Vol. 57, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1991, p. 1346-13530099-2240/91/051346-08$02.00/0Copyright C) 1991, American Society for Microbiology

Molecular Characterization of Three Small Isometric-HeadedBacteriophages Which Vary in Their Sensitivity to the

Lactococcal Phage Resistance Plasmid pTR2030tTAPANI ALATOSSAVAt AND TODD R. KLAENHAMMER*

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

Received 12 December 1990/Accepted 22 February 1991

Lactococcus lactis LMA12-4 is a pTR2030 transconjugant that has been used as an industrial starter culturebecause of its resistance to phages predominant in cheese plants. Plasmid pTR2030 interferes with susceptiblephages in this host strain via two mechanisms, restriction and modification (R/M) and abortive infection (Hsp).After prolonged use of LMA12-4 transconjugants in the industry, two different bacteriophages, designatednck202.+48 (4)48) and nck202.+50 (+50), were isolated which could produce plaques on LMA12-4 containingpTR2030. In this study, these two phages were characterized and compared with a third phage, nck2O2.+31(+31), which is susceptible to both the R/M and Hsp activities encoded by pTR2030. Phage +48 was notsusceptible to inhibition by Hsp, whereas +50 was unaffected by either the R/M or Hsp mechanisms. All threewere small isometric-headed phages, but small differences were noted between the phages in the structuraldetails of the tail base plate, susceptibility to chloroform treatment, and requirements for calcium infectivity.The phage genomes were all between 29.9 and 31.9 kb in length. Phages +31 and +48 harbored cohesive ends,whereas the phage +50 genome was circularly permuted, terminally redundant, and carried a putativepackaging initiation site. DNA-DNA hybridization experiments conducted between the phages revealed acommon region in +48 and +50 that may correlate with the resistance of the two phages to the Hsp-abortiveinfection induced by pTR2030. Phage +50 also harbored DNA sequences that shared homology to pTR2030 inthe region where R/M activities have been localized on the plasmid. Molecular characterization of the threephages localized regions within the genomes of the pTR2030-resistant phages that may be responsible forcircumventing plasmid-encoded Hsp and R/M defense mechanisms in lactococci.

The existence of plasmid-encoded phage defense mecha-nisms in lactococci is widely documented (16, 19, 28). Theseresistance mechanisms act at different stages of the phageinfection and include interference with adsorption, restric-tion and modification (RIM), and abortive infection (Abi orHsp). Genetic determinants for both R/M and Abi have beenidentified on selected phage resistance plasmids, such aspTR2030 and pKR5 (7, 12). Within the lactococci, it is nowapparent that combinations of phage defenses in a singlestrain such as Lactococcus lactis ME2 can confer a phage-insensitive condition to starter cultures used extensively inthe dairy industry (20).One of the most thoroughly characterized lactococcal

phage resistance plasmids is pTR2030 (10, 12, 13, 23, 31).This self-transmissible plasmid encodes both R/M and Hspfunctions; the latter is an Abi mechanism originally desig-nated as Hsp to describe the heat-sensitive reduction in burstsize and efficiency of plaquing of phage c2 during abortiveinfection on pTR2030 transconjugants (13, 23). Differentphage species appear to vary in their general susceptibility topTR2030. Jarvis and Klaenhammer (18) showed that all thesmall isometric-headed phages evaluated were completelyinhibited by pTR2030 transconjugants in five different hostbackgrounds. Plasmid pTR2030 also interferes with prolateand large isometric phages, but to a lesser degree. These

* Corresponding author.t Paper no. FSR90-22 of the Journal Series of the Department of

Food Science, North Carolina State University, Raleigh.t Permanent address: Department of Genetics, University of

Oulu, Linnanmaa, SF 90570 Oulu 57, Finland.

data suggested that phage which would most likely attackpTR2030 transconjugants in the industry would be primarilyprolate or large isometric phages.The phage resistance of lactococcal strains used in indus-

trial starter cultures is markedly improved after introductionof pTR2030 via conjugation (28, 30). pTR2030 transconju-gants have been used successfully in the dairy industry since1985. During this period, two phages were isolated fromindustrial sources that appeared resistant to interference bypTR2030. In this study, we have characterized these tWoindustrial phages and compared them morphologically andgenetically to a third phage that is completely inhibited bypTR2030. Molecular characterization of the three phages,which varied markedly in their susceptibility to inhibition bypTR2030, represents our initial efforts to elucidate geneticroutes whereby phages can evolve counterdefenses to plas-mid-encoded phage resistance mechanisms in lactococci.

