JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7573-7581 0021-9193/91/237573-09$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 23

Replication and Temperature-Sensitive Maintenance Functions of Lactose Plasmid pSK11L from Lactococcus lactis subsp. cremorist JASON S. HORNG, KAYLA M. POLZIN, AND LARRY L. McKAY* Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108-9999 Received 1 July 1991/Accepted 3 October 1991

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and TnS mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EBs than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.

the reasons for the differing abilities of L. lactis and L. cremoris strains to maintain pSK11L, we attempted to identify the pSK11L replication origin and regions involved in plasmid maintenance since these regions would be most likely to interact with host plasmid replication and maintenance functions. We report here that a 14.8-kbp PvuII fragment of pSK11L confers the replication ability and the temperature-sensitive pSK11L maintenance phenotype of L. lactis LM0230. We also report identification of several regions on this fragment which appear to affect pSK11L maintenance. Several of these regions affect pSK11L maintenance differently in L. lactis LM0230 and L. cremoris EB5.

Plasmid replication and stability have been extensively studied for several plasmids found in gram-negative species. These studies have led to identification of several plasmidencoded elements which mediate plasmid replication, control plasmid copy number, and stabilize plasmid inheritance (18, 24, 26, 30). However, little is known about host functions involved in these processes. One way to identify host factors and study their interactions with plasmid replication and stability factors could be to study plasmids which differ in replication and stabilization characteristics in closely related host strains. In addition, such studies could reveal ways in which changes in host proteins affect plasmid host range.

Lactococcus lactis subsp. lactis (L. lactis) and L. lactis subsp. cremoris (L. cremoris) have always been considered to be closely related and are distinguishable only by such characteristics as salt tolerance, maximum growth temperature, pH tolerance, and the ability to utilize arginine (11). The lactose plasmid from L. cremoris SK11, pSK11L (originally named pSK11 [10] and renamed pSK11L hereafter to reflect the lactose utilization ability encoded by this plasmid), is extremely stable in L. cremoris strains (8, 10). However, it has recently been shown in our laboratory that pSK11L displays a number of phenotypes in L. lactis LM0230 which are not observed in L. cremoris SK11 or EB5. Among these are plasmid instability and temperaturesensitive plasmid maintenance (10). To begin elucidation of

MATERIALS AND METHODS

Bacterial strains and plasmids. The L. lactis strains used in this study included LM0230, a plasmid-free, prophage-free derivative of C2 (9), and JF3216, an LM0230 transformant containing pSK11L, a lactose plasmid from L. cremoris SK11 (10). The L. cremoris strain used was EB5, a plasmidfree derivative of KR1 (3). Strains LM0230 and EB5 were grown at 32°C in M17 broth (33) supplemented with 0.5% glucose (M17-G). Transformants derived from these strains were grown at 25°C in M17-G containing erythromycin (Em) (10 jig/ml). Strain JF3216 was grown at 25°C in M17 broth supplemented with 0.5% lactose. The Escherichia coli plasmid used to clone the replication region of pSK11L was pVA891, a derivative of pACYC184 (4) containing the pAM,1 Emr-encoding gene (21). pVA891 and its derivatives were maintained in E. coli XL1-Blue (Stratagene, La Jolla,

Corresponding author. no. 19,179 of the contribution series of the Minnesota Agricultural Experiment Station, based on research conducted under project 18-62. *

