the bacteriophage resistance plasmid ptr2030 forms high-molecular-weight multimers in lactococci

PLASMID 25, 105-l 12 (1991)

The Bacteriophage Resistance Plasmid pTR2030 Forms High-Molecular-Weight Multimers in Lactococci’

COLINHILL,*LYNETTEA.MILLER,*ANDTODD R. KLAENHAMMER*,~.* Departments oj’*Food Science and tMicrobiology. Southeast Dairy Foods Research Center,

North Carolina State University, Raleigh, North Carolina 2769.5-7625

Received October 25, 1990; revised January 1, 199 1

Lactococcus lactis ME2 can transfer a 46-kb plasmid, pTR2030, which encodes abortive phage infection (Hsp) and restriction/modification (R/M) activities. pTR2030 can be detected as a monomeric plasmid in transconjugants at low copy number, but not in ME2. pTR2030-specific probes were cloned and used to determine the location of the element in ME2. No homology was observed between these pTR2030-specific probes and the CsCl-purified plasmid content of ME2. However, probes specific for pTR2030 hybridized strongly to a high-molecular- weight moiety, and not to chromosomal DNA, in total DNA isolated by a gentle lysis procedure. The absence of junction fragments indicates that pTR2030 forms high-molecular-weight multimers in lactococci. A phage-sensitive derivative of ME2, L. lactis N 1, is cured of pTR2030 and no longer possesses the high-molecular-weight species. When pTR2030 was reintroduced to N 1 via conjugation, an MEZ-like phage-insensitive phenotype was restored. pTR2030 could remain as a detectable monomeric plasmid in the N 1 transconjugants or could revert to the high-molecular-weight structure. 0 1991 Academic Press, Inc.

The conjugative bacteriophage resistance plasmid pTR2030 (46 kb) can be detected at low copy number in transconjugants result- ing from matings in which Lactococcus lactis ME2 is the donor (Sanders and Klaenham- mer, 1984; Sing and Klaenhammer, 1986; Steenson and Klaenhammer, 1986). Trans- conjugants are screened for acquisition, via donation, of the nonconjugative Lac+ plasmid pTR1040. Lac+ transconjugants are then scored for the phage resistance phenotypes associated with pTR2030, abortive phage infection (Hsp+) and restriction/modification (R+/M+) (Hill et al., 1989a,b, 1990; Jarvis, 1988; Jarvis and Klaenhammer, 1986). Linkage of the phage resistance and conjugative determinants to a 46-kb extra-

’ Paper 12296 of the Journal Series of the North Caro- lina Agricultural Research Service, Raleigh, NC 27695- 760 1. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Service of the products named, nor criticism of similar ones not mentioned.

’ To whom reprint requests should be addressed.

chromosomal element, pTR2030, has been confirmed by plasmid analysis of transconjugants, subcloning, and expression of both Hsp and R/M resistance mechanisms (Hill et al., 1989a,b, 1990). pTR1040 is mobilized from pTR2030-bearing transconjugants at higher frequencies than from ME2 (Klaen- hammer and Sanozky, 1985). This suggests that the mobilizing element may have under- gone alterations during the conjugal event which enhanced either its transfer ability or its ability to mobilize pTR 1040. However, due in part to plasmids of similar sizes in the original donor strain ME2, and in part to the presence of multiple homologies between pTR2030 and both ME2 chromosomal and plasmid DNA, the presence or absence of pTR2030 as a 46-kb element could not be confirmed previously in this background.

In this study we describe the construction and use of specific probes from pTR2030, including two internal fragments from the Hsp and R/M determinants, to identify an extrachromosomal location, but multimeric form, of pTR2030 in ME2. pTR2030 sequences

105 0147-619X/91 $3.00 Copyright 0 1991 by Academic Press. Inr All rights of reproduction tn any form reserved.

