APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1989, p. 1684-16890099-2240/89/071684-06$02.00/0Copyright © 1989, American Society for Microbiology

Vol. 55, No. 7

Localization, Cloning, and Expression of Genetic Determinants forBacteriophage Resistance (Hsp) from the Conjugative

Plasmid pTR2030tC. HILL,' D. A. ROMERO,2 D. S. McKENNEY,' K. R. FINER,' AND T. R. KLAENHAMMERl.2*

Departments of Food Science' and Microbiology,2 Souitheast Dairy Foods Research Center,

North Carolinca State University, Raleigh, North Carolina 27695-7624

Received 15 December 1988/Accepted 31 March 1989

Genetic determinants for a bacteriophage resistance mechanism (Hsp+) encoded by plasmid pTR2030 (46.2kilobases [kb]) were localized by mapping an 11.5-kb deletion that accompanied the transition of Lactococcuslactis LMA12-4 transconjugants (M. E. Sanders, P. J. Leonard, W. D. Sing, and T. R. Klaenhammer, Appl.Environ. Microbiol. 52:1001-1007, 1986) from phage resistance to phage sensitivity. The deleted 34.7-kbreplicon (pTR2023, Hsp-) retained its conjugative ability, demonstrating that the phage resistance andconjugal transfer determinants were genetically distinct. The Hsp region of pTR2030, which was containedwithin a 13.6-kb BglIH fragment, was cloned into the BamHI site of bacteriophage lambda EMBL3, and Hspwas subcloned into the Escherichia coli-Streptococcus shuttle vector pSA3. The recombinant plasmids pTK6and pTK9 were recovered in E. coli HB1I1 and contained a 13.6-kb insert in opposite orientations. L. lactisMG1363 transformants carrying pTK6 or pTK9 exhibited a significant reduction in plaque size, in addition toa slight reduction in the efficiency of plaquing for both prolate and small isometric phages. Phenotypic reactionsobserved for the recombinant plasmids suggest that pTR2030-encoded Hsp acts similarly against both prolateand small isometric phages. TnS mutagenesis was used to define the region essential for the expression of theHsp+ phenotype. Any of four insertions within a 3-kb region resulted in the loss of phage resistance, whereasa further 26 insertions outside this locus had no effect on Hsp expression. In vitro deletion analysis confirmedthat the 3-kb region contained all the information necessary for the observed resistance.

Genetic studies of bacteriophage-host interactions in lac-tococci have identified a variety of plasmids which interferewith the plaque-forming efficiency and burst size of lyticphages by an abortive-type process (3, 6, 10, 11, 16, 17, 22,25). Of these plasmids, pTR2030 was the first which wasidentified as a self-transmissible plasmid that correlated withabortive phage infections (17). Linkage of the conjugaltransfer ability (Tra) and the bacteriophage resistance mech-anism (Hsp) on pTR2030 has allowed evaluation of resis-tance phenotypes in different strains that are attacked bydifferent phage species (13, 29). In Lactocccus lactis andLactococcus cremoris transconjugants containing pTR2030,two distinct resistance phenotypes are exhibited, dependingon the species of phage used to challenge the strains (13).Hspt was originally described by Klaenhammer andSanozky (17) as a 10-fold reduction in the burst size of theprolate phage c2, which formed small plaques at 30°C. Inlater studies, complete resistance to small isometric phageswas demonstrated by pTR2030 transconjugants of 10 dif-ferent strains of L. lactis and L. cremoris (13, 14, 27, 29, 30).However, it was not known whether the different resistancephenotypes reflected the operation of more than one defensemechanism or identified a difference in the susceptibilities ofprolate and small isometric phages to a single mechanism.

Studies at the molecular level were initiated in order todefine the organization of the plasmid, identify the mecha-nisms of phage defense, and characterize the specific gene(s)and gene product(s) responsible for pTR2030-encoded phageresistance. The objective of this investigation was to localize

* Corresponding author.t Paper number 11388 of the Journal Series of the North Carolina

Agricultural Research Service, Raleigh, NC 27695-7601.

the region on pTR2030 that encodes genetic determinants forHsp. The expression of Hspt- by recombinant plasmids in L.lac tis is also described. The study established that hspdeterminants are confined to within 3 kilobases (kb) ofpTR2030.

