JOURNAL OF BACrERIOLOGY, Nov. 1992, p. 7463-7469 0021-9193/92/227463-07$02.00/0 Copyright C 1992, American Society for Microbiology

Vol. 174, No. 22

Molecular Characterization of a Second Abortive Phage Resistance Gene Present in Lactococcus lactis subsp. lactis ME2t EVELYN DURMAZ,1 DON L. HIGGINS,2 AND TODD R. KLAENHAMMERl.2,3* Departments of Food Science' and Microbiology2 and Southeast Dairy Foods Research Center,3 North Carolina State University, Raleigh, North Carolina 27695-7624 Received 18 May 1992/Accepted 21 September 1992 The fifth phage resistance factor from the prototype phage-insensitive strain Lactococcus lactis subsp. lactis ME2 has been characterized and sequenced. The genetic determinant for Prf (phage resistance five) was subcloned from the conjugative plasmid pTN20, which also encodes a restriction and modification system. Typical of other abortive resistance mechanisms, Prf reduces the efficiency of plaquing to 10-2 to 10-3 and decreases the plaque size and burst size of the small isometric-headed phage p2 in L. lactis subsp. lactis LM0230. However, normal-size plaques occurred at a frequency of 10-4 and contained mutant phages that were resistant to Prf, even after repeated propagation through a sensitive host. Prf does not prevent phage adsorption or promote restriction and modification activities, but 90%o of Prf4 cells infected with phage p2 die. Thus, phage infections in PrI cells are aborted. Prf is effective in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains against several small isometric-headed phages but not against prolate-headed phages. The Prf determinant was localized by TnS mutagenesis and subcloning. DNA sequencing identified a 1,056-nucleotide structural gene designated abiC. Prf+ expression was obtained when abiC was subcloned into the lactococcal expression vector pMG36e. abiC is distinct from two other lactococcal abortive phage resistance genes, abiA (Hsp+, from L. actis subsp. cis ME2) and abi416 (Abi+, from L. lad subsp. lactis fL416). Unlike abU4, the action ofabiC does not appear to affect DNA replication. Thus, abiC represents a second abortive system found in ME2 that acts at a different point of the phage lytic cycle.

The susceptibility of starter cultures to bacteriophage infection remains a problem in the cheese industry. Phenotypic and genetic analyses of natural phage resistance mechanisms have led to the identification of three categories of bacteriophage resistance in lactococci (21, 23, 39): interference with phage adsorption, DNA restriction/modification (R/M), and abortive infection resulting from interference with intracellular phage development at some point after normal injection and early viral gene expression (27). Genes encoding resistance mechanisms that abort phage infection are common in lactococci and are routinely associated with plasmid DNA elements (5, 9, 10, 28, 31, 33, 35, 45). Lactococcus lactis subsp. lactis ME2 is a prototype phageinsensitive strain which has been used successfully in the cheese industry (22). ME2 contains at least three plasmids which encode distinct phage defenses. The presence of plasmid pME0030 interferes with phage adsorption (37). Two selftransmissible plasmids, pTR2030 and pTN20, each encode at least one R/M system as well as a system that aborts phage infections (11, 15; this study). pTR2030 encodes the abortive infection mechanism Hsp, which appears to interfere with phage DNA replication in infected cells (12, 24). Since it was the first Lactococcus abortive infection gene to be cloned and sequenced, the hlsp gene will hereafter be designated as abiA (13). Confirmation of the hsp sequence was obtained when the DNA sequence of an analogous abortive resistance plasmid, pCI829, was found to be identical to that of abi4 (5). The present study defined and characterized an abortive resistance mechanism encoded by pTN20. This mechanism