106 HILL, MILLER, AND KLAENHAMMER

TABLE 1

BACTERIALSTRAINSANDPLASMIDS

Strain or plasmid Relevant characteristics” Source or derivation

L. lactis ME2 Nl NCK313 NCKI NCK274 NCK275 LM2345

E. coli DHI Plasmids

pBluescript pTRK73 pTRKlO0 pTRKlO1 pTRK144 pTR2030 pTR1040

Prototype phage-insensitive strain Phage-sensitive derivative of ME2 Lac-, $15, derivative of Nl MG1363 (pTR2030, pTR 1040), Hsp+, Lac+ NCK3 13 (pTR2030-HMW, pTR1040) Hsp, La? NCK3 13 (pTR2030-46 kb, pTR 1040) Hsp’, Lac+ spc4, ~$5 derivative of LM0230 Transformation host

AP’ Ap’, 456-bp internal hsp gene fragment Ap’, 2.0-kb Hind111 pTR2030: :pBluescript Ap’, 0.2-kb Hind111 pTR2030: :pBluescript Ap’, 1.4-kb Xba l-&l1 pTR2030 fragment Hsp+, R+/M+, Tra+ Lac+

Sanders and Klaenhammer, 1983 Sanders and Klaenhammer, 1983 This study Hill et al., 1989b Transconjugant, NCKl X NCK3 13 Transconjugant, NCK 1 X NCK3 13 Anderson and McKay, 1984 Hanahan, 1983

Stratagene, La Jolla, CA This study This study This study This study Klaenhammer and Sanozky, 1985 Klaenhammer and Sanozky, 1985

a Lac, lactose-fermenting ability; rif; resistance to 25 &ml rifampin; Hsp, phage resistance; spc, resistance to 300 &ml spectinomycin; Ap’, resistance to ampicillin; R/M, restriction and modification activity; HMW, high-molecular-weight form of pTR2030.

were shown to be absent in the ME2 derivative, Nl. The fate of pTR2030 upon introduction to Nl and its contribution to the phage insensitivity of ME2 are also examined.

MATERIALS AND METHODS

Bacterial Strains and Plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. Lactococcal strains were propagated at 30°C in M 17 broth (Terzhagi and Sandine, 1975) or Ml7 + 0.5% glucose where appropriate (M17G). Antibiotics were added in the following con- centrations: rifampin at 25 pg/ml and spectinomycin at 300 rg/ml.

Molecular Cloning and DNA Manipulations

General procedures for DNA manipulations, cloning, and transformation were es- sentially as described in Maniatis et al.

(1982). Treatment of plasmid DNA with ExoIII and mung-bean nucleases to generate deletions was performed as outlined in the protocol provided by Stratagene (La Jolla, CA). The cloning vector pBluescript (KS+) was also obtained from Stratagene. Esche- richia coli DH 1 was used as a transformation host for pBluescript and derivatives.

Lactococcal Total DNA Isolations

Plasmid DNA was isolated from lactococci as described by Anderson and McKay ( 1983) and purified by cesium chloride-ethidium bromide (CsCl-EB) density gradients. A gentle, nondenaturing protocol was employed in this study to isolate total DNA; 1.5 ml of an overnight culture was collected and resuspended in 250 ~1 of lysis solution (6.7% sucrose, 50 mM Tris, 1 m&r EDTA, pH 8.0). The suspension was warmed to 37°C before the addition of 10 ~1 of a 100 mg/ml stock solution of lysozyme (Sigma Chemical Co.,

pTR2030 FORMS HIGH-MOLECULAR-WEIGHT MULTIMERS 107

St. Louis, MO). After 15 min at 37°C SDS3 (20%) was added to a final concentration of 1.0%. The solution was incubated at 60°C after addition of 20 ~1 of Proteinase K (20 mg/ml, Sigma Chemical Co.) for 20 min. Two successive phenol extractions (3% NaCl- saturated) were followed by a single extrac- tion with chloroforrnisoamylalcohol (24: 1). The DNA was precipitated by the addition of 2 vol of cold 95% ethanol and resuspended in 500 ~1 of 0.3 M Na acetate. A second ethanol precipitation was performed and the result- ing DNA pellet was resuspended in 200 ~1 of sterile water. Approximately 5- 10 ~1 ( l-3 pg) was sufficient for restriction and hybridization experiments.