MATERIALS AND METHODSBacteria, bacteriophage, and culture conditions. The bac-

terial strains used in this study are listed in Table 1. L. lactisstrains were propagated at 30°C in M17 broth (31) or M17broth with 0.5% glucose (M17G) when appropriate. Bacte-riophages were propagated and titrated as described previ-ously (17, 31), except that cultures were grown to a finaloptical density at 600 nm of 0.5. For plaque assays at 39°C,cells were grown to an optical density at 600 nm of 0.5 at30°C, incubated at 39°C for 5 min, and held at 39°C for theremainder of the experiment. Escherichia coli strains werepropagated in LB broth (27) at 37°C with shaking. Whenrequired for selection, the following antibiotics were addedat the indicated concentrations, in micrograms per milliliter:for E. coli, kanamycin, 20; tetracycline, 10; chlorampheni-col, 20; for L. lactis, erythromycin, 10; spectinomycin, 300;and rifampin, 25.

Restriction enzyme analysis. Restriction enzymes and buff-ers were obtained from Bethesda Research Laboratories,Inc. (Gaithersburg, Md.). Restriction digestions were per-formed as described by Maniatis et al. (24). DNA fragmentsgenerated by either HindIII digests of bacteriophage lambdaor by HaeIII digests of bacteriophage XX174 (BethesdaResearch Laboratories) were used as molecular weight stan-dards.

Molecular cloning techniques. Plasmids were isolated fromE. coli by alkaline extraction (28) and from L. lactis as

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TABLE 1. Bacterial strains and plasmids used in this study

Strain or Relevant characteristics Source or derivationplasmid

L. lac-tisMG1363 Plasmid-free, transformation recipient, Lac Hsp- 9NCK2 MG1363(pTR2030) MG1363 transconjugantNCK210 LM2345(pTR2023) Transconjugant; this studyLMA12 Lac' Tra- parent 27LMA12-4 pTR2030; phage-resistant transconjugant of LMA12 27L442 pTR2023; phage-sensitive derivative of LMA12 27T-RS3 str-1, LM2301(pTR1040) 17LM2345 spc-4 rif-5 derivative of LM0230, plasmid-free. Hsp' 2NCK3 MG1363(pSA3) Transformant; this studyNCK6 MG1363(pTK6) Transformant: this studyNCK211 MG1363(pTRK18) Transformant: this study

E. (oliHB101 Host for E. (coli phage 4DH1 Lambda lysogen 12NCK11 HB101(pTK6) Transformant; this studyNCK270 DH1(pTRK18) Transformant; this study

PlasmidspTR2030 Hsp+ Tra', 46.2 kb 16pTR2023 Hsp- Tra', 34.7 kb This studypTR1O40 Lac', 72 kb 15pSA3 Cmr Emr Tc', 10.4 kb 7pTK6 Cm' Emr Hsp+, 24 kb pSA3::13.6-kb pTR2030 fragmentpTK9 Cmr Emr Hsp+, 24 kb pTK6, opposite orientationpTRK18 Cmr Em' Hsp+, 20.5 kb pTK6, with 3.5-kb deletion

described by Anderson and McKay (1). General proceduresfor DNA manipulations and cloning were essentially per-formed as described by Maniatis et al. (24). Materials andprotocols for bacteriophage lambda EMBL3 cloning andpackaging were from Promega Biological Research Products(Madison, Wis.). Bacteriophage DNA was isolated as de-scribed previously (28).

Conjugation and transformation. Conjugal matings andselection of Lac transconjugants were conducted as de-scribed previously (17, 26).

E. coli strains were transformed by a modification of theprocedure described by Curtiss (5). A fresh single-colonyisolate was inoculated into 5 ml of LB broth containing 10mM MgCl,. After 2 h of growth at 37°C with vigorousaeration, the cells were transferred to 100 ml of LB brothwith 10 mM MgCl2 and incubated until the optical density at550 nm was 0.5. Cells were harvested and suspended in 40 mlof transformation solution 1 (30 mM potassium acetate, 50mM MnCl, 100 mM KCI, 10 mM CaCl, and 15% glycerol[pH 7.01). The cells were placed on ice for 90 min and thenpelleted and suspended in 4 ml of transformation solution 2(10 mM MOPS [morpholine propanesulfonic acid; pH 7.0;Sigma Chemical Co., St. Louis, Mo.], 75 mM CaCl2, 10 mMKCI, and 15% glycerol). Samples of 200 ilI were frozenrapidly in liquid nitrogen and stored at -70°C. For transfor-mation, 200 .lI of competent cells was thawed on ice, mixedwith 10 .lI of DNA, and left on ice for 30 min. After the cellswere heat shocked for 5 min at 37°C, 0.9 ml of LB broth wasadded and the cells were incubated for 45 min before theywere plated onto LB plates containing 20 p.g of chloram-phenicol per ml.