MATERIALS AND METHODS

Bacteria, bacteriophage, and culture conditions. The bac-terial strains, bacteriophages, and plasmids used in thisstudy are presented in Table 1. L. lactis strains werepropagated at 30°C in M17 broth (35) with 0.5% lactose or0.5% glucose as appropriate. Phages nck2O2.4)31, nck202.448, and nck202.4)50 (designated 4)31, 4)48, and 4)50, respec-tively) were propagated on L. lactis NCK202 in M17 brothcontaining 10 mM CaCl2 as described previously (35). Allphages were first purified from single plaques on L. lactisNCK202, an isogenic derivative of LMA12 (12, 30) de-scribed originally as L2FA by Jarvis and Klaenhammer (18).

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BACTERIOPHAGES WITH SENSITIVITY TO pTR2030 1347

TABLE 1. Bacterial strains, plasmids, and bacteriophages

Strain, phage, orRelevant characteristicsa Reference or sourceplasmid

L. lactisNCK16 str-l Lac' R+/M+ transconjugant of NCK202; harbors pTN20 and pTR1040 8NCK17 str-l R+/M+, Lac- derivative of NCK16; harbors pTN20, cured of pTR1040 8NCK202 str-15 Lac- propagating host for bacteriophages, previously designated L2FA 12, 18NCK209 str-15 Lac' Hsp+ R+/M+ transconjugant of NCK202; harbors pTR2030 and 12

pTR1040

Bacteriophages4)31 Small isometric phage susceptible to inhibition by RIM and Hsp encoded by This study (12, 18)

pTR20304)48 Small isometric phage susceptible to R/M; resistant to Hsp encoded by pTR2030 This study (12)4)50 Small isometric phage, resistant to R/M and Hsp encoded by pTR2030 11

PlasmidspTK6 R+/M+ Hsp+, 13.6-kb BgIII fragment from pTR2030 cloned in pSA3 13pTRK18 R/M Hsp+, 3.5-kb deletion through R/M locus of pTK6 12pTR2030 R+/M+ Hsp+, 46.2-kb conjugative plasmid 12, 23pTR1040 Lac' Nisr, 72-kb plasmid 23pTN20 R+/M+ Tra+, 28-kb conjugative plasmid 8a str-1, str-15, resistance to streptomycin (1 mg/ml); Lac, lactose-fermenting ability; Hsp, abortive phage resistance; RIM, restriction and modification; Nis,

nisin resistance.

Bacteriophage assays. Titration of PFU and determinationof the efficiency of plaquing (EOP) were as described previ-ously (29). Modified phages were purified from singleplaques formed in cell lawns of the restrictive host anq aredesignated by suffixes denoting the last host used for prop-

agation. Determination of multiplicity of infection and phageburst size were as described previously (3, 22).Phage and phage DNA purification. Phages were concen-

trated from high-titer cell lysates with 10% (wt/vol) polyeth-ylene glycol 8000 in the presence of 0.5 M NaCl (37) andpurified in discontinuous CsCl gradients (2, 21). Purifiedphages were desalted with Centricon-30 microconcentrators(Amicon Division, Grace & Co., Danvers, Mass.) with 10mM Tris-HCl buffer (pH 7.0) supplemented with 10 mMMgCl2. Phage DNA was isolated from CsCl-purified phagesas described by Jarvis (14) with the following modifications:the protease treatment was omitted, and phage DNA wasdissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH8.0]) after ethanol precipitation.

Electron microscopy. Carbon-coated copper grids (150-mesh) were glow discharged before use to improve sampleand stain adsorption. The support film was prepared with 2%collodion (Polaron Equipment Ltd., Warford, United King-dom) in butyl acetate. Aqueous uranyl acetate (2%, pH 4.5)was used for negative staining. Grids were examined in a

