Vol. 58, No. 8

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1992, p. 2479-2484

0099-2240/92/082479-06$02.00/0 Copyright © 1992, American Society for Microbiology

Characterization of Lactococcus lactis Phage Antigens MATA1* Centre de Recherche de Biochimie et de Genetique Cellulaires du Centre National de la Recherche Scientifique, 31062 Toulouse, 1 and Transia, 69007 Lyon, 2 France C. SCHOULER,1 C. BOUET,2 P. RITZENTHALER,1 X.

DROUET,2 AND M.

Received 17 October 1991/Accepted 1 May 1992

Phage +197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing 13-galactosidase fused to epitopes of phage +197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac' phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to +197 showed that this epitope is well conserved in this genetic group.

Bacteriophages which infect lactococcal starter cultures remain a major problem in the dairy industry, since they inhibit or completely prevent acidification by the starter culture. The true magnitude of the problem is difficult to accurately assess, and many poor-quality cheese fermentations may be due in part to sublethal bacteriophage infection of the starter cultures. A number of measures to prevent phage infection, including implementation of hygienic procedures, rotation of starter cultures, isolation of resistant mutants, and formulation of media to suppress phage proliferation, have been instituted, with various degrees of success (9, 23, 24). Recently, the construction of strains improved for phage resistance has been achieved by transferring different types of phage resistance mechanisms with additive effects in the same strain (3, 7, 20). Another strategy to prevent and control phage development in dairy fermentation could be the early detection of phage contamination. To be efficient in industrial conditions, such a phage detection test must be very fast, easy to handle, and sensitive. To date, the tests used do not fulfill simultaneously these three requirements. Immunological detection of phages may be a way to achieve these goals. The availability of hybrid proteins containing ,-galactosidase fused to wellconserved phage epitopes should facilitate the development of an immunoenzymatic phage detection test using the enzymatic activity of P-galactosidase. The taxonomic differentiation of Lactococcus lactis phages is now well documented (2, 4-6, 14, 16, 18); in contrast, no information concerning the antigenic properties of phages of lactic acid bacteria is available. In a previous work, prolate-headed phages of L. lactis were classified in a unique DNA homology group named group I. The group is abundant, since 29% of the 101 phages analyzed belonged to this group (16). Phage 4)197 is one of the prototypes of group I. In this paper, the cloning of fragments encoding phage *

Corresponding author.

4)197 antigens to produce phage antigen-3-galactosidase fusion proteins is described. These proteins were used to raise polyclonal antibodies to the phage epitopes. The phage proteins containing these epitopes were identified. Crossreactions of the antiserum with related phages were also examined. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The Escherichia coli strains are MC1000 [A(ara-leu)7697 A(lac)X74 galU galK rpsL] and MH3000 (genotype same as that of MC1000 except ompR101) (27). MH3000 was used to construct LacZ+ clones, and MC1000 was used to test the productivity of hybrid proteins. The lacZ fusions were constructed in vector pORF2 (27). Both E. coli strains and pORF2 were obtained from T. Silhavy (Princeton University). Plasmids pTZ18R and pTZ19R (10) were used for sequencing. AluI or RsaI restriction fragments from phage 4)197 were inserted into pORF2 opened at its SmaI site, resulting in plasmids pOR14, -16, -77, -89, -127, and -130 (RsaI inserts) and plasmids pOA17, -64, -74, and -79 (AluI inserts). S17 was the L. lactis propagating strain of phage 4)197. Lactococcal strain IL1403 and plasmid pIL253 were described previously by Simon and Chopin (22). L. lactis phages were 4)29, 4)30, +44, 4)45, and 4)48 (18); P001 (2); and c6A (15). All of these phages except 4)48 (group II) were classified in DNA homology group I (16, 18). The Streptococcus thermophilus phage 4)91 was used as the control (17). Phage production and purification were performed as described previously (25). DNA manipulation procedures. The construction of recombinant DNA clones and DNA-DNA hybridizations were carried out by standard procedures (19). The nucleotide sequences of both strands of the fused genes, upstream from the lacZ junction, were determined by the dideoxy chain termination method (21) with the Sequenase kit (United States Biochemical Corp., Cleveland, Ohio). A synthetic primer (17-nucleotide oligomer) in the 5'-terminal part of 2479

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SCHOULER ET AL.

lacZ was used. For the sequencing of subclones in plasmid pTZ18R, M13 primers were used. Pulsed-field gel electrophoresis was performed with the LKB-Pharmacia Pulsaphor system on a 1% agarose gel in 0.05 M TBE (1 M TBE is 1 M Tris, 1 M boric acid, and 20 mM EDTA). Phage DNAs were laid directly in the wells, and the running conditions were 240 V for 10 to 14 h with a 1-s pulse time. Purification of fusion proteins. The hybrid proteins were purified by the method described by Ullman (26), based on affinity chromatography in the presence of a high salt concentration. This step was followed by gel filtration on a Superose 12 high-pressure liquid chromatograph (Pharma-

cia).