t Report

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Calif.) grown at 37°C with shaking in LB broth (22) supplemented with chloramphenicol (25 ,ug/ml) or Em (30,ug/ml). DNA manipulations. L. lactis plasmid DNA was isolated by the method of Anderson and McKay (1). E. coli plasmid DNA was isolated by either an alkaline lysis procedure (22) or the rapid small-scale lysis procedure of Rodriquez and Tait (31). Large-scale plasmid isolations were followed by DNA purification through a CsCl-ethidium bromide density gradient. Restriction enzymes were purchased from Life Technologies, Inc. (Grand Island, N.Y.) or Boehringer Mannheim Biochemicals, Inc. (Indianapolis, Ind.), and digestions were performed as recommended by the manufacturers. Agarose gel electrophoresis was performed by using Tris-acetate-EDTA buffer (pH 8.0) (22) at 4.0 V/cm, followed by staining in ethidium bromide (0.5 jig/ml). DNA ligations were performed with T4 DNA ligase (Life Technologies, Inc.), and the ligated DNA was introduced by electroporation with a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) into either E. coli as recommended by the manufacturer or into LM0230 or EB5 as described by Feirtag et al. (10). When necessary, deletion derivatives of pKMP1 were constructed in E. coli and then electroporated into LM0230 and EB5. Southern hybridizations. Southern transfers were performed as described previously (29), by using Nytran membranes (Schleicher & Schuell, Inc., Keene, N.H.) prepared as suggested by the manufacturer. Probe labeling, hybridization, and detection were conducted by using the Genius kit (Boehringer Mannheim Biochemicals) as recommended by the manufacturer. DNA fragments used as probes were isolated by using DEAE-cellulose (Schleicher & Schuell) as described previously (34). TnS mutagenesis of pKMP1. Tn5 mutagenesis was performed essentially as described by de Bruijn and Lupski (6), by using bacteriophage X467, which carries TnS inserted into the rex gene, to transduce E. coli MC1061 (23) harboring pKMP1. The Tn5 insertions in pKMP1 were localized by restriction enzyme digestion with ClaI, HindIII, BamHI, and when necessary, XhoI. Inactivation of pKMP1 replication functions by TnS was determined by electroporation of pKMP1::TnS into LM0230 and noting the number of Emr transformants. The stability of Tn5 insertions during transfer from E. coli to L. lactis was verified by restriction digestions. Time course elimination of pKMP1 from L. lactis LM0230. A single L. lactis LM0230(pKMP1) colony from an M17G-Em (10 ,g/ml) plate incubated at 21°C was inoculated into 10 ml of M17-G-Em (10 ,ug/ml) broth and grown overnight at 21°C. The cells were harvested by centrifugation at 4,315 x g for 10 min, washed with deionized, distilled H20 to remove the Em, and suspended in 10 ml of fresh M17-G broth. A 10-2 dilution was then made in M17-G broth, and 1 ml (approximately 107 cells) was inoculated into each of four flasks containing 100 ml of M17-G broth, two of which also contained Em (10 ,ug/ml). One set of cultures (one with and one without Em) was grown at 25°C, and the other was grown at 37°C. Samples were withdrawn hourly from both sets of cultures, plated on M17-G agar, and incubated at 30°C. One hundred colonies were then picked from each set of plates to M17-G-Em (10 jig/ml) plates to determine the percentage of Emr cells in the cultures at each time point. Stability assays of pKMP1 and its deletion derivatives. Strains to be tested were taken from frozen stock cultures, inoculated into M17-G-Em (10 ,ug/ml) broth, and grown at 25°C to approximately 109 cells per ml. Input viable counts were determined by plating on M17-G plates, and 10-5