JEOL 100 CX Scantem electron microscope at a magnifica-tion of x36,000.DNA purification. Plasmid and chromosomal DNAs were

isolated as described by Anderson and McKay (4) with themodifications described previously (34) and then purified inCsCl-ethidium bromide equilibrium gradients (22). Ethidiumbromide was removed with CsCl-saturated isopropanol in 30mM Tris-HCl-5 mM EDTA-50 mM NaCl (pH 8.0), and theDNA was concentrated through a Centricon-30 microcon-centrator with TE buffer.DNA analysis and radioisotope labeling. Restriction en-

zymes were used according to the manufacturers instruc-tions. DNA fragments were analyzed as described by Mani-atis et al. (23) in 0.7% or 1.0% agarose gels (SeaKem MEagarose) in 90 mM Tris-90 mM boric acid-2 mM EDTA (pH

8.0). Phage or plasmid DNAs were labeled with 35S-dCTP(specific activity, 1,400 Ci/mmol; New England Nuclear,Boston, Mass.) with a nick translation kit (no. 5000; Amer-sham Corp., Arlington Heights, Ill.). Radioactively labeledprobes were recovered in pre-packed Sephadex G-50 col-umns (Boehringer Mannheim). Specific activities of theDNA probes were estimated at 2 x 108 cpm/Iug of DNA.DNA-DNA hybridization. DNA fragments were separated

on agarose gels and capillary transferred to GeneScreen Plusnylon membranes (NEN Research Products, Boston, Mass.)as described previously (32). The membranes were washedwith 2x SSC (lx SSC is 150 mM NaCl plus 15 mM sodiumcitrate) and air dried. Prehybridization, hybridization, andmembrane washes were conducted in an Omniblot System(American Bionetics, Inc., Emeryville, Calif.). Hybridiza-tion solutions (10 ml per 11- by 15-cm pouch) contained 25%formamide (deionized), 6x SSC, 1% sodium dodecyl sulfate(SDS), lx Denhardt solution (25), and 50 p,g of denaturedsalmon sperm DNA per ml. "S-labeled DNA probes (totalactivity, 2 x 107 to 4 x 107 cpm) were denatured and addedto hybridization reactions, which were conducted for 20 h at430C. After hybridization, two washes (first wash in 350 mlof lOx SSC-0.01% SDS; second wash in 350 ml of lx

TABLE 2. EOP of phages 4)31, 448, and 4)50 on L. lactisNCK202 and NCK209(pTR2030)

EOPPhage' NCK209NCK202 (pTR2030)

4)31NCK202 1.0 <10-94)48NCK202 1.0 1.1 x lo-3b048NCK209 1.2 1.04)48NCK209 and NCK202 1.0 1.3 x 10-3b4)5ONCK202 1.0 0.94)5ONCK209 1.2 1.0

a Modified phages are designated by suffixes indicating the last host used forpropagation of the phage.

b Heterogeneous plaque size.

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1348 ALATOSSAVA AND KLAENHAMMER

ck 202-31 0nck202-48 0nck2O2-50FIG. 1. Electron micrographs of negatively stained L. lactis phages: (A) 431, (B) 448, (C) +50. Bar, 100 nm.

SSC4.1% SDS) were conducted at 45°C. After air drying,the membranes were exposed to XR-Omat X-ray film(Kodak, Rochester, N.Y.) at room temperature.

RESULTS

Phage reactions on pTR2030 transconjugants. Phages 4)48and 4)50 were provided by M. E. Sanders (Miles, Inc.,Marschall Products Division). These phages were detectedat industrial facilities where strain LMA12-4, a pTR2030transconjugant (30), was being manufactured or used. In ourlaboratory, these phages were propagated on L. lactisNCK202, which is a phage-sensitive indicator strain that isisogenic to the industrial strain LMA12-4.The reactions of 4)31, 4)48, and 4)50 are shown in Table 2.

The small isometric phage 4)31 is completely inhibited whenpTR2030 is present in this host background. EOPs of <10'on NCK209 for phage 431 are consistent with previousreports describing 4)31 interactions with TL2F1 (18, 31) andLMA12-4 (30). Phage 4)48 was restricted by NCK209 to an

1.0

0

0.1

0 5 10 15 20Ca2+ concentration (mM)

FIG. 2. Effect of Ca2+ concentration on the EOP of 031 (0), 448(A), and +50 (O) on L. lactis NCK202.

