixed and pipetted into a chilled 0.2-cm cuvette. An electrical pulse was delivered to the cuvette with the Gene Pulser apparatus 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 hlv genes, were subcloned from the XEMBL3 recombinant Hlv3 into M13 mpl8 and mpl9. These two fragments were also cleaved with AluI, RsaI, Sau3A, TaqI, HindIII-SphI, and Sau3A-SphI, and the resulting subfragments were cloned into M13 mpl8 and mpl9 (50). HJ1 and HJ4 insert DNA was also ligated with EcoRI-cleaved M13 mpl8 and mpl9. DNA for sequencing was prepared from the phage by a procedure published by International Biotechnologies, Inc. (New Haven, Conn.). Sequencing of both DNA strands was carried out by the method of Sanger et al. (38) with [35S]dATP (NEN Research Products, Boston, Mass.) and the Sequenase DNA sequencing kit (United States Biochemical Corp., Cleveland, Ohio). A number of site-specific primers were synthesized with a Pharmacia Gene Assembler oligonucleotide synthesizer (Pharmacia, Piscataway, N.J.) to sequence regions for which no suitable subfragments were available. Individual sequences were analyzed for overlaps and organized into a contiguous sequence (18). Restriction sites and predicted amino acid sequences were determined by using programs written by Mount and Conrad (7, 31). The DNA sequence was subjected to a base-preference analysis with the UWGCG computer program Testcode (9, 11). Molecular weights and pls were calculated with the program PeptideSort (UWGCG). The GenBank and the NBRF protein sequence data bank were searched for similar DNA or amino acid sequences by using the program of Wilbur and Lipman (48). Nucleotide sequence accession number. These data have been submitted to GenBank under accession no. M30121. RESULTS Characterization of Xgtll clones reactive with polyclonal antibody to helveticin J. Bacteriocin-reactive IgG, isolated from antiserum made against helveticin J, was employed to screen 350,000 clones representing several Xgtll libraries of total DNA isolated from L. helveticus 481-C. Two phages, designated HJ1 and HJ4, produced positive signals. The inserts from these two clones hybridized to each other and to total genomic DNA isolated from L. helveticus 481, the producer of helveticin J. The clones did not hybridize to DNA from L. helveticus 103 or 1846 or L. jugurti 1244, which either produce a bacteriocin different from helveticin J or do not produce a bacteriocin. E. coli Y1089 containing HJ1 or HJ4 lysogens produced large amounts of ,-galactosidase fusion proteins. The HJ4 fusion protein was slightly larger than the HJ1 fusion protein. This would be expected, since HJ4 has a 600-bp insert, whereas HJ1 has a 350-bp insert. Helveticin J activity was not detected when the titers of the lysates were determined against the sensitive indicator L. bulgaricus 1489.