L. lactis protoplasts were transformed as described byKondo and McKay (20) with the following modifications.Plasmid DNA (1 to 4 .g) was mixed with an equal volume of2x SMM buffer (19), and the mixture was added to 100 p.1 ofprotoplasts. The protoplast-DNA mixture was transferred to

1.5 ml of cold 30% polyethylene glycol 8000 (J. T. BakerChemical Co., Phillipsburg, N.J.) and mixed by gentleinversion. The transformation mixture was held on ice for 3min and then transferred to a 37°C water bath for 5 min. Themixture was poured into a centrifuge tube containing 10 mlof SMMB buffer (20) and mixed by inversion. Protoplastswere recovered by centrifugation at 12,000 x g; washed with10 ml of SMMB; and suspended in 0.9 ml of a solutioncomposed of 0.5 ml of SMMB, 0.2 ml of 2x SMM, and 0.2ml of M17G. Following incubation for 1 h at 30°C, cells wereadded to a soft agar overlay and plated onto M17G platessupplemented with 0.5 M sucrose and 10 pLg of erythromycinper ml. Transformants were scored after a 7-day incubationat 300C.

Electroporation of L. lactis MG1363 was performed asdescribed by Luchansky et al. (23), except that M17G wasused as the growth medium.Tn5 mutagenesis. Tn5 mutagenesis was performed as

described by de Bruijn and Lupski (8). Lambda467 (lambdab221 rex::TnS c1857 Oam29 Pam8O) was used at a multiplic-ity of infection of 10 to deliver Tn5 (Kmr) to E. coli DH1(lysogenic for lambda) containing pTRK18 (Cmr Em'r Hsp+).Cmr Kmr colonies were harvested, and the plasmid DNAwas isolated for transformation to E. coli. The position ofTn5 in pTRK18 clones was determined by restriction en-zyme digestions of plasmids isolated from the Cmr Km' E.coli transformants.

In vitro transcription and translation. A procaryotic DNA-directed, cell-free, coupled transcription-translation systemwas used for in vitro transcription and translation, as spec-ified in the protocol supplied by the manufacturer (Amer-sham Corp., Arlington Heights, Ill.). Plasmid DNA waspurified through cesium chloride-ethidium bromide gradientsand was used in the reaction in which proteins were labeledwith L-[35S]methionine. One-third of the reaction mixturewas analyzed on sodium dodecyl sulfate-15% polyacryl-

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amide gels (21). After electrophoresis, the gels were proc-essed by fluorography in Amplify (Amersham Corp.) andexposed to XR-OMAT films (Eastman Kodak Co., Roches-ter, N.Y.).

RESULTS

Genetic segregation of Hsp and Tra. Sanders et al. (27)have reported that serial transfers of L. lactis LMA12-4(pTR2030, 46.2 kb, Hsp+) at 37°C generate a phage-sensitivevariant (L442, Hsp-) that carries a deleted form of pTR2030,which is designated here as pTR2023 (34.7 kb). L. lactisL442 (Lac' Hsp-, pTR2023) was examined for conjugalability and compared with the responses exhibited byLMA12-4 (Lac' Hsp+, pTR2030) and the phage-sensitiveparent LMA12 (Lac'). L. lactis LMA12 was incapable ofdonating Lac' in conjugation experiments with LM2301.When matings were conducted with LMA12-4(pTR2030) andL442(pTR2023) as conjugal donors, Lac+ transconjugantswere recovered at frequencies of 6 x 10-7 and 2.4 x 10-6per donor cell, respectively. The 11.5-kb deletion inpTR2030, which correlated with the loss of phage resistance,did not eliminate or suppress conjugal transfer functions.Lac+ transconjugants generated from matings with theLMA12-4(pTR2030) and L442(pTR2023) donors were Hsp+and Hsp-, respectively.