was discovered after subcloning of pTN20 inactivated the R/M+ phenotype of this plasmid. The residual phage resistance was manifested by reduction in efficiency of plaquing (EOP) and heterogeneity in plaque size of the small isometric phage p2. This phage resistance mechanism was designated Prf, for phage resistance five, and is the fifth mechanism characterized from the phage-insensitive strain ME2. Subcloning of Prf. Plasmid pTN20 in toto was subcloned into the 6-kb gram-positive, origin probe shuttle vector pSA34 (38). Plasmid pSA34 (Table 1) is a derivative of pSA3 (6), which confers erythromycin resistance in L. lactis and does not contain a gram-positive origin of replication. pTN20 DNA isolated from L. lactis cells by the method of Anderson and McKay (2) was partially digested with HindIII and ligated with HindIII-digested pSA34 DNA. The ligation mix was transformed into L. lactis subsp. lactis LM0230, using protoplast transformation as described by Kondo and McKay (26). Erythromycin-resistant transformants (1.5 p,g/ ml) were selected and evaluated for conjugal ability and for resistance to phages p2 and c2. Conjugation experiments were carried out on milk-glucose plates as described by McKay et al. (29). The 34-kb recombinant plasmid pTRK97, a pTN20::pSA34 chimera that was self-transmissible (Tra+) in conjugation experiments with L. lactis, did not confer any resistance to phage c2 or exhibit R/M activity against phage p2 (data not shown). The presence of pTRK97 did, however, cause phage p2 to plaque at an EOP of approximately 10-2, with heterogeneous plaque sizes. To localize the phage resistance determinants, pTRK97 was partially digested with EcoRV, religated, and transformed into LM0230. One transformant contained an 11.4-kb plasmid, designated pTRK99, which was composed of pSA34 plus 5.4 kb of pTN20 DNA.

* Corresponding author.

t Paper FSR92-13 of the Journal Series of the North Carolina

Agricultural Research Service, Raleigh.

7463

J. BA=PEIOL.

NOTES

7464

Strain, phage, or plasmid

Bacteria L. lactis subsp. lactis ME2 LM0230 NCK199 NCK426 NCK203

TABLE 1. Bacteria, plasmids, and phages Relevant characteristic(s)a pTN20, pTR2030 Conjugal recipient, str-1, plasmid-free

Source or reference

25

LM0230(pTRK99) LM0230(pTRK319) Conjugal recipient, str-15

11, 30 This study This study 15

L. cremonis subsp. lactis M43A

Conjugal recipient, Strr

41

E. coli MC1061 DH5a

Transformation host Transformation host

17 GIBCO-BRL

11 20 19

ml2r UL36

Small isometric phage for LM0230 Small isometric phage for LM0230 Small isometric phage for NCK203, susceptible to inhibition by R/M and Hsp encoded by pTR2030 Small isometric phage for NCK203, susceptible to R/M, resistant to Hsp encoded by pTR2030 Small isometric phage for NCK203, resistant to R/M and Hsp encoded by pTR2030 Small isometric phage for M43A Small isometric phage for NCK203

skl c2 lambda 467

Small isometric phage for LM0230 Prolate phage for LM0230 Xb221 rex::TnS cI857 Oam29 Pam8O

Bacteriophages p2 jjS0

+31 4)48

4)50

1 1 41 S. Pandian, University of Laval, Qu6bec, Canada 32 36 7

Plasmids pTN20 pTRK97

11 R+/M+ Tra+ Prf+, 28 kb This study R-/M- Tra+ Prf+ Emr, 34 kb This study R-/M- Tra- Prf+ EmT, 11.4 kb pTRK99 44 Lactococcal expression vector, Emr, 3.7 kb pMG36e 38 Shuttle vector, Cmr Emr Tcr, ori- (gram positive), 6 kb pSA34 6 Shuttle vector, Cmr Emr Tcr, 10.4 kb pSA3 Stratagene Cloning vector, Apr, 2.9 kb pBluescriptII KS+ str and StrT, streptomycin resistance; EmT, erythromycin resistance; Tcr, tetracycline resistance; ori, origin of replication; ApT, ampicillin resistance; Cmr,

a

chloramphenicol resistance; R/M, restriction/modification; Tra+, conjugal transfer ability; Prf, phage resistance five.