J. BACTERI'OL.

Both ,B-galactosidase monoclonal antibody and helveticin J antibody bound to the fusion protein bands present in lysates obtained from isopropyl-,-D-thiogalactopyranosideinduced E. coli Y1089 carrying the HJ1 or HJ4 lysogens. Signals produced by the bacteriocin IgG binding to the fusion proteins were faint in comparison with the signal generated by the antibody binding to helveticin J. The signal was also faint on immunoblots of fusion proteins purified via a P-galactosidase immunoaffinity column. This phenomenon is most likely due to the fusion proteins displaying fewer antigenic epitopes as compared with those displayed by helveticin J, and hence fewer antibody molecules bound to the hybrid proteins. Affinity purification of antibodies recognizing recombinant protein. One strategy for testing whether a Agtll recombinant encodes the particular protein is to use the phage clones to affinity purify antibody from the screening IgG (41). The clone-purified antibody is then used to probe immunoblots of the protein in question. HJ1 and HJ4 were employed to affinity purify a fraction of the screening IgG that bound antigenic epitopes of the fusion proteins produced by each respective clone. The wild-type Xgtll was employed as a negative control in the affinity purification of helveticin J antibody. Very faint signals were obtained when wild-type Xgtll-purified IgG, representing P-galactosidase-reactive antibodies, was probed against immunoblots of HJ1 and HJ4 fusion proteins. No signal was detected against membraneimmobilized helveticin J. HJ1- or HJ4-purified IgG produced signals with both HJ1 and HJ4 fusion proteins. HJ4-purified IgG produced a faint signal with helveticin J, and an even weaker signal was produced by the HJ1-purified IgG. The difference in signal intensity is logical, since the HJ4 clone is larger than the HJ1 clone. Therefore, the HJ1 and HJ4 fusion proteins shared some of the same antigenic epitopes as those displayed on helveticin J. Fusion protein antibody studies. The ,-galactosidase fusion protein produced by the lysogen E. coli Y1089-HJ4 was purified via a ,B-galactosidase immunoaffinity column. The purified fusion protein was injected into rabbits to produce polyclonal antibody specific for epitopes expected to be present on the HJ4-encoded portion of the fusion protein. P-Galactosidase-specific antibody was removed from the total IgG fraction with an agarose column containing immobilized P-galactosidase. A high-affinity antibody was obtained that gave strong signals when bound to membraneimmobilized helveticin J. To aid in clone verification, an immunoblot of partially purified helveticin J, P-galactosidase, and the HJ1 and HJ4 fusion proteins was probed with the HJ4-specific antibody lacking the P-galactosidase-reactive portion of IgG. A duplicate protein blot was stained to detect protein bands. The antibody bound and produced signals with the HJ1 and HJ4 fusion proteins and the 37,000-Da protein band corresponding to helveticin J (Fig. 1). The antibody also produced signals with a triplet of bands of approximately 43,000 Da in the partially purified bacteriocin preparation. These bands were not detected previously with the antibody that was made against denatured helveticin J. Possibly, the second antibody made against the fusion protein detected another form of the bacteriocin. No signals were detected in the P-galactosidase lane (Fig. 1), indicating that all of the n-galactosidase-reactive IgG had been successfully removed from the screening antiserum. These results unequivocally demonstrated that the portion of the fusion proteins encoded by HJ1 and HJ4 shared similar antigenic epitopes with helveticin J.