Localization of the Hsp region on pTR2030. A physicalcomparison of pTR2023 with pTR2030 was initiated in orderto localize the Hsp region. To facilitate this comparison,both plasmids were introduced into a plasmid-free back-ground. Lac+ Hsp+ transconjugants of L. lactis MG1363were constructed as described previously (29). A Lac-derivative (NCK2) was isolated; this derivative retainedpTR2030 (Hsp+) but was cured of pTR1040 (Lac+). WhenpTR2023 mobilized the Lac plasmid from L442, only cointe-grated Lac+ plasmids were recovered in transconjugants(data not shown). To isolate intact pTR2023 without cointe-grate formation, a Lac- derivative of L442 was isolated anda three-way mating was performed in which pTR2023 wastransferred first to T-RS3 (pTR1040, Lac+) and then toplasmid-free LM2345 (Spcr Rif"). Transconjugants were se-lected on lactose indicator plates containing spectinomycinand rifampin. Plasmid profiles showed that both pTR2023and pTR1040 were present in transconjugants. pTR1040 wassubsequently cured, resulting in NCK210, which containedonly pTR2023.

Restriction enzyme analysis of pTR2023 indicated that thedeletion in pTR2030, and presumably the region involved inHsp, was completely contained within a 13.6-kb BglII frag-ment (Fig. 1).

Cloning the Hsp region from pTR2030. Repeated attemptsto clone the 13.6-kb BglII fragment in E. coli or Bacillutssubtilis by using pGK12 (18) were unsuccessful.The eventual cloning strategy used was as follows. BgiII

fragments from pTR2030 were ligated into the BamHI site ofbacteriophage lambda EMBL3. The 28.6- and 4-kb frag-ments were too large or small, respectively, to yield viablelambda clones (15). The 13.6-kb BgIII fragment was of theappropriate size for successful cloning into the stuffer regionof lambda EMBL3. Of 10 plaques that were examined, 9contained inserts of 13.6 kb; the 10th plaque did not containan insert. Characterization of one of the clones, lambdaNCK8Hsp, verified that the entire 13.6-kb BgllI fragment ofpTR2030 was present in the recombinant phage. LambdaNCK8Hsp was used as a source of DNA for further restric-tion mapping and subcloning to plasmid vectors.

Bgl

Ava

FIG. 1. Circular restriction map of pTR2030. The inner lineindicates the approximate location of the deletion which gave rise topTR2023.

Lambda NCK8Hsp was digested with Sall (two Sall siteswere immediately adjacent to the BamHI cloning site), andthe 13.6-kb fragment was isolated for subcloning into theSall site of pSA3 (7) and pBR322 (23). All efforts to clone the13.6-kb Sall fragment into pBR322 were unsuccessful. How-ever, this fragment was efficiently cloned in E. coli in bothorientations by using the E. coli-Streptococcus shuttle vec-tor pSA3. pTK6 (Fig. 2) and pTK9 contained the insert inopposite orientations and were chosen for further study.

Expression of Hsp by recombinant plasmids. pSA3 and therecombinant plasmids pTK6 and pTK9 were introduced intoL. lactis MG1363 by protoplast transformation. pSA3 andpTK6 were stable in both E. coli HB101 and L. IactisMG1363 under antibiotic pressure. L. Iactis NCK6 andNCK9 were challenged with a prolate phage (c2) and twosmall isometric phages (p2 and skl) to compare the efficiencyof plaque formation and plaque morphologies against thoseof L. Iactis MG1363 and NCK2(pTR2030). Phage reactions

Ava

Xho l/Ava -

Pvu 11

Sac

Pvu 11

FIG. 2. Circular restriction map of pTK6. The inner line indi-cates the approximate location of the deletion which gave rise topTRK18.