J. BACTERIOL.

dilutions were then made into M17-G and grown at 25, 32, 37, and 39°C for L. lactis LM0230 and 25, 32, and 37°C for L. cremoris EB5 for 24 to 48 h. Final viable counts were then determined for each temperature by plating on M17-G agar. The percentages of Emr colonies for both the input and final plates were determined by picking 100 colonies from the viable-count plates to an M17-G-Em (10 ,ug/ml) plate and scoring the number of Emr colonies. All plates were incubated at 30°C. The plasmid loss rate per generation was determined with the formula given by Roberts et al. (30). DNA sequence analysis. Double-stranded DNA sequencing (5) was performed by the dideoxynucleotide chain termination method of Sanger et al. (32) by using [ot-355]dATP (New England Nuclear, Boston, Mass.) and the Sequenase version 2.0 DNA sequencing kit (United States Biochemical, Cleveland, Ohio). Synthetic primers 15 to 20 nucleotides long were prepared by a 391 PCR-Mate EP DNA synthesizer from Applied Biosystems, Inc. (Foster City, Calif.). DNA sequence data were analyzed with IntelliGenetics sequence analysis software (release 5.4) run on a SUN workstation. Nucleotide sequence accession number. The GenBank accession number for the DNA sequence of the pSK11L replicon is M74446. RESULTS Physical and genetic map of pSK11L. To determine the physical relationship between the replication, stability, and temperature-sensitive maintenance regions of pSK11L and other pSK11L genetic loci, a restriction map of pSK11L was prepared and the positions of known genetic loci were determined (Fig. 1A). pSK11L was 47.3 kbp long. The replication region and regions affecting stability were localized on the basis of the studies described below for the 14.8-kbp PvuII fragment of pSK11L cloned in pKMP1. The lactose operon was determined to be within the 13-kbp Bcll fragment by subcloning (25). One end of the operon was found to extend beyond the BgIII site because of the inability to recover Lac' transformants when the smallest BglII fragment was deleted from pSK11L. The other end of the operon included the 4.3-kbp XhoI fragment, since this fragment encodes the phospho-,-galactosidase gene of the operon (27). The large size of the pSK11L lactose operon is in accordance with previously characterized lactococcal lactose operons which have been shown to contain not only the three genes of the lactose phosphoenolpyruvate phosphotransferase system but also the tagatose-6-phosphate pathway genes and an open reading frame (ORF) with an undetermined function (35). pSK11L contained two lactococcal insertion elements. Southern hybridization using the internal 0.7-kbp DraI fragment of ISSJ (29) (data not shown) revealed that pSK11L contained one copy of ISSJ and a region with homology to ISSJ located on opposite sides of the lactose operon (Fig. 1A). Southern hybridization with the internal 0.7-kbp XbaI fragment of IS981 (32) (data not shown) revealed that pSK11L also contained a copy of this insertion element immediately upstream of or slightly overlapping the lactose operon (Fig. 1A). Cloning of the pSKllL replication origin. pVA891 is a derivative of E. coli plasmid pACYC184 (4) containing the pAM31 Emr gene which is expressed in lactococci (21). Several attempts to transform pVA891 into L. lactis LM0230 verified that this plasmid does not replicate in this host, as no Emr transformants were recovered. To clone the replication origin, pSK11L was digested separately with Clal, XbaI,

LACTOSE PLASMID pSK11L FROM L. LACTIS SUBSP. CREMORIS

VOL. 173, 1991

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FIG. 1. (A) Restriction map of pSK11L and locations of known genetic elements. (B) Restriction map of pKMP1. The single line in panel B indicates pVA891; the striped block in panel B indicates the 14.8-kbp PvuII fragment from pSK11L. Abbreviations: ori, replication region; ts, temperature-sensitive maintenance; Lac, lactose operon; stb, regions contributing to plasmid stability; (EC), E. coli; EmR, Emr; CmR, chloramphenicol resistance. (PvuII) indicates that additional PvuII sites were not mapped. The arrows indicate the orientation of the open reading frames.

and PvuII and shotgun cloned into pVA891 and Emr LM0230 transformants were selected. No transformants were recovered by using ClaI fragments, despite two transformation attempts. Two transformants were recovered from the XbaI cloning experiment, but these contained no detectable plasmid DNA and were not examined further. However, one clone, which contained the 14.8-kbp PvuII fragment of pSK11L ligated to pVA891, was recovered from the PvuII cloning experiment. This plasmid was named pKMP1 (Fig. 1B) and presumably carried the replication region of 0kb

pSK11L. The cloned 14.8-kbp PvuII fragment did not appear to encode the temperature-sensitive inhibition of host growth function previously reported by Feirtag et al. (10), as L. lactis LM0230 cells carrying pKMP1 were able to grow at 38 to 40°C. Localization of the pKMP1 replication region by deletion analysis and TnS mutagenesis. A number of deletion derivatives of pKMP1 were constructed in E. coli and screened for the ability to replicate in L. lactis LM0230 (Fig. 2). Successful transformation of LM0230 with a deletion derivative 10

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FIG. 2. Localization of the pKMP1 replication region by deletion analysis and TnS mutagenesis. The solid single lines indicate plasmid DNA remaining after deletion. Filled circles represent Tn5 insertions unable to replicate in L. lactis LM0230, and open circles represent Tn5 insertions able to replicate in LM0230. The ability of each deletion derivative to replicate in L. lactis LM0230 (rep) is shown as a minus or plus sign on the right. The deduced minimum replication region is flanked by vertical dashed lines. Restriction sites: P, PvuII; E, EcoRI; H, HindIlI; Ev, EcoRV; X, XbaI; A, AvaI; Bc, BclI; Sc, ScaI; Nh, Nhel; C, CMaI; Nd, NdeI; Se, SpeI; B, BamHI; S, Sall; N, NcoI.