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charged amino acid (histidine) was present within the aminoterminal region of the signal peptide. However, this region carried a net negative charge, a property that would be unusual for signal peptides. ORF2 and ORF3 were separated by a small noncoding region of 30 bp. A putative ribosomal binding site (13) was located 7 bp upstream of the initiation codon of ORF3. The -- _*1,002-bp ORF3 could encode a hydrophilic protein with a pI of 6.03 and a molecular weight of 37,511, which is very close to the weight of helveticin J of 37,000 as predicted by SDS-polyacrylamide gel electrophoresis (16). The L. helveticus DNA inserts in HJ1 and HJ4 were contained within _m, ORF3; HJ4 (515 bp) was located at nucleotide positions 2314 to 2828 (Fig. 2). The smaller HJ1 insert (338 bp) was internal to HJ4 and resides between nucleotides 2491 and 2828. Searches of NBRF and GenBank sequence data libraries did not identify any polypeptides with significant sequence homology to the predicted products of ORF2 or ORF3. A 312-bp noncoding region separated ORF3 and ORF4. A putative ribosomal binding site lay 5 bp upstream of the FIG. 1. Lanes A through E show an SDS-polyacrylamide gel protein 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 noncoding carbonic anhydrase, 30,000; from top to bottom); B, partially region could serve as the -10 promoter site. However, a -35 region was not evident within the same stretch of DNA purified helveticin J; C, P-galactosidase; D, HJ1 fusion protein; E, P t HJ4 fusion protein. Lanes F through I show an SDS-polyacrylamide gel protein blot probed with HJ4 fusion protein IgG: F, partially region, or perhaps ORF4 is a continuation of the ORF2purified helveticin J; G, P-galactosidase; H, HJ1 fusion protein; I ORF3 putative operon. HJ4 fusion protein. The arrow indicates a 37,000-Da protein band Expression of helveticin J. Attempts to clone the 5.5-kb corresponding to helveticin J. HindIII fragment derived from X recombinant Hlv3 in the high-copy-number E. coli vector Bluescript M13 were unAttempts to demonstrate neutralization or precipitation of successful due to plasmid rearrangements in the transforhelveticin J with antibodies specific for the bacteriocin or mants. A smaller 4-kb BglII fragment of the same L. helvefusion 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. Restriction analysis. 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 J XEMBL3 was constructed and probed with the insert DNA activity was not detected in broth supernatants or cell from 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 into hybridized 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 colonies these fragments were cloned into phage M13 in both orienrevealed three classes of transformants. Class I transfortations. Smaller subfragments of the EcoRI and HindIII mants, obtained with strains NCK88, NCK64, ADH, and fragments, which could readily be sequenced, were gener1750, were visually devoid of pTRK135 but exhibited a Cmr ated with restriction enzymes and subcloned into M13. Also, phenotype. Deleted forms of pTRK135 were present in the the 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 seNCK64, and NCK89. Several L. acidophilus NCK64 transformants in class III appeared to have the intact plasmid. quenced (Fig. 2). The gene organization of the sequenced region is shown in Fig. 3. Open reading frame (ORF) 2 and After repeated transfers of the L. acidophilus NCK64 class ORF3 have been completely sequenced and are possibly III transformants through MRS broth containing chloramcotranscribed. 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 NCK247 sequence has been determined. DNA sequence analysis by (pTRK135), was plated on MRS agar containing chloramthe Testcode program (11) predicted that a 1,400-bp region phenicol and overlaid with an indicator lawn of L. lactis NCK252 to detect bacteriocin-producing colonies. The macontaining ORF2 and ORF3 has a high probability of being a coding region. jority of colonies exhibited zones of growth inhibition in the indicator lawn. A colony producing a zone of inhibition ORF2 consisted of 315 bp and possibly encodes a protein of 11,808 Da with a pl of 6.81. Upstream of ORF2, putative (NCK249) and a colony lacking a similar zone (NCK248) were isolated and examined for bacteriocin-producing abil-10 and -35 promoter regions (37) could be identified (Fig. 2). The putative -35 region was identical to corresponding ity. The isolate NC}K249 gave rise to a homogeneous population of colonies with inhibition zones (Fig. 4). The "noregions in promoters from E. coli (37). A possible ribosomal zone" phenotype of NCK248 was also stable. Plasmid DNA binding site (13) was located 5 bp upstream of the initiation examination of NCK249 and NCK248 revealed that NCK249 codon for ORF2. The putative protein encoded by ORF2 carried the intact pTRK135, whereas NCK248 had incurred contained an amino terminus with characteristics of a signal a deletion in the recombinant plasmid. Total plasmid DNA peptide (46, 47), because it contained a stretch of nonpolar was isolated from NCK249 and used to transform E. coli amino acids that preceded a glycine residue. A positively A

B

C

D

E

F

G

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ORF 1 -> 1 G AAT TCC AAA GAT GCA GAT CCT ATT TAT GTA GGA AAA AAC AAC 43 N V K N Y P I D A D G K S N 44 TAC AAA TAT GCT TTA ACG CAT TAT GAA ACC TTC AAG GGC AAG K K F T E Y Y Y L T G A K H

85

1848 GTA GTA GAC GGA CTA GAA GCT AAA GGT TTT AAA GTG GAA GAC 1889 V

V

D

G

L

E

A

128 GAA AAG ATA GTC AGA TTC CAC GGC AAA ATT AGT GGT GCT CCA 169 G P V R F G K I S A K I H E 170 TTG TAC TTA GTG GCT TCT AAG GAT AAG AAG TAC AGC TGC TGG 211 S C D K K Y K W A S Y V L L

N

F

K

V

E

D

E

N

S

S

K

GAAGATAGAGATTTTTTC2AGGTTTT

1979

*

ORF 3 -> 1980 ATT ATG AAG CAT TTA AAT GAA ACA ACT AAT GTT AGA ATT TTA 2021 L R I N V T T E N L H K M

2022 AGT CAA TTT GAT ATG GAT ACT GGC D D T G F S 0 M

2064 AAA GGC AAT GTA GGT TCA 212 ACT ACG CAA GCA ATG CTT CAA TAT TAT TAC TTC AAT AGC AAG 253 K Y F N S Y Y Q L A T T Q M

K

G

N

V

G

S

AAT 295

R

K

G

A

T

T

TAT CAA GCA GTA GTT CAA

Y

Q

A

V

V

2063

0

AAA TAT GTA TAT GGA TTA CAA CTT 2105 L L K Y V Y G Q

2106 CGC AAA GGT GCT ACT ACT ATC TTG 254 GGG ATG CGC GGA GTA GTA AAT CCA TTG AAG AGA ATT GCT P L K R I A N V V R G M G

G

1890 CAA GAT GTA AAG GAT ATT TTT GCA AAG GTC GCA AAA ATT ATT 1931 I A K I D I F A K V K Q V D 1932 AAT GAA AAT TCT TCT AAG TAG