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TABLE 2. Phage reactions on L. lactis MG1363,NCK2(pTR2030), NCK6(pTK6), and NCK211(pTRK18)

at 30 and 39oCa

Efficiency of Avg plaqueStrain Phage plaquing at: size (mm) at:

300C 39°C 30°C 39°C

MG1363 c2 1 1 3.5 4.5ski 1 1 1.5 1.5

NCK2(pTR2030) c2 0.3 0.3 0.2 3.5ski <10-9 0.1 0.2

NCK6(pTK6) c2 0.5 0.33 1.0 3.5ski 0.1 0.05 ppb 0.2

NCK211(pTRK18) c2 0.5 0.4 1.5 3.5ski 0.25 0.05 PP 0.2

a Efficiencies of plaquing and plaque sizes were determined over threetrials.

b pp, Pinpoint plaques (<0.2 mm).

for pSA3 transformants were identical to those of the paren-tal strain L. lactis MG1363. Table 2 shows that at 300C thepresence of pTK6 correlated with a significant reduction inplaque size for skl and c2 phages; the efficiency of plaquingwas reduced slightly to levels of 0.5 to 0.74, respectively.Similar reactions were observed for phage p2 (data notshown). At 39°C the transformants showed an increase inplaque size for all three phages. The efficiency of plaquingand plaque size were the same regardless of the orientationof the insert (pTK6 versus pTK9; data not shown). Pheno-typic differences were apparent when phage reactions werecompared between clones carrying pTR2030 (NCK2) or therecombinant plasmids (NCK6 and NCK9). First, the plaquesizes of phage c2 on the pTK6 and pTK9 transformants werenot as small as they were on pTR2030-containing strains.Second, while p2 and skl formed small plaques on NCK6and NCK9, these phages did not form plaques onNCK2(pTR2030) at 30°C.

skl was not observed to form plaques on Hsp+ strainswhen glucose was omitted from the plating medium andoverlay. The plaque size against MG1363 was also reducedunder these conditions.

E. coli HB101 and its derivatives containing pSA3 (NCK3)and pTK6 (NCK6) were evaluated for resistance against aseries of bacteriophages: T2, T3, T4, T5, T6, T7, and lambdavir. All bacteriophages were propagated through HB101prior to use. In standard plaque assays, no inhibition ofplaquing or reduction of plaque size was detected.TnS mutagenesis of pTRK18. pTRK18 is a spontaneous

deletion derivative of pTK6 that was isolated in L. lactisMG1363; it lost 3.5 kb of the pTR2030-derived region (Fig.2). The remaining 10-kb insert encoded a heat-sensitivereduction in plaque size which was similar, although notidentical, to that of pTK6 (Table 2). pTRK18 was chosen asa target for TnS mutagenesis because of the reduced amountof pTR2030 DNA that was to be mutagenized.

Following mutagenesis the position of TnS in 44 indepen-dent isolates was determined. Thirty Tn.5 insertions areshown in Fig. 3A. In 25 cases the insertion occurred withinthe pTR2030 region. Initially, six plasmids (pTRK38,pTRK39, pTRK40, pTRK44, pTRK48, and pTRK52) wereintroduced into L. lactis by protoplast transformation orelectroporation. The transformants were challenged withphage skl to determine the Hsp phenotype. The phenotypic

A 3 34 3837 3 4143 48 55531j 33 35 s 5ii559

' I4 47 484 54817CAT:^l.l4:M 4951tgI II I^.

Ava 1 BamHIIlEcoR1 Sal 1 EcoR1

I Eco>R1EcoR E

B

)knotAI---I Ava I

SaN1Nru 1

prEXK6 +pTR362

pTFrlK84+ pTflK88

FIG. 3. (A) Linear restriction map of the 14-kb AvaI fragment ofpTRK18 showing the positions of 30 Tn5 insertions (numbers abovethe map). (B) Deletion derivatives of some of the plasmids shown inpanel A. The line marks the extent of the deletion. The Hspphenotype of each deletion plasmid is indicated by the plus or minussign.

reaction encoded by five of the six plasmids was indistin-guishable from that encoded by pTRK18 (Table 2). Theremaining plasmid, pTRK48, did not confer any detectablephage resistance on L. lactis. An additional five plasmidswith TnS insertions flanking that of pTRK48 (Fig. 3A) wereintroduced into L. lactis in order to determine the extent ofthe indispensable region surrounding position 48. Three ofthe five plasmids (pTRK47, pTRK49, and pTRK50) wereHsp-, while pTRK46 and pTRK51 gave the Hsp+ pheno-type. It was concluded that the region of approximately 3 kbbetween positions 46 and 51 is essential for the expression ofthe Hsp phenotype (Fig. 3A).