Southern Hybridization

Plasmids and restriction fragments were separated on 0.8% agarose gels, depurinated (0.25 N HCl for 15 min), capillary-transferred to GeneScreen Plus nylon membrane, and hybridized as recommended by the manufac- turer (NEN Research Products, Boston, MA). DNA was labeled with [32P]dCTP by the random primer protocol (Amersham In- ternational, UK) and hybridizations were performed at 68°C. Three washes were uti- lized: 2~ SSC, 0.5% SDS for 5 min at room temperature; 2~ SSC, 0.1% SDS for 5 min at room temperature; and 0.1 X SSC, 0.5% SDS at 68°C for 2 h. The membranes were ex- posed to XR-Omat X-ray films (Kodak, Rochester, NY) at -70°C with intensifting screens.

Bacteriophage Sensitivities

Adsorption studies were performed as described by Sanders and Klaenhammer ( 198 1). Inhibition of cell growth by phage 4 18 was examined by adding 10 ml of M 17G, 0.1 ml of an overnight culture, and 50 ~1 of 1 M CaCl, to a sterile cuvette. After incu- bation at 30°C for 30 min, 0.2 ml of 4 18 was

3 Abbreviations used: SDS, sodium dodecyl sulfate; EB, ethidium bromide; HMW, high molecular weight.

added. Cell growth was monitored by mea- suring the optical density at 600 nm (OD,,) of the cultures at selected time intervals.

Conjugation Experiments

Plasmids were transferred on solid surface agar matings as previously described (Klaen- hammer and Sanozky, 1985). Spot matings were performed by overlaying 10 ~1 of recipient cells (late log phase) with 20 ~1 of donor (late log phase) on a nonselective agar plate. After growth for 18 h, the cell mixture was streaked on selective agar. Transconjugants were evident as large yellow colonies appear- ing within the smaller white streak (Steele and McKay, 1989).

RESULTS

L. lactis Nl Is Cured of pTR2030

L. lactis N 1 is a derivative of ME2 that has been cured by growth at high temperatures of the resident 46-kb plasmid pME0030 (Sanders and Klaenhammer, 1983). Correla- tive evidence has linked pME0030 with inter- ference of phage adsorption. Nl exhibits an unusual clumping phenotype, typically associated with highly conjugative plasmids (An- derson and McKay, 1984; Gasson and Da- vies, 1980). However, N 1 transfers pTR 1040 (Lac+) at extremely low frequencies (<lo-’ per input donor) and pTR2030 is never detected in N 1 -derived transconjugants (Hig- gins et al., 1988). Both plasmid and total DNAs from ME2 and N 1 were probed with a pBluescript derivative (pTRK10 1) which contains a 0.2-kb Hind111 fragment of pTR2030 cloned in a shotgun experiment. No homology to the Nl plasmid or total DNA nor to the ME2 CsCl-purified plasmid complement was detected (Fig. 1). Therefore, in addition to the loss of pME0030, L. lactis Nl is missing at least part of the pTR2030 genome.

To assess the extent to which pTR2030 sequences were missing from Nl, three addi- tional pTR2030 fragments were cloned and


FIG. 1. Southern hybridization of L. lactis ME2 and N 1 DNA with “P-labeled pTRKl0 1. Lane 1, pTR2030- Aval/BglII; lane 2, pTR2030-EcoRl; lane 3, uncut ME2 plasmids; lane 4, uncut Nl plasmids; lane 5, ME2 total DNA-EcoR 1; lane 6, N 1 total DNA-EcoR 1.