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

K

I

L

CGT GGT TAC CGT GGA AGT 2147 S R G R G Y

N

2148 AAA ATT AAT AAC CCT ATT CTT GAA TTA TCT GGT CAA GCA GGT 2189 296 AGA AGT GCT GAT AAG AAT ATT ATT AGT CTA AAG K L S I I K N A S D R

AAT AAA CAA 337 N K Q

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

K

I

N

N

P

I

2190 GGT CAC ACA CAG ACA TGG T T 0 G W H 2232 ATT AAT GGT GAA

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

I

N

G

E

L

E

510

511

TAGAAAAACTGATGCATCTAAGCTATGAAGCTAGGAGCATCAGTTTTTTGTTA

563

564

CATTAGATCGTCATTGTCTTTAAAAAGTTGCTTGGTTGCAGCGGTCATTCTTT

616

617

CGGGGATGCCATAGTTCTGCATAAAGGTTCCTGGTTCTTGAATGTAGTCACCA

669

670

TTTTTGCAGGAATAGTCGTAAATAATCTTAAGAGAAAATAGATCTGCTTCCTC

722

723

TTCTTCCGAGTTTTGTCTTGAAAAGCTAGGCCAATAGGCGATACCTGAATCAC

775

S

G

Q

A

G

GAA AGA GCA GGT CAA TGG TTT ATA GGT GTT 2273 V F I G G A R E 0 W

HJ4-> 2274 AAA CCA TCG AAA ATT GAA GGA AGC

422 CAA CTT AAG AAA GAT AAC AAT ATT GGC GTT GAA GGC GAC AAC 463 N D E V I N G N K G K D L Q 464 TTG CTT TTG TTT GGT TTT TAA AGTGAAAAAKTACGTTAAAAGAACA * F F L L G L

L

GAA TTT GCT GGT GAT CGT AAA GAC 2231 D G D R K A F E

K

P

S

K

I

E

G

S

2316 CAA ATT GCA AGA GTT GAT CTT AGA R L D V R A I 0 2358 TAT TCA S Y

AAA ATT ATT TGG GCA AAG 2315 K K I I A W AAT CAA ATG

N

0

M

GGA G

CCT CAT 2357

P

H

AAT ACT GAC TTT CCT CGA TTA TCC TAC TTG AAT CGC 2399 R L S Y L N R P F T N D

2400 GCC GGT TCT AAT CCA TTT GCT GGT G A F P N S G A

AAT AAG ATG ACG CAT GCC 2441 A N K T H M

2442 GAA GCC GCA GTA TCA CCT GAT TAT ACT T Y D P S V A A E HJ1-> 2484 ACT GTT GMA AAT AAC TGT ATT GGT CAT G I C N N E V H T

AAG TTT TTA ATT GCT 2483 A L I F K TTT ACT ATA TAC AAT 2525 N T I Y F

2526 TTA GAT ACA ATT AAT GAA AAA CTT GAT GAA AAG GGA AAT AGT 2567 N S K G E D L K E N I T D L 2568 GAA GAT GTT AAT CTC GAA ACT GTT AAA TAC GAA GAT AGT TTT 2609 F S D E Y K V T E L N V D E 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

2610 ATC ATT GAT AAT TTA TAT GGT GAT GAT AAT AAT TCT ATT GTA 2651 I V N S N D D G Y L N I D I 2652 AAT TCA ATT CAA GGG TAT GAT TTG GAT AAT GAT GGA AAT ATT 2693 I N D G N D L Y D I G S N 2694 TAT ATT TCC AGT CAA AAA GCG CCA GAT TTT GAT GGC TCT TAT 2735 Y S G D F D P 0 A K Y S S I 2736 TAT GCA CAT CAT AAG CAG ATT GTT AAG ATT CCA TAT TAT GCT 2777 A Y Y P I I V K 0 K H A H Y 2778 CGG TCT AAA GAA AGC GAA GAC CAA TGG AGA GCT GTA AAT TTA 2819 L V N N R A Q D E S E K S R 1638 ATG GAT ATT CAT GAT TAC GTT GAA TTG ATA GCT TTA GCG TTT 1679 F A Y V L A I L E D H M D I 1680 TGG GTT ATT AGT GTT GTA AGT GTT GGT ATC TTG AGT CAT GTT 1721 V V S S V L V I V H S G I W