pTRK99 had lost conjugative ability (Tra-) and lacked R/M activity but still encoded some resistance to phage p2. Bacteriophage resistance. The phage resistance phenotype conferred by pTRK99 was characterized by assaying the reactions of phage p2 on L. lactis subsp. lactis NCK199 (Table 1). For preparation of bacteriophage lysates and determination of PFU per milliliter, M17 glucose broth or agar was used as described previously (42) except that in determining the phage titer, 0.3 ml of log-phase cells (optical density at 600 nm [OD6.] = 0.5) was used in each assay. The EOP was obtained by dividing the phage titer on the test strain by the titer on a phage-sensitive, but otherwise homologous, host. Phage p2 formed heterogeneous-size plaques on NCK199 at an EOP of 10-2 to 10'. Plaque morphologies for phage p2 on an NCK199 lawn ranged from normal (about 1.7 mm) to small (pinpoint). Normal-size plaques were detected at an EOP of 10-4. The normal-size plaques were picked, propagated on LM0230, and plaqued again on NCK199. These phages formed normal-size plaques at an EOP of 1.0 on NCK199, indicating that they were unaffected by the presence of pTRK99, even following propagation through the sensitive

host. A similar situation was reported by Gautier and Chopin (10), who described the abortive infection plasmid pIL105, which caused turbid plaque formation and lowered the EOP of isometric-headed phage 66, while clear mutant plaques occurred at an EOP of 10-4. The p2 phage mutants resistant to pTRK99 were not characterized further except that a comparison of restriction enzyme digestion patterns of phage p2 and the mutant phages revealed that there had not been detectable additions, deletions, or rearrangements (data not shown). Small p2 plaques on a lawn of NCK199 occurred at an EOP of 10-2 to 10-3, were pinpoint in size, and were difficult to enumerate. Individual small plaques picked from NCK199 lawns (p2.NCK199 small) and assayed directly back on NCK199 continued to form heterogeneous plaques at EOPs of 10-1 to 10-2. In a third round of plaquing, phages isolated from plaques on the lawn of the sensitive host (p2.NCK199 small.LM0230) plaqued at EOPs of 10-1 to 10-3 on NCK199. This progressive increase in EOP was similar to that reported for the prolate-headed phage c2 during repeated plaquing on HID600 (45). Ward et al. proposed that a

VOL. 174, 1992

NOTES

10

9. -

LM0230

E 84 L.

0~ 0

0

7

6

NCK199

) -r

~O_ O 8-

T-

30 45 60 75 Time (min) FIG. 1. One-step growth curves for phage p2 on L. lactis subsp. lactis LM0230 and NCK199(pTRK99). 0

15

heritable mutation occurs in phage exposed to the abortive resistance plasmid in HID600. The phage resistance phenotype encoded by pTRK99, which reduces the EOP and plaque size of phage p2, was designated Prf+. Prf is not an R/M mechanism, because plaque size and EOP are reduced for phage p2 regardless of whether the phage has been propagated on Prf+ or Prfcells, with the exception of the resistant phage mutants described above. Adsorption assays (36) revealed that phage p2 adsorbed to NCK199 at 91.4%, compared with 97.1% adsorption to LM0230 without pTRK99. Center-of-infection assays (40) were conducted on log-phase cells (OD6.J = 0.8). The number of plaques formed by the infected cells on a lawn of the phage-sensitive host defined the numbers of centers of infection. The efficiency at which centers of infection formed (ECOI) was obtained by dividing the number of centers of infection for the test strain by the number of centers of infection for the homologous phage-sensitive strain. The burst size for the phage on a test strain was determined as follows: (phage titer at the end of the single step growth curve initial titer)/initial titer. The ECOI for p2, first propagated through LM0230, on NCK199 was 8 x 10-2 (Fig. 1). The average burst size of phage p2 after 45 min was 63 in LM0230 and 7 in NCK199. Thus, while the Prf phenotype does not markedly affect phage adsorption, it reduces the proportion of infected cells which release phage and limits -