Following electroporation of pTRK48 and pTRK49 into L.lactis, a number of Emr Hsp+ revertants were recovered.Restriction analysis of the plasmids showed that preciseexcision of TnS from the Hsp- clones was accompanied bythe restoration of the Hsp+ phenotype. This indicates thatthe loss of the Hsp+ phenotype in pTRK48 and pTRK49 isdirectly linked to the presence of Tn5 at those loci and not toa secondary, undetected mutation.Hsp is encoded at a single locus. The possibility remained

that the region identified by Tn5 mutagenesis was only one ofa number of loci on the 10-kb insert of pTRK18 which wasresponsible for phage resistance. This possibility was elimi-nated by construction of deletion derivatives in which theDNA on either side of the 3-kb region was removed. Thiswas achieved by using the internal Tn5 restriction sitesBamHI and NruI, which are unique in pSA3. To removeDNA from the left-hand side of the Hsp region (as shown inFig. 3A), plasmids were digested with BamHI and religated.The ligation mixture was transformed to E. coli by selectingfor Cmr. Since both Tn5 and pSA3 contained a single BamHIsite, clones were obtained in which the DNA between theTnS insertion point and the vector was deleted. In a similarfashion the DNA on the right-hand side of the insert wasremoved by using NruI. Six deletion derivatives were con-structed in E. coli (Fig. 3B) and introduced into L. lactisMG1363 for evaluation of the Hsp phenotype. The resultsconfirmed that DNA flanking the Hsp region is not essentialfor the expression of phage resistance.

In vitro transcription and translation. The proteins derivedfrom a number of the plasmids described above were ana-