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indicated that the deletion had not inactivated the lactococcal replication region. Failure to recover Emr transformants implied that the lactococcal replication region had been disrupted. As shown in Fig. 2, deletion of the region to the left of the Scal site (pKMP1-SP) and deletion of the small 0.7-kbp SpeI fragment (pKMP1-Se) did not prevent replication. However, deletion of the region to the left of the NheI site (pKMP1-Nh) and deletion of the small 0.8-kbp NdeI fragment (pKMP1-Nd) abolished replication in LM0230. Therefore, the replication region was localized to the 2.3-kbp Scal-Spel fragment of pKMP1. This was confirmed by subcloning this fragment into pVA891 and transforming LM0230 to Emr with the resulting plasmid (pKMP1-SS). Tn5 mutagenesis was then used to confirm the location and further localize the pKMP1 replication region (Fig. 2). One hundred and two TnS insertions into pKMP1 were mapped, of which 58 occurred in the 14.8-kbp PvuII fragment. Forty-two insertions were in the 6.9-kbp XbaI-ClaI fragment, and two were in the adjacent 2.6-kbp ClaI fragment overlapping the replication region. A 2.4-kbp region overlapping the replication region was devoid of TnS insertions. The reasons for the uneven distribution of TnS insertion sites and the failure to obtain TnS insertions in the 2.4-kbp region are unknown. Twenty-eight pKMP1::TnS derivatives were screened for the ability to replicate in LM0230 by attempting to recover Emr LM0230 transformants containing these plasmids. The presence of pKMP1::TnS in Emr transformants was verified by restriction digestion analyses. As indicated in Fig. 2, only TnS insertions in the 2.3-kbp ScaI-SpeI fragment abolished pKMP1 replication ability. However, the insertion just to the right of the Scal site did not affect replication, indicating that the replication region did not span the entire 2.3-kbp region but began slightly to the right of the ScaI site. These results verified that the pKMP1 replication region was in the 2.3-kbp ScaI-Spel fragment. Transformation of LM0230 with certain pKMP1::Tn5 derivatives with insertions in the replication region did produce Emr transformants; however, the frequency was significantly lower than pKMP1 with Tn5 insertions in other regions and these transformants contained either no detectable plasmid DNA or a deleted and/or rearranged form of pKMP1 (data not shown). DNA sequence analysis of the pSK1lL replicon. The entire 2.3-kbp ScaI-SpeI fragment was sequenced in both directions and found to contain two complete ORFs (ORFi and ORF2) and one incomplete ORF (ORF3) (Fig. 3A). ORFi was short (381 bp), and its 5' region did not contain sequences resembling the lactococcal ribosome-binding site (RBS) and promoter sequences (20). ORFi did not appear to be necessary for pSK11L replication, since TnS insertions within this region did not inactivate pKMP1 replication (Fig. 2). ORF3 was preceded by the lactococcal RBS and promoter sequences; however, its coding sequence was truncated at the Spel site. Truncation or deletion of ORF3 did not inactivate replication but did affect plasmid stability in both L. lactis LM0230 and L. cremoris EB5, as demonstrated below. The overall organization (panel B) and the DNA sequence (panel C) of ORF2 and its 5' upstream region are shown in Fig. 3. ORF2 is 1,155 bp long and has the capacity to encode a polypeptide (designated Rep) of 385 amino acids with a molecular weight of 45,639. The putative Rep protein is necessary for pSK11L replication, as TnS insertions within its coding region abolished the pKMP1 replication ability in L. lactis LM0230. ORF2 was preceded by the canonical RBS and -10 and -35 promoter sequences of lactococci (20). In