used to probe ME2 and N 1 DNAs. pTRKlO0 was also constructed by shotgun cloning HindIII-digested pTR2030 DNA in pBlue- script. pTRK73 is a pBluescript derivative containing a 456-bp fragment internal to the abortive infection gene, hsp (Hill et al., 1990). A third probe, pTRK144, consists of a 1.4-kb Xba 1 -Bcl 1 fragment from within the R/M determinants, also cloned in pBlue- script. The approximate positions of the probe fragments are indicated in Fig. 2. All three probes were labeled and hybridized against CsCl-EB-purified and total DNAs from ME2 and N 1. Similar results were obtained for pTRK 100, pTRK73, and the meth- ylase gene (data not shown). No homology was detected in either case against Nl DNA or to the CsCl-EB-purified plasmid fraction of ME2. This indicates that the entire pTR2030 element has been lost in Nl .

pTR2030 Is a High-Molecular- Weight (HA4 W) Molecule

In all cases, using single or multiple probes, identical patterns of homology were obtained against both the pTR2030 plasmid isolated from otherwise plasmid-free transconjugants and the ME2 total DNA preparations (data not shown). This result implies that pTR2030 is not integrated into the chromosome in ME2, since such an event would generate junction fragments. Such fragments were never observed, even on prolonged ex- posure of the autoradiogram, suggesting an extrachromosomal location for pTR2030.

When undigested total ME2 DNA was slowly electrophoresed into the gel (25 V for 16 h) and probed with pTRKl44, a faint signal which migrated only a short distance into the gel was observed (Fig. 3, lane 4’). This signal was obviously separate from the chromosomal fraction, which migrated consider- ably further into the gel (Fig. 3, lane 4). Treat- ment with BamHl resulted in a slight in- crease in the amount of signal detected, including a signal which comigrates with lin- earized pTR2030 DNA. Digestion with Xhol, which cuts once within pTR2030, re-

Xhol ! Aval

FIG. 2. Restriction site map of pTR2030, showing the approximate locations of the probes used in this study. A, pTRK73; B, pTRK144; C, pTRK100; D, pTRK101.


sults in the complete loss of the HMW moiety and the appearance of a 46kb band consistent with linear pTR2030 (lane 6’). The amount of signal in lane 6’ is significantly higher than the amount detected in lanes 4’ and 5’, even though identical amounts of DNA were loaded in each case. It appears that digestion with an enzyme that cuts within pTR2030 was necessary for the DNA to enter the gel. Since no junction fragments were observed, and no homology to CsCl- EB-purified DNA was detected (Fig. 3, lanes I’, 2, and 3’) we conclude that the HMW DNA is composed of pTR2030 genomes repeated in tandem.

Fate of pTR2030 upon Introduction to Nl

A Lac- variant of N 1 cured of pTR 1040 (Lac+) was selected and marked with rifampin resistance ($15); the derivative was designated NCK3 13. L. lactis MG1363

FIG. 3. Composite photograph of agarose gel and auto- radiograph of total and C&l-EB-purified DNA from L. lactis ME2 probed with “P-labeled pTRK144. Lanes 1 and 1’, uncut ME2 plasmids; lanes 2 and 2, ME2 plasmids cut with BamH 1; lanes 3 and 3, ME2 plasmids cut with Xho 1; lanes 4 and 4’, uncut total ME2 DNA; lanes 5 and S, total ME2 DNA cut with BumH I ; lanes 6 and 6, total ME2 DNA cut with Xhol Size standards were also present on the gel and autoradiogram (not shown).

FIG. 4. Composite photograph of agarose gel and auto- radiograph of CsCl-EB-purified DNA from L. lactis ME2 and derivatives probed with ‘*P-labeled pTRK 144. Lanes 1 and l’, ME2; lanes 2 and 2’, Nl; lanes 3 and 3’, NCK275; lanes 4 and 4’, NCK274; lanes 5 and 5’, control pTR2030 DNA.