Preparation of E. coli extract used for SDS-PAGE and enzyme-linked immunosorbent assay (ELISA). E. coli strains containing the recombinant plasmids were grown in LuriaBertani medium with 100 ,ug of ampicillin per ml until the culture reached an optical density at 600 nm of 0.8 to 1. The lacZ fusion-containing extracts were prepared from these cultures as described by Peterhans et al. (13). This preparation shall hereafter be referred to as the fusion protein crude extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli (8), and the gels were silver stained by the method of Oakley et al. (12) or stained with Coomassie blue or amido black. Antiserum production. Polyclonal rabbit antisera were prepared as follows. Rabbits were inoculated subcutaneously with a purified phage preparation (300 ,ug of proteins per inoculation), fusion proteins purified by affinity chromatography (100 ,ug of proteins per inoculation), or fusion proteins excised from preparative SDS-polyacrylamide gels. In this last case, the gel band was crushed by sonication. Before inoculation, the preparations were emulsified in Freund's complete adjuvant. Three successive injections of antigen in Freund's incomplete adjuvant were given, after 10, 20, and 40 days. Rabbits were bled 8 days after the last inoculation. The class immunoglobulin G (IgG) antibodies were purified from the antisera on a protein A column. PAb174 is the polyclonal antibody prepared against phage 4197; the sera prepared against the fusion protein encoded by plasmid pOA17 were designated PAb219 (protein purified by chromatography) and PAb220 (protein excised from

SDS-polyacrylamide gel). ELISA. Highly-binding Nunc microplates were coated overnight at 4°C by incubating 100 ,u1 of a solution of IgG

from PAb174 in each well at a concentration of 1 ,ug/ml in 50 mM carbonate buffer, pH 9.6. After the IgG coating, the wells were washed with 0.05% Tween 20-phosphate-buffered saline (PBS) and blocked with 200 ,u1 of 1 M glycine-100 mM sodium carbonate buffer, pH 8.4. After the blocking step, 100 p.1 of fusion protein crude extracts was added to each well. After incubation for 2 h at 37°C, the plates were washed and an enzyme substrate (200 p.1 of 2.5 mM o-nitrophenyl-l-thio-,-D-galactopyranoside-1 mM MgCl2-10 mM 3-mercaptoethanol per well) was added. The optical density at 405 nm was measured after 30 min at 37°C. Western blotting (immunoblotting). Proteins fractionated on SDS-polyacrylamide gels were electrophoretically transferred onto nitrocellulose sheets. The transfer was done in Tris-glycine-methanol buffer (25 mM Tris HCI [pH 8.3], 192 mM glycine, 20% [vol/vol] methanol) at 0.8 A for 2 h with an LKB 2005 Transphor unit. The nitrocellulose membranes were first treated with 0.05% Tween 20-PBS-5% (wt/vol) nonfat dry milk overnight at 4°C to reduce nonspecific binding. The IgG antibodies from polyclonal antibodies (0.5

H E

E

E

C

E

P

EjV

H~~~E

1

10

1~~~~~~~~~E

20

pOA17 pORl4 pOA79

I

or

FIG. 1. Restriction map of phage 4197 and location of antigenic determinant coding sequences on the 4197 genome. Restriction sites: B, BglI; C, ClaI; E, EcoRI; EV, EcoRV; H, HpaII; P, PvuII. Open boxes, restriction fragments which hybridized to the cloned AluI or RsaI phage restriction fragments coding for phage epitopes. The scale (in kilobases) is given under the restriction map.

to 1 ,ug/ml) diluted in 1% nonfat dry milk-0.05% Tween 20-PBS were incubated with the blots for 3 h at 37°C. To detect the antigen-antibody complexes, the paper strips were incubated 30 min with horseradish peroxidase conjugated to goat anti-rabbit IgG, diluted 1:50,000. After each step, unbound reagents were removed by five washes (6 min each) with 0.05% Tween 20-PBS. The blots were finally treated at room temperature with a solution of 0.06% 4-chloro-1naphthol, 0.01% 3,3'-diaminobenzidine, 20% (vol/vol) methanol, and 0.02% hydrogen peroxide in PBS. The developed blots were rinsed with water and dried.

RESULTS Restriction map of phage +197. The DNA from phage 4197 was refractory to restriction by numerous endonucleases: HaeII, MluI, SacII, HindIII, SmaI, SalI, XhoI, BglII, Sau3A, and BamHI. In contrast, AluI, DdeI, Hinfl, RsaI, and TaqI frequently cut the phage DNA. The phage genome size determined by pulsed-field gel electrophoresis was estimated to be 23 kb. Cleavage sites were localized relative to each other by double digests. The EcoRI, EcoRV, BglI, ClaI, HpaII, and PvuII restriction map was constructed (Fig. 1). Phage 4197 DNA was able to produce spontaneously concatemers as detected in pulsed-field gel electrophoresis, suggesting that its genome contains cohesive ends. Construction and identification of fusion proteins. Fusion proteins of phage 4197 proteins and ,B-galactosidase were constructed with vector pORF2 (27). This plasmid allows cloning of DNA fragments upstream from the lacZ gene and downstream from the 5' end of the ompF gene containing the promoter, the transmembrane signal sequence, and the first 12 amino acids of the mature OmpF protein of E. coli; ompF and lacZ are out of frame, and insertion of an open reading frame (ORF) of the correct length results in a lacZ+ gene that produces a trihybrid OmpF-ORF-0-galactosidase fusion protein. Digestion of 4197 DNA by AluI or RsaI generated fragments smaller than 2 kb. 4197 DNA was digested separately by these two endonucleases, and the resulting fragments were cloned into the SmaI cleavage site of pORF2. The constructs were transformed into strain MH3000. Among the 3,500 clones analyzed, about 7% had a Lac' phenotype. The Lac' clones were screened for their ability to produce a hybrid 3-galactosidase that contains phage antigenic determinants by ELISA with PAb174, a polyclonal serum against purified phage 4197.

LACTOCOCCUS LACTIS PHAGE EPITOPES

VOL. 58, 1992

TABLE 1. Analysis by ELISA of fusion proteins produced in crude extracts of strain MH3000 containing the recombinant plasmidsa

a

1 2 3

b 1 2 3

2481

c

1 2 3

OD405b

Plasmid

>2 >2 >1 to 1 to 0.2 to 0.2 to 0.2 to 0.2 to 0.2 to

Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile t...
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