7465

the average number of viable phage progeny released per cell. Cell survival after infection was assayed by the method of Behnke and Malke (3). The'aYerage number of surviving cells following phage challenge at a multiplicity of infection of 10 was 2.6% for LM0230 and 13% for NCK199. Therefore, Prf significantly reduces the EOP and the ECOI of a phage, while only slightly improving the rate of cell survival after infection. These phenotypes collectively indicate that Prf is an abortive phage resistance mechanism. Prf expression against other phages and in other Lactococcus strains (Table 2). The prolate phage c2 was not affected by Prf. However, Prf was effective against the small isometric-headed phages JJ50 and skl in the LM0230 background. Plasmid pTRK99 was introduced into L. lactis subsp. lactis NCK203 and L. lactis subsp. cremoris M43A by electroporation. Electroporation was carried out as described previously (8) on overnight cells after two washes in sterile distilled water and resuspension in water with 10% glycerol. Cells prepared by this procedure were stored in aliquots at -70°C. Electroporation in the Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) was performed with 40 ,J of cells and 1 to 5 ,ul of DNA in 0.2-cm cuvettes at 2.0 kV, 200 fl, and 25 ,uF; average time constants were 4.0 to 4.4 ms. In NCK203, a derivative of an industrial starter strain (15), Prf was effective against phage UL36, a phage isolated from cheese whey in Canada (31a). Prf did not, however, interfere with the large isometric phage 949 or with a number of industrial phages that attack NCK203: phages 4)31, 448, and 450. In strain M43A, a derivative of the industrial strain M12R, Prf lowered the EOP of phage ml2r to 10-4. Therefore, Prf is expressed in both L. lactis subsp. lactis and L. lactis subsp. cremoris strains and is active against some, but not all, of the small isometric-headed phages tested. It is interesting that phage p2, which is subject to Prf action, is not affected by R/M (LlaI) on pTR2030, while phage 431, which is not affected by Prf, is highly susceptible to inhibition by both R/M and Hsp (24). Strain ME2 contains both Prf (pTN20) and Hsp (pTR2030) and therefore contains abortive infection mechanisms which collectively are effective against all the small isometric phages tested thus far, with the exception of phages 4)48 and 4)50. Phages 4)48 and 4)50 are mutant phages which have been isolated from cheese whey since the introduction of starter cultures containing pTR2030 into the dairy industry in 1984 (1, 14). Replication of phage p2 DNA. The intracellular replication of p2 phage DNA was followed by extraction of total cell DNA at timed intervals after phage infection as described by Hill and coworkers (12, 24). Samples were withdrawn from

TABLE 2. Reactions of various phages on three strains containing pTRK99 Prf+ strain containing pTRK99

L. lactis subsp. lactis LM0230

Phage

c2 p2

jSO skl NCK203

+31

048

+50

L. lactis subsp. cremoris M43A

UL36 949 ml2r

EOP"

Phage morphology/speciese

Prolate/c2 Small isometric/936 Small isometric/936 Small isometric/936 Small isometric/P335 Small isometric/P335 Small isometric/P335 Small isometric/P335 Large isometric/949 Small isometric/not determined

a From reference 18. b Plaques were normal size where EOP = 1.0 and pinpoint where EOP < 1.0. Plaques of Prf' mutant phages

1

10-2_10-3 10-3

10-3 1 1 1

1O-3 1

10-4

occurring at lower frequencies were normal size.

7466

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NOTES

(a)

2

1

A

3

4 5

6 7 8 9

10

pSA34

(b)

A

1

2

3

4 5

6 7 8 9 10

FIG. 3. Map of pTRK99. Hatched section indicates pSA34. Numbered boxes indicate the sites of TnS insertions.

4S~~~~~~~~~~~~~~~~~~~~~~~4

DNA replication (12, 24). The mechanism of action of Prf is not yet known.

FIG. 2.