2862 GTT GAA AGC ATC CAA ATT ATT GGT GAG AAT CAT TGT TAC TTA 2903 Y L C I I N S E H 0 G I E V 2904 ACT GTT GCA TAT CAT TCT AAA AAT AAA GCG GGT GAA AAT AAA 2945 K E N G A K N K S Y H A V T

3043 TATTGAAATTCATCGCTTTTTATTTTTAATTAAATTATTGGATATACTTATAATA 3097 3098

TATATTGCTGGATATATTGCTGGGATAAGAGTAAAATAATTATAGGCATTATTTC 3152

3153 TAAATTAAAAGGACAATTATTATGATAAAAAACAAGATTATATCAGCTTCAATTG 3207

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

3208 CAGCATTAATGGCTGTAAGTCCAGTGCTTCCACTTAGCTCACAGGCTCATACGGT 3262 ORF 4 -> 3263 TCAAGCTGCAGATAATTCTGTCAOAMMCAGTT ATG CAT AAT TCA ATT 3311 I S N H M

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

3312 GCT TAT GAT AAA GAT GGC AAT TCA ACA GGT CAA AAG K 0 T S N K G D Y G A D

1806 AGT TTA GCC AGC GAT CAA AAA ATC AAT CGG ATT GTC AAT GCA 1847

3354 GCT TAC GGA TC Y A G

S

L

A

S

D

Q

K

I

N

R

I

V

N

A

TAT TAC 3353 Y Y

3364

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.

LACTOBACILLUS BACTERIOCIN GENE

VOL. 172, 1990 ORF4 (N-term.)

ORF2 ORF3

ORFI (C-term.)

6345

-_

I~ R

a

S

a

I

B

R _

I

I

I

I

0

1

2

3

--------- --------.---------I 5 4 ---------

.

I

6

]cb

) and contiguous (---- ) L. helveticus 481-C DNA. The entire FIG. 3. Location of ORFs and restriction sites in the sequenced ( 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 obtained. EcoRI and SphI digests of the recombinant plasmid verified that the pTRK135 insert consisted of fragments identical in size to those of the BgIII fragment cloned from L. helveticus 481-C genomic DNA (Fig. 3). When NCK249 was cured of pTRK135, the bacteriocin-producing phenotype disappeared. Plasmid pGK12 in L. acidophilus NCK64 failed to 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 putative Hlv+ clone, NCK249(pTRK135). A, NCK249(pTRK135) supernatant before heat; B, NCK249(pTRK135) supematant after heat (100°C, 15 min); C, 481-C supernatant before heat; D, 481-C supernatant 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 phenotype. Since L. acidophilus NCK64 is a Laf Laf' derivative of L. acidophilus NCK88 (Laf' Laf') (32), we considered the possibility that latent lactacin F genes might be activated upon introduction of pTRK135. Therefore, experiments were conducted to confirm that helveticin J was the bacteriocinogenic substance responsible for the formation of the inhibition zones. Indicator strains were screened via direct antagonism assays for sensitivity to NCK249(pTRK135). Inhibition of growth was detected only among indicator strains that were sensitive to helveticin J (16). Sensitive strains included L. helveticus 1846, Lactobacillus jugurti 1244, L. bulgaricus 1373 and 1489, and L. lactis 970. Insensitive strains included L. leichmannii 4797, L. acidophilus 11694 and NCK89, and L. fermentum 1750. These latter Lactobacillus strains are sensitive indicators for lactacin F (32). Lactacin F is heat resistant (32) as compared with helveticin J, which is heat sensitive (16). Titers of supernatants of MRS broth cultures of L. acidophilus NCK88 and NCK249 and L. helveticus 481-C were determined before and after a 15-min, 100°C heat treatment. The NCK88 supernatant retained 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 inhibitory activity after the heat treatment (Fig. 4b). The narrow activity spectrum and heat sensitivity of the bacteriocin, as compared to corresponding lactacin F characteristics, proved that L. acidophilus NCK249 harboring pTRK135 produced helveticin J. DISCUSSION To our knowledge, this was the first attempt to clone a bacteriocin gene by employing the expression vector Xgtll. Adequate protein characterization and purification of helveticin J had been accomplished (16) to allow production of an antibody probe. Also, the suspected chromosomal location of the helveticin J gene (16) necessitated the screening of a library representing the entire genome of this organism. Immunological, nucleotide sequence, and genetic data demonstrated that the cloning strategy was successful. The cloned fragment contained an ORF (ORF3) that potentially encodes a 37,511-Da protein, whose molecular weight would be close to that of helveticin J (37,000) as estimated from SDS-polyacrylamide gel electrophoresis. Database searches for proteins with similar amino acid sequences did not indicate that the predicted product of ORF3 was similar to other bacteriocins. The ice nucleation protein produced by Pseudomonas syringae (14) had the highest homology scores when compared with the predicted product of ORF3. This is due to imperfect repeats of a consensus octapeptide within the ice nucleation protein sequence that lined up with glycine