(pTR1040, pTR2030) was used as a donor to transfer Lac+ to NCK3 13 recipients. Of 14 randomly chosen Lac+ transconjugants, 10 had acquired pTR2030 in addition to pTR 1040. The presence of pTR2030 was de- termined by probing Sau3A-digested total DNAs from the transconjugants with pTRK73 (data not shown).

All 10 pTR2030-containing transconjugants transferred the La? phenotype in second-round spot matings. Two NCK3 13 transconjugants bearing pTR2030 and pTR1040 were randomly chosen for more detailed analysis of the fate of pTR2030 upon reintroduction to the NCK3 13 background. In the case of NCK275, homology to pTRK73 was observed to a ca. 46-kb band in the CsCl-EB-purified fraction (Fig. 4, lanes 3 and 3’). Therefore, the plasmid can be maintained and isolated as a monomer in the NCK3 13 background. However, we were unable to exclude the possibility that a portion of the pTR2030 content in NCK275 existed in the HMW form. The second transconjugant, NCK274, showed little homology between pTRK73 and CsCl-EB-purified cova-


lently-closed-circular (CCC) DNA (Fig. 4, lanes 4 and 4’), but gave the expected homology when the total DNA fraction was probed (data not shown). In this transconjugant, we conclude that pTR2030 returned to the HMW form and was not clearly detected as a monomeric plasmid. In solid surface agar matings, NCK275 and NCK274 transferred the Lac+ phenotype to a Lac- recipient, LM2345, at frequencies of 2 X 10e3 and 2.5 X 1 Om3 per input donor, respectively. These frequencies were in excess of those observed from ME2 (2 X 10e4). In the absence of pTR2030, the parental strain Nl failed to transfer Lac at frequencies greater than 1 O-‘.

Phage Resistance Phenotypes of NCK274 and NCK275

Both ME2 and Nl are resistant to phage 418 at 30°C (Sanders and Klaenhammer, 1983). However, Nl is sensitive to the phage at 40°C. Modified 418.Nl (propagated through Nl at 40°C) can inhibit the growth of Nl at 30°C but not of ME2 (Fig. 5). The NCK3 13 transconjugants NCK274 and NCK275 were challenged with 418.Nl to determine the contribution of pTR2030 to the phage resistance exhibited by ME2 compared with that exhibited by Nl. The pTR2030 transconjugants, NCK275 and NCK274, were resistant to lysis by 4 18.N 1 (Fig. 5), suggesting restoration of the phage-insensitive condition of L. lactis ME2. N 1 adsorbs phage $I 18 at 99%, whereas ME2 only adsorbs 50% of $18 (Sanders and Klaenhammer, 1983). The pTR2030 transconjugants of NCK3 13 also adsorb 418 at 99%, confirming that pTR2030 plays a major role in the phage insensitivity of ME2 without affecting phage adsorption.

DISCUSSION

The conjugative plasmid pTR2030 has been introduced by conjugation to a number of industrially important strains of lactococci in which it provides an extremely effective barrier against phage attack (Klaenhammer, 1987; Sanders, 1988; Sanders et al., 1986).

I I I I I I I

1 2 3 Time ih)

5 6

FIG. 5. Growth curve of Lactococcus lactis ME2, N 1, NCK275, and NCK274 at 30°C in the presence of q5 18.N 1. Phage was added to a final titer of 10’ after 1 h. n , ME2; 0, Nl; A, NCK275/NCK274.

Although many of the properties of this plasmid have been investigated, its role and location in the original source strain, L. lactis ME2, had not been elucidated. In this study we have shown that pTR2030 is present in ME2 in a HMW form. The HMW pTR2030 molecule is not amenable to CsCl-EB purification procedures. This may reflect the large size of the pTR2030 multimer or may indi- cate that the molecule is not supercoiled. In either event, a gentle nondenaturing protocol is essential to detect the HMW form, and its presence can be confirmed only through use of hybridization techniques. The absence of junction fragments, the large size of the HMW DNA, and the relatively high copy of pTR2030 sequences compared with chromosomal fragments detected in hybridization analysis (data not shown) strongly suggest that the HMW DNA is composed of multiple copies of pTR2030 repeated in tandem. Em- pirical estimates of the number of copies of


pTR2030 per unit chromosome suggest that between three and five genomes are repeated in tandem (data not shown). Similar results have been described for certain B. subtilis plasmids which can form HMW molecules upon insertion of foreign DNA (Gruss and Erlich, 1988). In that instance it was pro- posed that the HMW forms resulted from unresolved replication intermediates and were dependent on a rolling circle mode of plasmid replication.