(a) Phage p2

DNA

replication

in L

lactis

in the presence

absence of pTRK99. Lanes 1 and 2, DNA from uninfected cells: lanes 3 to 10, DNA from cells prior to infection (lanes 1 and 2) and after phage infection at 15 min (lanes 3 and 4), 30 min (lanes 5 and 6), 45 min (lanes 7 and 8), and 60 min (lanes 9 and 10). MspI digests of phage p2 DNA (lane A), LM0230 (lanes 1, 3, 5, 7, and 9), and LM0230 containing pTRK99 (lanes 2, 4, 6, 8, and 10). (b) Same gel as in panel a probed with 32P-labelled phage p2 DNA. or

NCK199 and LM0230 broth cultures at 15, 30, 45, and 60 min after infection with phage p2. The total cell DNA was extracted from cell pellets, equilibrated by OD, and restricted with MspI, and the fragments were separated in an electrophoresis gel (Fig. 2a). The amount of phage DNA was further analyzed by in-gel DNA-DNA hybridization, using 32P-labelled phage p2 DNA as a probe as described by Tsao et al. (43). Gels for use in in situ agarose gel hybridization were first dehydrated between two pieces of filter paper under vacuum on a Bio-Rad Model 583 gel dryer at room temperature and then air dried overnight at room temperature without vacuum. The dried gels were treated with denaturation and neutralization buffers as appropriate for Southern membrane hybridization (34); this was followed by standard prehybridization and hybridization steps. Visual examination and autoradiography of the electrophoresis gel showed no apparent or consistent difference between the LM0230 and NCK199 infected cells in the amount of phage DNA being replicated over the time course of the experiment (Fig. 2b). Therefore, Prf acts at some point in the lytic cycle following phage DNA replication. In contrast, the first abortive gene cloned and sequenced in lactococci, abiA4, which encodes Hsp, has been shown to interfere with phage

Localization of the Prf determinant on pTRK99. A restriction map of pTRK99 identified an EcoRV fragment (1.3 kb) and a HindIII fragment (3.8 kb) separated by 300 bp within the 5.4-kb region cloned from pTN20 (Fig. 3). Each of the major fragments was subcloned, but neither fragment alone expressed Prf when introduced into LM0230. Therefore, TnS mutagenesis was used to localize Prf on pTRK99. TnS mutagenesis was conducted with Escherichia coli MC1061, using lambda 467 (lambda b221 rex::Tn5 c1857 Oam29 Pam8O) as previously described (7, 16). Thirty E. coli MC1061 transformants containing pTRK99 with TnS insertions were examined. Of these, four contained TnS insertions in the EcoRV fragment (insertions 14, 15, 23, and 24) and eight contained TnS insertions within the HindIII fragment (insertions 1, 8, 9, 11, 12, 17, 22, and 26) (Fig. 3). These 12 plasmids were electroporated into LM0230, and the transformants were tested for resistance to phage p2. TnS insertions at positions 14, 15, and 23 were Prf- (EOP of 1.0); the remaining insertions were Prf+ (EOP of 10-3, small plaques). These data localized the Prf genetic determinants within the 1.3-kb EcoRV and adjacent 300-bp fragments, which is the region between insertions 24 and 9. Sequencing. The 1.3-kb EcoRV fragment of pTRK99 was subcloned into pBluescriptll KS+ (Stratagene, La Jolla, Calif.) and transformed into E. coli DH5a, with blue and white color selection using IPTG (isopropyl-3-D-thiogalactopyranoside) and X-Gal (5-bromo-4-chloro-3-indoly-3-D-galactopyranoside) (34). Double-stranded DNA was sequenced on both strands by using the 7-deaza-dGTP kit for Sequenase (U.S. Biochemicals, Cleveland, Ohio) following the manufacturer's instructions. Synthesized 17-mer oligonucleotide primers were obtained from U.S. Biochemicals and from Bio-synthesis, Inc. (Denton, Tex.), and used to "walk" along the template DNA. The sequencing reaction mixtures were electrophoresed on a 2010 Macrophor electrophoresis unit (Pharmacia-LKB, Piscataway, N.J.). The sequence