EOP of 10-3, and host-dependent phage replication wasobserved after propagation of 4)48 through NCK209 (datanot shown). pTR2030 encodes R/M activities that affect theEOP of phage 4)48 (12). Plaques formed by 4)48 on NCK209were slightly variable in size. This was not attributed to aHsp-dependent reduction in plaque size, since plaque heter-ogeneity on NCK209 was no longer detected after modifica-tion of $48. In contrast to phages 4)31 and 448, phage 4)50plaqued equally well and without alteration in plaque mor-phology in the presence or absence of pTR2030. These dataand other observations made concurrently in our laboratory(12, 31) indicate that phages 4)48 and 4)50 are not inhibited byHsp, 450 is not inhibited by Hsp or R/M, and 431 is sensitiveto inhibition by both mechanisms.

Morphological and physiological characteristics of phages.Figure 1 shows that all three phages are small isometricphages with head diameters of 50 to 55 nm and long, flexible,noncontractile tails. The tail length of phage 4)50 was 105nm, about 25% shorter than the tails of 4)31 and 4)48, whichwere 130 to 140 nm long. Base plates were present on phage4)48 and 4)50 but absent on 4)31. In addition, phage 4)48 hasa single tail fiber, about 30 nm in length, that extends fromthe base plate (Fig. 1B).

All three phages required calcium for infectivity (Fig. 2).The minimum calcium concentration for maximum plaquingefficiency was 3 mM for 4)31, 6 mM for 4)48, and 10 mM for4)50. Calcium requirements could not be replaced withmagnesium (data not shown).Table 3 shows that chloroform treatment was detrimental

to all three phages. The infectivity (percentage of the initialPFU per milliliter) of phage suspensions was reduced sub-stantially upon chloroform treatment: >50% reduction for4)31 and >85% for 4)48 and 4)50. The presence of calciumduring chloroform treatment accentuated infectivity lossesfor all three phages. The two phages with tail base plates,4)48 and 4)50 (Fig. 1B and C), were more sensitive tochloroform treatment and required higher levels of Ca2+ formaximum EOP (Fig. 2).

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TABLE 3. The effect of chloroform on the infectivity of L. lactisphages 4)31, 4)48, and 4)50 in the presence and absence of calcium

Infectivity remainingePhage after chloroform treatmentb

No CaCl2C +10 mM CaC12

4)31 43 6 15 2448 14 3 2 14)50 12 3 3 2

a Percentage of initial PFU per milliliter remaining ± standard deviation (n= 5).

b Chloroform (5%, vol/vol) was added to the phage suspensions andvortexed for 60 s. The aqueous phase was removed and diluted, and the titerwas determined on L. lactis NCK202.

c Phage suspensions were in M17G broth without added CaCl2.

One-step growth curves and burst size experiments wereconducted with the sensitive indicator strain, L. lactisNCK202 (data not shown). For all three phages, cell lysisbegan 45 min after infection at 30°C. Burst sizes wereestimated at 125 + 12 progeny for 4)31, 136 + 30 progeny for4)48, and 49 + 5 progeny for 4)50.

Characterization of phage genomes. The genomes ofphages 4)31, 4)48, and 4)50 are double-stranded DNA, aswould be expected for tailed bacteriophages. Figure 3 showsphysical maps for the three phages constructed with sevenrestriction endonucleases. Genome sizes were estimated at31.9, 31.1, and 29.9 kb for phages 4)31, 448, and 450,respectively. The restriction maps identified single cuttingsites for BamHI and BglII in all three phages. Sall also cutphages 4)31 and 4)48 only once but failed to cut 4)50.Cohesive (cos) ends were detected in phages 431 and 4)48 byidentification of restriction fragments that were subject toheat-inducible dissociation. The types and positions of re-striction sites near the cos ends of the two phages werenearly identical (Fig. 3). In contrast, 4)50 did not harborcohesive ends. Upon digestion with BamHI, PvuII, or BglII,a pattern of subfragments was detected. At least five sub-fragments were generated upon digestion with BamHI with a

0

onck 202-3131.9 kb

onck 202-4831.1 kb

onck202 5029.8 kb

0

10

10

0nck202-50 pvu 1l

Hind21 29.8 kb 7.511

22.1~~~~~~~~~~~~~~~~~~~~~~~~~1

FIG. 4. Partial restriction map of 4)50 genome in circular form.The region homologous with plasmid pTR2030 is indicated by theblack box. pac indicates the putative initiation site for the first roundof packaging of 4)50 DNA. The arrow indicates the direction ofpackaging and the amount of terminal redundancy (TR).