6346

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JOERGER AND KLAENHAMMER

residues in ORF3. The glycine residues in the putative bacteriocin gene also exhibit some periodicity, and 48% of the total glycines in the protein can be found within the first 100 amino acids. This finding may be significant, since a number of colicins also contain a higher percentage of glycines at the amino-terminal end of the protein (35, 49). This has led to the prediction of ,-turns and ,-sheets at the amino-terminal ends, which are thought to be involved in the uptake of colicin molecules into the target cell. Interestingly, the amino terminus of the putative protein encoded by ORF3 contains a pentapeptide similar to a consensus sequence found among TonB-dependent colicins and outer membrane proteins involved with iron siderophore and vitamin B12 transport (40). Uptake of these colicins across the outer membrane is dependent on TonB protein function. The consensus pentapeptide begins within the first 23 residues of the mature polypeptide and consists of an acidic amino acid followed by an almost invariant threonine and then by two uncharged amino acids and an invariant valine. The ORF3 pentapeptide Glu-Thr-Thr-Asn-Val starts at the sixth amino acid and matches the consensus sequence. However, it is not obvious what function this pentapeptide might have in a protein produced by a gram-positive bacterium, since they have no outer membrane. The apparent location of the helveticin J structural gene (ORF3) within an operon is reminiscent of the genetic organization of many colicin genes (26). The function of the ORF2-encoded protein product is unknown; however, the features of the amino terminus might permit its secretion from the cell. Since helveticin J is hydrophilic in nature, one can speculate that ORF2 encodes the immunity protein that binds to helveticin J and facilitates its export from the cell. Some colicins have been shown to be released as a complex with their immunity protein (35). Expression of the helveticin J gene could not be demonstrated in E. coli. Due to the adenine and thymine-rich composition of the bacteriocin gene as compared with E. coli DNA in general, there is a strong preference for codons composed of the two bases. Some of these codons are used infrequently by E. coli and are recognized by only minor tRNA species. Therefore, the expression of adenine- and thymine-rich genes appear to be limited by the availability of tRNA in E. coli. Gamier and Cole (12) reported and discussed a similar scenario, where they unsuccessfully tried to express the Clostridium perfringens bacteriocin BCN5 in E. coli. The recombinant plasmid pTRK135 was successfully introduced into L. acidophilus NCK64. The 4-kb BglII insert originating from L. helveticus 481-C conferred on L. acidophilus NCK249 the ability to produce helveticin J as judged by inhibitory spectrum and heat lability. Future efforts to establish the function of the ORF2encoded protein product and the completion of the DNA sequence analysis of ORFI and ORF4 should yield more information concerning the regulation of helveticin J synthesis, export, and uptake. Such data will facilitate the cloning of related bacteriocin genes and will promote the construction of food-grade cloning vectors carrying bacteriocin phenotypic markers. An understanding of the genetic organization and regulation of Lactobacillus bacteriocin genes will expand our capabilities to engineer novel protein antimicrobial agents for food and dairy products.