NOTES

VOL. 174, 1992 RBS

ATCATATAGCAAg-AGGATTTTATATGTCAGAAAAGAAAAATACAAAAGGCAGTCCCATT

60

TATATGAAAAAAAGTTTTTGGATTCCGACAATTATATTTGTTGTATTTGTATTTGTGTTT

120

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GTAATGCTATTGAAATTACCTAGTTTTGGATTATGGTATGGAGCAAACGTTAAAGATAAA V

M

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1020

D

ATAATGAATTTCAAATCCTTTGTGTCAGCCGATTCGTAATTTCAGAAGCAAAGTTCTTGG I

960

K

AAACGAAAAGATTATGAGAAGAAATTAAAAAGTATTGATAATATAGCAGAATTTGAGGAT K

900

P

GAAATTATTCAAGACGATTTATCAATCTTTATTAACGATAATGAAGGTAAAAATATAAAA E

840

T

AATTTATTTGGAGATGAAAAAGATTTTAAAATTGACCAACATTTCGTAATTCCTAAACCC N

780

I

CTTATCAATTCTCTCTATGTTAAGAGGGGCTTGGGACTGGGTATTGAATTAATAGGAACT L

720

D

TATAAAAATTATATAGGAATATTGCGGACTCAGCTATCTTCTGAAGAACTAGTTGTAATA Y

660

F

CACAGAATATTGAAGAGCCTAAACAAAAGATTTGACGAAAAGAAATTAGATGAAAGTGAT H

600

L

GTTATTTCCCGACAATTTGATGATGTTTATAATAAAATGAGCTCATACTTTAAAATATTT V

540

I

ACATATAACAGTTCAAAATATAAAGGCAAGAATTTAACTAAAGAGCAAAAAGTAGAATTA T

480

D

ATAGGAAATGGGCTGGATTTTCGATTTAATTTATTTGAAAGTAATCAATGGTTTTCTATT I

420

L

TATAAGAGGGCAATTAACGACAAACATGGCAACTCATTTATAAATGATTACAATATAGAT Y

360

L

TTAGACCTATTTAAAAAAGAACAAAATAAATCAGAAACAATAAAAGCGATAAGTTTTTTG L

300

N

CAAGAGGAGTCGAAGAAACTTAATAATTCTGATAGTGCTAATAGAGAGTTTTATAGTTTA Q

240

L

ATTGCTCCTATACTAACTTTTTTGGTCTTCATGAACACATTAAGTATACAGAAAAAAAAT I

180

K

GTTAGTCCAAATTCAATCAATATCAGTAGAGTCGAGCTTTTAAAAATATTAATATCTTTA V

7467

1080

-

AGATTATGCAAATGTTGGCTACAATGCTACAAAGGGACAGTACTTCTATGGATGTAAATG

1140

TCATGCTTTAGTCAGTGAATCAGGCTATGTCATAGACTACACAATTACTCCTGCTTCAAT

1200

GGCTGATAGTTCAATGACCGAGGAAGTGTTGAGTCAATTTGGGACACCAACAGTCCTTGG

1260

1271 AGATATGGGAT FIG. 4. Sequence of 1,271-bp EcoRV fragment encoding abiC. The putative ribosomal binding site (RBS) is indicated.

information was processed and analyzed by PC/Gene (IntelliGenetics, Inc., Mountain View, Calif.). The DNA sequence, shown in Fig. 4, contained a large open reading frame (ORF), from nucleotides 25 to 1056. A putative ribosomal binding site (GAGGAG) was identified 6 bp upstream of the start codon. The predicted product of this ORF, designated abiC, is a protein of 344 amino acids. No upstream expression signals were identified. In fact, the EcoRV fragment which was sequenced ended 12 nucleotides upstream from the putative ribosomal binding site. Two putative transmembrane helices were identified in the amino

acid sequence deduced from the DNA sequence. The helices are located near the amino terminus of the predicted protein, at amino acids 18 to 44 and 65 to 86. No significant homology was found between the deduced amino acid sequence and those in the SWISS-Prot 20 data bank, February 1992 release. Comparison of the DNA sequence with that of the abortive gene hsp (abiA) sequenced from pTR2030 (13) showed no significant homology. In addition, the DNA sequence showed no homology with that of abi416, from IL416 (3a, 4). Cloning and expression in pMG36e. The TnS insertions