J. BACTERIOL.

addition, an upstream TG sequence conserved in lactococcal promoters was separated by 1 bp from the -10 sequence. As illustrated in Fig. 4, the overall organization of the pSK11L replicon is remarkably similar to that of pCI305, an 8.7-kbp cryptic plasmid from L. lactis UC317 (13). As in pCI305, the sequence upstream of the pSK11L rep gene (designated ori; nucleotides 1 to 288) did not encode a polypeptide but contained several salient structural features typical of a replication origin. These included an 11-bp imperfect direct repeat (Ia and Tb; nucleotides 125 to 149) separated by 3 bp and another 22-bp sequence tandemly repeated three and one-half times (IIa to IId; nucleotides 205 to 283). All of the 22-bp repeats were perfect, except for the second set, which contained a single mismatch at position 1 (T replaced by C). Similar repeated structures have been implicated in mediation of plasmid incompatibility and regulation of plasmid copy number (18, 19, 26). pSK11L and pCI305 replicons shared 59% DNA sequence identity and 54% amino acid identity for the Rep protein (data not shown). In pCI305, two potential hairpin loop structures were found upstream of repB; one involved the -10 and - 35 promoter regions, and the other separated the -10 promoter sequence from the RBS (13). No similar stable stem-loop structures were possible in the corresponding regions of pSK11L, although two imperfect inverted repeats were identified, one of which flanked the -10 promoter region and the other of which flanked the -35 promoter region. On the other hand, pSK11L contained a stable stem-loop structure (nucleotides 90 to 138; AG = -11.8 kcal [1 cal = 4.184 J]/mol) which included the first set (Ta) of the 11-bp direct repeat. The pSK11L replicon also shared 51% DNA sequence identity with pRAT11 (data not shown), a low-copynumber kanamycin resistance plasmid from Bacillus subtilis (16). pRAT11 also contains three and one-half direct repeats of a 24-bp sequence in its ori (repA) region. Maintenance of pKMP1 is temperatu're sensitive. Maintenance of pSK11L has been shown to be temperature sensitive in L. lactis LM0230 (10). To determine whether pKMP1 encodes the temperature-sensitive maintenance phenotype and the plasmid replication function of pSK11L, the time course elimination of pKMP1 was measured at 25 and 37°C (Fig. 5). In the absence of Em, the proportion of Emr cells decreased at about the same rate at both temperatures, resulting in 82 and 83% Emr cells after three generations at 25 and 37°C, respectively. However, after 3 generations, the percentage of Emr cells decreased faster in the 37°C culture than in the 25°C culture, resulting in 38 and 72% Emr cells, respectively, after 8.3 generations. It was possible that the decrease in the proportion of Emr cells was due to overgrowth by Ems cells present in the initial inoculum (approximately 10%) rather than to the loss of pKMP1. To rule out this possibility, cultures grown in the presence of Em were also assayed for percent Emr cells. As Em (10 ,ug/ml) inhibited growth of Ems cells (data not shown), the proportion of Ems cells should decrease during growth in the presence of Em unless additional Ems cells are produced by plasmid loss during growth of the Emr cells. For LM0230(pKMP1) grown for 8 h at 25°C in the presence of Em, the proportion of Ems cells fluctuated between 9 and 13%. At 37°C in the presence of Em, the proportion of Ems cells increased from the initial 8% to 40% after 8 h (data not shown). These results indicated that Ems cells were generated during growth of LM0230(pKMP1) and supported the conclusion that the decrease in percent Emr cells in the absence of Em was at least partially due to pKMP1 instability rather than solely to overgrowth by Ems cells present in

VOL. 173, 1991

LACTOSE PLASMID pSK11L FROM L. LACTIS SUBSP. CREMORIS

A.

1.1 kb

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413

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512

521

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90

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80

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170

160

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ATCGGGTATC AAGTCATGGC TAAATAATCA

120

ACTAGCAATT CGGGTATTTT AMAAAAAGAA AATTGGGACT CCTTAGAGTC

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60

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200

230

240

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temperature-sensitive maintenance phenotype as well as the plasmid replication function of pSK11L. Regions of pKMP1 contributing to plasmid stability and temperature-sensitive maintenance in L. lactis LM0230. Regions affecting plasmid stability in LM0230 were identified by comparing the loss rates of pKMP1 and its deletion derivatives from L. lactis LM0230 during growth at 25°C (Fig. 6). These results implicated several regions as potentially being involved in stabilization or destabilization of pKMP1 in LM0230. Deletion to the first BclI site (pKMP1Bc) had no significant effect on plasmid stability. However, deletion of three regions, 1 (pKMP1-A), 3 (pKMP1-Se), and 5 (compare loss of pKMP1-SPH with that of pKMP1-SP), resulted in a two- to threefold increase in plasmid loss, suggesting that these regions stabilize pKMP1 in LM0230. A region which potentially destabilizes pKMP1 in LM0230 was also identified. Deletion of region 2a (pKMP1-E) resulted in a plasmid significantly more stable than pKMP1. Regions of pKMP1 involved in temperature-sensitive