ACKNOWLEDGMENTS

Support of this investigation was provided by the National Dairy Promotion and Research Board and by the North Carolina Dairy Foundation. We thank P. M. Muriana for providing strains and sharing the electroporation protocol for L. acidophilus NCK64, NCK88, and NCK89; L. Morelli for suggestions on electroporation of L. helveticus, M. A. Conkling, H. M. Hassan, and P. M. Foegeding for insightful discussion and guidance; P. E. Bishop for the use of instrumentation to synthesize primers; and R. D. Joerger for assistance with VAX sequence analysis programs. LITERATURE CITED 1. Anderson, D. G., and L. L. McKay. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. 2. Barefoot, S. F., and T. R. Klaenhammer. 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl. Environ. Microbiol. 45:1808-1815. 3. Birnboim, H. C., and J. Doly. 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Buchman, G. W., S. Banerjee, and J. N. Hansen. 1988. Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J. Biol. Chem. 263:1626016266. 5. Bullock, W. O., J. M. Fernandez, and J. M. Short. 1987. XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotech-

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19. Kaletta, C., and K. D. Entian. 1989. Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product. J. Bacteriol. 171:1597-1601. 20. Kekessy, D. A., and J. D. Piguet. 1970. New method for detecting 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 rapid characterization of plasmid deoxyribonucleic acid. Appl. Environ. Microbiol. 35:592-600. 22. Kleeman, E. G., and T. R. Klaenhammer. 1982. Adherence of Lactobacillus species to human fetal intestinal cells. J. Dairy Sci. 65:2063-2069. 23. Kok, J., J. M. B. M. Van Der Vossen, and G. Venema. 1984. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl. Environ. Microbiol. 48:726-731. 24. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly 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 plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus, and Propionibacterium. 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.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 27. Manfioletti, G., and C. Schneider. 1988. A new and fast method for preparing high quality A DNA suitable for sequencing. Nucleic Acids Res. 7:2873-2884. 28. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: 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:315422. 30. Messing, J., B. Gronenborn, B. Mueller-Hill, and P. H. Hofschneider. 1977. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindIII fragment of the lac regulatory region in M13 replicative form in vitro. Proc. Natl. Acad. Sci. USA 74:3642-3646. 31. Mount, D. W., and B. Conrad. 1984. Microcomputer programs for graphic analysis of nucleic acid and protein sequences. Nucleic Acids Res. 12:811-817. 32. Muriana, P. M., and T. R. Klaenhammer. 1987. Conjugal transfer of plasmid-encoded determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Appl. Environ. Microbiol. 53:553-560. 33. Murray, N. E., W. J. Brammer, and K. Murray. 1977. Lambdoid 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. The nonidentity of porcine N-acetylglucosaminyltransferases I and II. J. Biol. Chem. 256:11477-11482.

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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 DNA techniques: an introduction, p. 48-49. Addison-Wesley Publishing Co., Reading, Mass. 37. Rosenberg, M., and D. Court. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet. 13:319-353. 38. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain elongation inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 39. Scherwitz-Harmon, K., and L. L. McKay. 1987. Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of BclI fragments 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: consensus pentapeptide among tonB-dependent colicins and receptors. J. Bacteriol. 169:3350-3357. 41. Snyder, M., and R. W. Davis. 1985. Screening X-gtll expression libraries 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 among DNA 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. 40:722756. 44. Upreti, G. C., and R. D. Hinsdill. 1975. Production and mode of action of lactocin 27: bacteriocin from a homofermentative Lactobacillus. 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 lactococcal bacteriocin plasmid. Appl. Environ. Microbiol. 55:11871191. 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 sequences. Nucleic Acids Res. 12:5145-5164. 48. Wilbur, W. J., and D. J. Lipman. 1983. Rapid similarity searches 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 for colicin 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. Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13 mpl8 and pUC19 vectors. Gene 33:103-110. 51. Young, R. A., and R. W. Davis. 1985. Immunoscreening X-gtll recombinant DNA expression libraries, p. 29-41. In J. K. Setlow and A. Hollaender (ed.), Genetic engineering, vol. 7. Plenum Publishing Corp., New York.