difference in size between the fragments of 2.2 + 0.3 kb.These data indicated that 4)50 harbors a packaging initiationsite (pac) and is a circularly permuted phage with a terminalredundancy of 2.2 kb (Fig. 4). At least five rounds ofpackaging occurs from each concatemeric intermediate;therefore, circular permutation may extend over at leastone-third of the 4)50 genome.DNA homology between phages and with pTR2030. The

DNA relatedness between the three phages was evaluatedby hybridization of total DNA from one phage to restriction

20 30 kb

CAN Eco RI

040 Eco RV

V&V Pvu 11

*8. Hind III

30 kb20

FIG. 3. Restriction maps of 4)31, 4)48 and 4)50 DNAs. In the map of 4)50 only the two HindIll sites corresponding to the largest HindIIIfragment are presented. All other HindIII sites are omitted. Cohesive ends (cos) and putative packaging initiation sites (pac) are indicated.BamHI, BglII, and Sall endonucleases are indicated with letters; each has only a single digestion site. EcoRI, EcoRV, PvuII, and HindIIIendonucleases have several digestion sites in each phage DNA, and these sites are indicated by symbols. Closed symbols and boxed lettersindicate identical (+ 0.1-kb accuracy) distance of the corresponding restriction sites to the cos site in 4)31 and +48 DNAs.

al 11 Sal rmHI |

co$ TTtISYT Cos

Sal |am Ml1 egi 11

Cols tCt ? f T Tlos

BgI 11 Bam Hi

pac ~~~~~~~~~~~~~~~a

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1350 ALATOSSAVA AND KLAENHAMMER

H El12.8 17.4

Sal I Bam Hi23.5 27.9

El27.6

0nck202 31

0/31.9COS

0nck2O2- 48

0nck202-50

Sal I

8.8

H H8.0 10.3

l

16V8 22.4 23.3 Bar HI

16.1 El.2 El.2 26.9

H H H El116.1 21.2 23.2 26.4

1 1~~

lKI 1

-~~~~~~~~~~~~~~~~~~~~~~

EV 0/31.9

Cos

Bgl II

30.1

0/29.8 2.0 18.0 0/29.8paC El pac

FIG. 5. Genetic homology among 4)31, 4)48, and 4)50 based on Southern hybridization analysis. Moderate stringency conditions wereemployed allowing mismatches up to about 25%. Darker stripes indicate strong hybridization; lighter thin stripes indicate weakerhybridization. Regions without stripes indicate no homology when tested the phage DNA probes. DNA probes: (-) 431, (%) 4)48, (S)4)50.

fragments of the other two phages immobilized on mem-brane filters. Figure 5 summarizes the results of the hybrid-ization experiments. Phages 4)31 and 4)48 shared homologyacross the length of the genome, but strong homology wasevident in the region surrounding the cos sites of the twophages. This homology was anticipated by noting the simi-larity in restriction sites mapped for the two phages aroundthe cos site (Fig. 3). About 55% of the 4)50 genome wasunique, sharing no homology with 4)31 or 4)48 genomes overmap positions ranging from 0 to 17 kb. Homology betweenphages 4)31 and 4)48 with 4)50 DNA was limited to regions of13 + 1 kb that mapped between positions of 16.8 and 29 kbin all three phage genomes. Within this region, both phages4)48 and 4)50 contained a 2-kb area (map positions of 21.2 to23.2 kb in 448 and 21.8 to 23.9 kb in 4)50) which was notpresent in the genome of phage 4)31.

Plasmid pTR2030 DNA was also used to probe the threephage genomes for regions of homology. There was no

homology detected for phage 4)31 or 4)48, but DNA fromphage 4)50 hybridized with pTR2030 DNA. A 2.5-kb regionof phage 450, located between the EcoRV and BamHI sitesat a map position of 24 to 26.5 kb (Fig. 4), was homologouswith pTR2030. Further hybridization experiments revealed

that phage 4)50 did not hybridize with pTR2023, a derivativeof pTR2030 with an 11.5-kb deletion. Contained within this11.5-kb region on pTR2030 are the genetic determinants forHsp+ and R+/M+ (10, 12, 13). To determine whether theregion of phage-plasmid homology could be further local-ized, hybridization experiments were conducted with pTK6(Hsp+ R+/M+) and pTRK18 (Hsp+ R-/M-). pTK6 is arecombinant plasmid carrying the 11.5-kb phage resistanceregion of pTR2030, and pTRK18 is a deletion derivative ofpTK6 that is deficient in R/M activities (12, 13). Phage 450hybridized to pTK6 but not to pTRK18 (data not shown).This suggests that phage 4)50 contained sequences that werehomologous to those found near the R/M region defined onpTR2030.