7468

NOTES

J. BACTERIOL.

Dr-aI

Sacl

Ac1 IraI

1,Dral Sspl

I I

DraI

SC£I RsaI Fok I

A

RBS prf

produces resistant phage. Likewise abortive resistance and adsorption resistance genes are leaky to the extent that phage can escape the system. When the three types of mechanisms are combined, either in a single strain or in a rotation of strains containing separate mechanisms, phage escaping restriction are "trapped" by the abortive mechanism, resulting in cell death without the release of viable phage (23). The use of this strategy of stacking distinct mechanisms in a genetically engineered strain, or series of strains used in a rotation, will significantly increase the number of phage-resistant strains which are available for long-term use by the dairy industry. Nucleotide sequence accession number. The sequence of the 1,271-bp EcoRV fragment encoding abiC has been deposited in GenBank under accession number M95956.

FIG. 5. The 1.3-kb EcoRV fragment from pTRK99 inserted into the EcoRV site of the multiple cloning site of pMG36e.

This work was funded by Agricultural Research Service Project NCO 2168 and, in part, by Haarman and Reimer, Inc., Food Ingredients Division. We thank Marie-Christine Chopin for conducting DNA-DNA homology comparisons between abiC and abi416 and for sharing sequence data prior to publication.

localized the Prf+ determinant within the abiC ORF. Hownoted above, an L. lactis subclone containing the 1.3-kb EcoRV fragment which was sequenced did not express Prf+ when ligated into pSA3. Since this fragment does not encode a promoter for the ORF, we subcloned the 1.3-kb EcoRV fragment into the lactococcal expression vector pMG36e (44) to determine whether Prf+ would be expressed with a functional promoter in place. The recombinant plasmid, pTRK319, was recovered in E. coli DH5a and analyzed (Fig. 5). Restriction mapping with AccI confirmed that the fragment was in the proper orientation for expression of Prf from the pMG36e promoter. Plasmid pTRK319 was successfully electroporated into LM0230, and its presence in the transformant (NCK426) was confirmed by Southern hybridization using pMG36e as a probe. The EOP of phage p2 on NCK426 was 10-, with heterogeneous plaque sizes. Mutant Prf-resistant phages were again detected from normal-size plaques, and phages from small plaques continued to plaque at reduced EOPs, with variable plaque sizes, in further rounds of plaquing. The EOP of p2 on LM0230 containing pMG36e was 1.0. Expression of Prf in LM0230(pTRK319) confirmed that the abiC gene encodes Prf. The promoter on the expression vector was necessary to express Prf since the native expression signals were not contained within the 1.3-kb EcoRV fragment. Conclusions. The abortive infection mechanism encoded by abiC is distinct from those encoded by abiA (hsp) and abi416. Comparison of the DNA sequences of the three genes and DNA-DNA hybridization indicate that no homology exists between the genes. Therefore, Prf is a third distinct Lactococcus abortive gene. It is logical to assume that resistance mechanisms which induce abortive infections could target any one of numerous points in the lytic cycle. This is substantiated by the fact that Hsp and Prf act at different points in the cycle. It is likely that further research into lactococcal abortive resistance genes will reveal that a number of completely different mechanisms are involved. The fact that five or more distinct systems are stacked in the naturally occurring phage-insensitive strain ME2 indicates that new phage-resistant strains may be designed in a similar fashion. An R/M system alone can be overcome by the phage, since modification, occurring even at low frequency,

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