The ability of pTR2030 to form multimers is significant from a number of standpoints. Normal plasmid-phenotype linkages are not immediately apparent and could lead to in- correct presumptions of a chromosomal location for the phage resistance genotypes, despite the conjugal nature of the phenotype. This phenomenon would also lead to unsuc- cessful attempts to isolate extrachromosomal HMW elements through CsCl-EB prepara- tive techniques. Recently, Kaletta and En- tian (1989) also described a conjugal nisin- producing element which was “nearly immo- bile on 0.7% agarose gels,” but was visible after endonucleolytic cleavage. In addition, Steele and McKay ( 1986) were able to conju- gally transfer nisin production determinants but were unable to detect plasmid DNA in transconjugants. These authors concluded that the phenotype was plasmid-coded, but was present in a form which prevented isolation by conventional plasmid purification techniques.

We have shown that pTR2030 can exist as a monomeric plasmid or in a HMW state in the NCK313 background. The transfer frequency of the Lac+ phenotype from NCK274 (HMW pTR2030) is identical to that of NCK275 (monomeric pTR2030), suggesting that the maintenance of HMW molecules is not a prerequisite for conjugation. The higher rate of transfer of pTR 1040 from NCK275 and NCK274, compared to that of ME2, presumably reflects a higher rate of transfer by pTR2030 from the NCK3 13 background. This is most probably a result of the clumping phenotype exhibited by Nl and its derivatives, which is often accompanied

by increased conjugation rates in lactococci (Gasson and Davies, 1980; Walsh and McKay, 198 1).

It is noteworthy that Nl retains a high de- gree of phage insensitivity, despite the loss of the adsorption plasmid pMEOO30 and the absence of the pTR2030-encoded restriction/ modification and abortive infection mechanisms linked to pTR2030. Nl continues to show a highly phage-insensitive state, including an active restriction and modification system, localized to the 20-MDa plasmid pTN20 (Higgins et al., 1988), and a heat-sensitive reduction in plaque size which resem- bles the Hsp mechanism encoded by pTR2030. This suggests that there are at least five distinct phage resistance mechanisms in ME2, two R/M systems of different specifici- ties (one on pTR2030, one on pTN20), two heat-sensitive abortive infection mechanisms, and a mechanism that interferes with phage adsorption. We have shown that pTR2030 plays an active role in the increased resistance to phage 4 18 exhibited by ME2 over N 1. Therefore, the major difference in phage sensitivities between ME2 and Nl, originally ascribed only to the loss of pME0030 (Sanders and Klaenhammer, 1984), is also due to the absence of pTR2030 in the sensitive derivative.

It is not immediately obvious why pTR2030 should form HMW DNA in prefer- ence to a monomeric plasmid. It may be a consequence of an unresolved replication in- termediate, but this remains to be confirmed. The isolation of isogenic backgrounds, NCK274 and NCK275, in which pTR2030 can be maintained in both forms, will allow us to assess the possible phenotypic and geno- typic benefits or disadvantages of either situa- tion.

ACKNOWLEDGMENTS

This work was supported by the Molecular Biology Program, U.S. Department of Agriculture, under Agree- ment 87-CRCR-l-2547 and, in part, by the Biotechnol- ogy Products Division of Miles Inc., Elkhart, Indiana.

112 HILL, MILLER, AI

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Communicated by Gary Dunny

the bacteriophage resistance plasmid ptr2030 forms high-molecular-weight multimers in lactococci

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