A

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GAT CM GCT

782 ACG Thr

TTT CAT Phe His

the inoculum and that the degree of instability was greater at 37°C than at 25°C. pKMP1, therefore, appeared to encode a

GCA

GTT

485

GCT TTT TTC AGA ATA GAG AM Ala Phe Phe Arg Ile Lys Glu Lys

728

CGT Ile Arg

RBS

and deduced amino acid

sequence of the pSK11L replication region. The three identified ORFs are shown in panel A. The truncated ORF 3 is indicated by a broken arrow. The overall organization of the pSK11L replicon is depicted (not drawn to scale) in panel B. Arrows with striped heads indicate the 11-bp direct (Ia and Ib) and 22-bp tandem (Ila to Ild) repeats. Arrows with solid heads indicate inverted repeats. The -10 and -35 regions of the rep promoter and the RBS are depicted as striped, solid, and open blocks, respectively. The DNA and predicted amino acid sequences for ORF2 (putative Rep protein) are shown along with its upstream region in panel C. The 11-bp direct repeats (Ia and Ib) and the three and one-half tandem repeats (Ila to Ild) are indicated by arrows below the sequence. Arrows above nucleotides indicate inverted repeats. Also shown are the promoter sequences (-10 and -35) and the RBS for ORF2. sequence

476

575

ATA

GTGTTTCGTG TTATTATTTA TTTAAATCAT AAAAGGAGTG GATT -10

FIG. 3. DNA

280

449

440 TCA

GAT

AAAACTGTG TATAGTCCTA

llC

Ilb

Ila

270

431

His Asn Asp Leu Ile Ser Ser Val Ala

MAT GAT AMA CAT CGT CGT Asp Asn Asp Lys His Arg Arg

674

AGT TCA Ser Ser

CGG ATA GTA CCT ATA CCT TAT GTT GAG TGG MAT GAT TAT MYT AMA GTT TTG Arg Ile Val Pro Ile Pro Tyr Val Glu Trp Asn Asp Tyr Asn Asp Lys Val Leu

210

AGGGGTAACA GGATTATAGT

lb 220

Glu Gln

140

CCTTAATT ATTTTATATY _1

190 AGTGTTTGCA

GA CA

395

386

AAA CAA ATA CAG GAA ATA Lys Gln Ile Gln Glu Ile AGC

ATT

GAT

620

C.

AAC GAT TTA

467

AMA ATG Leu MET

ACA

Arg Asn

CAT

GAG

GCA

Arg Lys Val Ala Glu

*EF-3

377

368

ATG CAA AAG ATA GAC ACA GGA GAA AGAAAT

7577

Leu

1421

MAT GAT Asn Asp

1466

CTT

MT

Asn

GM ACG Glu Thr

TTA Leu

MA

MGO

Lys Lys

MT

Asn

1475

TTG AGT TCA AYU Ser Arg Leu Ser

7578

J. BACTERIOL.

HORNG ET AL.

A. Sri

_-Rjjj4-57bp-_ 5I-lE-3hp_Ejsbp-*

I

pCI3S5 5-IIj.4--3hbp

PSKlII

Plasmid pSKI I pCl3US

Designatlio In

pSKI I

lb

pEISOS

lib

I Ic lid

11

lib

Iic

Dlb

llec

IIIdl-*-a

lid |*-Sbp-

r

5

*NI sequence

5'-T1ETTETfTT-3' 5-TTUTITEm-3' 5TETUTETITNT-3' _ 5 -1TETUTm-3

pSKI 1 pC 1305 pSKI 1

5'-TITISTCCTICIIIIIICTGTS-T

pCl3O5

T-TINTUTUCCICIIIIIIIIICT6T6-3

pSKI 1

5'-TRTE6TCC1ICIUEIRICT6T6-3

5-TIT1TICCUUCIIIIIICTETS-3 S'-CSTa6TCCTSCSUEIIECT6T6-3

pC30O5

T-CTIlClCCECIIIIIIIICTETM-3

pSKI I

5-TETSI6TCCIIC8I-3'