Susceptibility of phage +50 to other R/M systems. Phage4)50 was evaluated to determine whether it was susceptibleto R/M systems different in specificity from pTR2030-en-coded R/M. The R/M plasmid pTN20 (8) was introduced intoL. lactis NCK202 via conjugation (7a). L. lactis NCK17(pTN20) restricted phage 4)50 as well as phages 4)31 and 4)48.The EOPs for the three phages on NCK217(pTN20) rangedfrom 10-5 to 10-6. Therefore, factors that are responsible for

Bgl II9.9

H8.3

I I I00,

.NN

...bl I

I I I I 1- I I--A -:3

I F-I I I

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BACTERIOPHAGES WITH SENSITIVITY TO pTR2030 1351

the resistance of 4)50 to pTR2030-encoded R/M do notprotect this phage from other R/M systems.

DISCUSSION

In this study, three phages that were virulent for anindustrial starter strain but exhibited different reactions inthe presence of pTR2030 were characterized. Phage 431 is avirulent phage that attacks the parental industrial starterstrain, LMA12. Earlier studies demonstrated that whenpTR2030 was introduced via conjugation into L2FA (18), anisogenic background for the industrial strain L. lactisLMA12 (30), the transconjugants were completely resistantto phage 4)31. Phages that formed plaques on pTR2030transconjugants of LMA12 were not detected in industrialwhey samples at that time (30). The appearance of two newphages that formed plaques on LMA12-4 provided our firstopportunity to evaluate phage counterdefenses that presum-ably occurred after the introduction of phage-resistantpTR2030 transconjugants into the commercial arena. Thethree phages were characterized phenotypically into thefollowing three distinct classes relative to their susceptibilityor resistance to the R/M and Hsp defenses encoded bypTR2030: sensitive to both mechanisms (4)31), resistant toboth mechanisms (4)50), and sensitive to inhibition by R/Mbut not Hsp (4)48).The three phages were identified as small isometric-

headed phages (1) with genomes ranging from 30 to 32 kb insize. These characteristics are typical of lactococcal phagesin groups a and b of Jarvis (15, 16) and group III of Prevotset al. (27). In this study, we did not evaluate their geneticrelatedness to other lactococcal phages, since final designa-tion of type phages are still forthcoming from the interna-tional group on lactococcal phage taxonomy (13a). Fromearlier studies (18, 23) showing that pTR2030 is generallymore effective against small isometric phages than prolatephages, it was expected that prolate phages would be thefirst disruptive species to appear industrially. This was notthe case, suggesting that this host background may notsupport proliferation of prolate-type phages. Jarvis et al. (17)have indicated that the host background of phage resistanttransconjugants is a major factor that determines the effec-tiveness of plasmids that inhibit phage. Noting the appear-ance of the two small isometric phages 4)48 and 4)50 in thisstudy, we would further suggest that the host backgroundselected will dictate the types and numbers of phage speciesthat will appear and, therefore, the genetic potential forphage to establish counterdefenses against lactococcalstrains engineered for phage resistance.Although similar in gross morphology, phages 4)31, 4)48,

and 4)50 showed differences in tail length and base platestructures. Optimal calcium concentration for plaquing alsovaried among the three phages, with phage 4)50 requiringthe highest levels to achieve maximum EOP. The phagesalso varied considerably in their sensitivity to chloroformand the degree to which the presence of calcium accentuatedthe effects of chloroform. It is not clear whether there is adirect relationship between calcium binding and chloroformsensitivity, but these parameters are noteworthy as distin-guishing characteristics of the three phages. The calciumrequirement for phage infectivity is common to phages oflactic acid bacteria (33, 36), but the molecular basis of thisdependence is not yet understood. Further studies on calci-um-dependent steps such as adsorption and DNA injectionand penetration are needed. Work in this area could poten-tially expand phage defense strategies that are directed

specifically against the calcium binding requirements oflactococcal phages.Genomic comparisons of the phages revealed significant