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1 2 3 4 5 6 kD

92

469

_0 30

~~~~~~~~~cat

kn

14

FIG. 4. In vitro transcription-translation products derived from

pSA3 (lane 1), pTK6 (lane 2), pTRK18 (lane 3), pTRK38 (lane 4),

and pTRK48 (lane 5). Molecular size markers are shown in lane 6.

Symbols: > (lane 2), additional products from the pTR2O3O insert:,>

(lanes 3 and 4), 40-kilodalton (kD) protein: > (lane 5), truncated

20-kilodalton product (see text). The pSA3 c(it and Tn5 kan gene

products are also indicated.

lyzed by a coupled transcription-translation assay (Fig. 4).

The products derived from the cloning vector pSA3 are

shown in Fig. 4, lane 1. pTK6 (Fig. 4, lane 2) contained at

least five additional bands which were presumably derived

from the cloned 13.6-kb pTR2O3O fragment. Four of these

products were not detected when pTRK18 was the templateDNA (Fig. 4, lane 3), indicating that these products are

linked to the 3.5-kb region deleted from pTK6 and are not

involved in the expression of the Hsp phenotype. The

introduction of Tn5 at four locations outside the Hsp region

(positions 38, 39, 44, and 52; Fig. 3A) had no effect on the

banding pattern except for the presence of a novel protein

resulting from the kan gene of Tn5 (pTRK38 is shown in Fig.

4, lane 4; the other plasmids are not shown). The Hsp-plasmid pTRK48 was missing the fifth pTR2030-derivedproduct of approximately 40 kilodaltons (Fig. 4, lane 5),

thereby implicating this protein in the expression of the

resistance phenotype. A novel 20-kilodalton product was

detected in lane 5 of Fig. 4 and may represent a truncated

form of the missing 40-kilodalton protein.

DISCUSSION

Sanders et al. (27) have previously identified a deleted

form of pTR2O3O which correlated with the loss of phageresistance in L. lacdts. In this study the deleted plasmid

pTR2023 was identified and characterized, and the location

of the Hsp region was revealed. The entire region coveringthe deletion responsible for the loss of Hsp' was success-

fully cloned, first into bacteriophage lambda EMBL3 and

then into the shuttle vector pSA3 in both orientations.

Expression by L. la-i transformants harboring recombi-

nant plasmids verified that Hsp' determinants are locatedwithin the 13.6-kb BglIIl fragment of pTR2030. Furtherlocalization of Hsp to an essential 3-kb region was achievedby using Tn5 mutagenesis. Deletion constructs confirmedthat a unique 3-kb region is involved in the expression of Hspdeterminants. The mutations were total, in that no interme-diate phenotypes were observed. However, it remains pos-sible that either one of the flanking insertions (insertions 46and 51) is within the hlsp structural gene(s), but in a nones-sential region. Therefore, the four insertions which eliminatethe Hsp phenotype may delineate only the essential codingsequence.

Transcription-translation assays implicated the involve-ment of a 40-kilodalton protein in the Hsp phenomenon,since this was the only pTR2030-derived product which wasaffected by a Tn5 insertion responsible for the loss of the Hspphenotype. This protein is unlikely to be the sole product ofthe Hsp region, since it cannot account for the observed sizeof the 3-kb essential region determined by TnS mutagenesis.

In E. coli the presence of the recombinant plasmidsprovided no defense against the phage lambda vir nor tophages T2, T3, T4, T5, T6, or T7. In L. lactis MG1363 bothprolate and small isometric phages formed small plaques ontransformants containing pTK6 or pTK9. The phage reac-tions were the same regardless of the insert orientation,providing evidence that the pTR2030-derived DNA carriedits own promoters. The central location of the Hsp regionwithin the 10-kb insert and the in vitro deletion resultsfurther support this hypothesis. The phage resistance phe-notypes exhibited by the transformants were similar, but notidentical, to those exhibited by strains containing pTR2030.The level of resistance imposed by pTK6 and pTK9 wasweaker than that imposed by pTR2030, since plaque sizes forphage (2 at 30°C were not reduced to the same extent aswere those detected for the transconjugant NCK2 containingthe intact plasmid pTR2030. The weaker phenotype ex-pressed by NCK6 and NCK9 was also evidenced by failureof the recombinant plasmids to direct the complete inhibitionof plaque formation by small isometric phages.

In spite of the lower levels of expression exhibited by theclones when compared with that exhibited by pTR2030, theentire structural gene requirements for Hsp were cloned andwere found to be contained within the 3-kb region. This issupported by the fact that full expression of Hsp (equal tothat of pTR3030) has been observed in pTK6 cointegrateswith conjugative plasmids that do not themselves expressphage resistance (D. A. Romero and T. R. Klaenhammer,manuscript in preparation). This strongly suggests that theexpression of Hsp may be affected to some degree by thereplicon on which it is located. Perhaps copy number and,consequently, gene dosage are the critical factors or perhapsanother, as yet undetermined, parameter plays an importantrole in controlling the level of expression.

Laible et al. (22) recently identified a 23-megadaltonplasmid, pKR223, that was correlated with a reduction inplaque sizes for prolate and small isometric phages. Theresults reported for pKR223 and pGB301 clones carrying the17-kb HpaII fragment are very similar to those reported forpTR2030 (16). Although small differences in efficiencies ofplaquing, plaque sizes, and heat sensitivity responses couldbe emphasized, it appears that the genetic determinantsresponsible for Hsp-directed abortive phage infections maybe similar for pTR2030 and pKR223.The reactions observed in this study indicate that the

mechanism encoded by the 3-kb Hsp region of pTR2030provides similar resistance to both prolate and small isomet-

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CLONING PHAGE RESISTANCE DETERMINANTS FROM pTR2030 1689

ric phages. The variation in the degree to which plaque sizeis reduced appears to reflect the proportionate strength ofone mechanism against different phage species that are moreor less susceptible to inhibition by the Hsp mechanism.Alterations in gene dosage and expression levels of pTR2030hsp determinants are being examined to determine theeffects on the efficiency of a mechanism that appears to abortinfection by small isometric and prolate phages. pTR2030 isresponsible for an even wider range of phenotypic re-sponses, ranging from a reduction in burst size (Hsp) tocomplete inhibition of plaque-forming ability. Given thedefinition of the hsp locus and considering the range ofpTR2030-encoded phenotypes, it is likely that a seconddefense mechanism encoded on pTR2030 contributes to thetotal phage resistance imposed by the plasmid on lactococci.

ACKNOWLEDGMENTS

This work was supported by the Molecular Biology Program,U.S. Department of Agriculture, under agreements 85-CRCR-1-1727and 87-CRCR-1-2547 and, in part, by the Biotechnology Group ofMiles, Inc., Elkhart, Ind.We thank M. E. Sanders for providing L. lactis L442(pTR2023)

and alerting us to the important deletion events that occur inpTR2030. We thank L. R. Steenson for efforts to construct the initialpTR2030 map and R. B. Sanozky-Dawes for technical assistance.We also thank J. Ferretti, M. Gasson, J. Kok, L. McKay, and G.Venema for providing us with strains, phages, and cloning vectorsthat were essential to the conduct of this study.

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

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