differences between the three. The most striking differenceis that two types of packaging mechanism are suggested.Two of the phages (4)31 and 4)48) contain cohesive endscharacteristic of the cos-type packaging mechanism oflambda. Alternatively, 4)50 is a circularly permuted andterminally redundant phage (overlap extension of 2.2 + 0.3kb) that should use a headful packaging (pac-type) mecha-nism similar to that of phage P22 of Salmonella typhimurium(5, 6, 26). Different packaging mechanisms, however, arenot responsible for the relative sensitivity or resistance ofthese phages to the Hsp or R/M defenses encoded onpTR2030.The phage genomes of 4)31 and 4)48 showed some homol-

ogy over their entire length and nearly identical restrictionmaps near their cohesive ends (Fig. 3 and 5). The pac phage4)50 was not related to either cos phage in over 50% of itsgenome. However, in the 11- to 13-kb region where the threephages shared some homology (map positions at >16.8 kb inFig. 5), both the 4)48 (cos) and 4)50 (pac) phages contained a2-kb region that was absent in phage 4)31. These data suggestthat this region (21.2 to 23.2 kb in 4)48, 21.8 to 23.9 kb in 4)50)is responsible for the insensitivity of phages 4)48 and 4)50 tothe Hsp-induced abortive infection by pTR2030. Subsequentstudies in our laboratory (11) have identified and sequencedthe phage 4)50 origin of replication from this region. SincepTR2030 appears to inhibit phage DNA replication afterinjection (9, 31), we believe that the pTR2030-hsp geneproduct (10) is ineffective against phages that harbor repli-cation regions similar to those of phages 448 and 4)50.However, the mode of action of the hsp gene product and thespecific interactions that may occur with phage replicationloci or functions remain to be defined.Of the three phages, only phage 4)50 was not susceptible to

restriction by pTR2030-encoded R/M. The resistance ofphage 4)50 to restriction was not a generalized phenomenon,since the R/M system encoded by pTN20 (8) was effective on4)50 at the expected efficiency. DNA-DNA hybridizationsbetween phage 4)50 and pTK6 revealed a significant regionof homology that mapped to the location of R/M determi-nants on pTR2030 (12). The homology between phage 4)50and pTR2030 suggested that the phage may have acquiredmethylase sequences from the R/M region of pTR2030.Subsequently, the region of identity between pTR2030 andphage 450 has been sequenced. It was demonstrated thatthe phage acquired 1,273 bp of identical sequence fromthe amino domain of a bifunctional methylase LlaI encodedby pTR2030 (lla). This portion of the truncated methylaseis active; therefore, the phage self-methylates in anyhost supporting its growth. There are a variety of mecha-nisms by which phages have evolved antirestriction systems(24). None of these, however, has occurred through thedirect acquisition of methylases or active portions thereoffrom bacterial R/M systems. It is highly significant that, withthe industrial use and manufacture of pTR2030 transconju-gants, acquisition of a functional portion of the bacterialmethylase constitutes one counterdefense. It will be impor-tant to investigate further the different routes through whichphages can adapt to lactococci engineered with R/M phagedefenses.

Definition of these three classes of phage have provided a

unique opportunity to examine the defense mechanisms oflactococci at the molecular level and begin to identify geneticroutes through which phages circumvent these defenses.

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1352 ALATOSSAVA AND KLAENHAMMER

The understanding of the molecular biology of lactococcalbacteriophages is accelerating at an unprecedented rate (16).With the current availability of the genetic tools to constructstrains that are insensitive to phages now in the industry,unique cultures can be introduced into the most dynamicphage-host ecosystem known and create a window throughwhich we can view molecular adaptation of lactococcalbacteriophages. Ultimately, this information will be criticalto the genetic design of phage-insensitive starter cultures andstarter systems that are capable performing long enough inthe industry to make their development worthwhile.

ACKNOWLEDGMENTS

This work was supported by a U.S.D.A. Molecular BiologyProgram agreements 87-CRCR-1-2547 and, in part, by the Biotech-nology and Dairy Products Divisions of Miles, Inc., Elkhart, Ind.T.A. was supported, in part, by a grant from the Finnish CulturalFoundation and VALIO Finnish Co-operative Dairies Associationand thanks colleagues at North Carolina State University for theirhelp and hospitality.We thank Mary Ellen Sanders for providing phages 4+48 and 050

for this study and Colin Hill for helpful suggestions and discussion ofunpublished data.

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