pC1305

3b

repm

5-CETUTECCAIC-3

B. PCl305

5-TmT-1s6bp-T.TRhTS-60bp

pSKI I

5-

80866I6*-4

hp 83 bp

repi, I 15 bp

6T6TT4- 1l7bp-*TIIIT4- 13 bp -_ 11166164- 6 bp-* rep, 1155 -35 Res -to

3'

pbp[-

FIG. 4. Comparison of the DNA sequence and organization of the pSK11L replicon with that of pCI305. Panel A details the organization and DNA sequence differences between the pSK11L and pCI305 replication origins (ori). Nucleotide differences in the direct (Ia and Ib) and tandem (Ila to Ild) repeats are underlined. Panel B compares the organization of the rep promoter regions and RBSs of pSK11L and pCI305.

70-

|

60

-

40

-

30

.

.

0

4

2

No. of

6

8

generatio

FIG. 5. Time course elimination of pKMP1 from L. lactis LM0230 during growth at 25 and 37°C without Em. The number of generations and the percentage of Emr cells were determined as described in Materials and Methods.

maintenance were determined by comparing the loss frequencies of pKMP1 and its deletion derivatives at 25, 32, 37, and 39°C (Fig. 6). No significant difference between loss frequencies at 25 and 32°C was observed (data not shown). However, plasmid loss rates increased an average of 6-fold at 37°C and at least 10-fold at 39°C compared with loss frequencies at 25°C for all plasmids except pKMP1-C and pKMP1-SS. Therefore, the temperature required for expression of the temperature-sensitive maintenance phenotype in L. lactis LM0230 appeared to lie between 32 and 37°C. Whether pKMP1-SS also displays temperature-sensitive maintenance was uncertain, since pKMP1-SS was unstable, even at 25°C. In contrast, pKMP1-C, although slightly less stable than pKMP1 at 25°C, showed no significant increase in loss frequency at 39°C. pKMPl-C differed from pKMP1 and other deletion derivatives in that it lacked region 4 (1.2-kbp ClaI-HindIII fragment). These results suggested that region 4 is responsible for temperature-sensitive maintenance of pKMP1 in L. lactis LM0230. Regions of pKMP1 contributing to plasmid stability and detection of temperature-sensitive maintenance in L. cremoris EB5. Examination of the loss rates of pKMP1 and its deletion derivatives from L. cremoris EB5 during growth at 25°C (Fig. 6) revealed that except for plasmids lacking region 2b (pKMP1-N) or 2d (pKMP1-SP), pKMP1 and all of the pKMP1 derivatives tested were more stable in L. cremoris EB5 than in L. lactis LM0230. Deletion of several regions had different effects in EB5 than in LM0230. Deletion of

VOL. 173, 1991

LACTOSE PLASMID pSK11L FROM L. LACTIS SUBSP. CREMORIS E

Pi II

-

pKMP 1

-I

Ev S H Sc HI I I -

-

Il -

Bc

x

c

a

NIESC

'EH EIA ...

-

.

Nhl

Se

Se

NdN

..

: :

::

::

~1

aa:::::

~I

:::

C Il

'

Regions affecting stsbilitg in LM10230

7579

: 3:

::

H

p

I

~~~~~~~ ~~SCALE-1 khl : 4 :

I

X loss/generation :

Regions affectiag stabilitg In EB5

::

2a:h::2d:

:

5

L110230 252..

pKMP I pKMP 1 -X pK11P 1 -Bc pKMP 1 -A

.

37

EB5 39

25-

37

.:

3.3

24.6 p37.3

0.02 0.05

2.8

21.0 337.6

0.4

0.8

..

..

2.5

ND

23.0

ND

ND

9.2

32.3 a35.1

3.6

8.7

pKMP 1 -SX pK1P 1 -E

9.5

24.6 >37.0

3.4

4.8

1.4

1 0.5 >26.9

0.22 0.74

pKMP I -N pK11P l-B pKMP I -BS

0.98

ND

ND

ND

pKMP I -E pKMP 1 -SP pKMP 1 -Se pKMlPI-SBH pKMP 1 -C pKMlPI-SPH pKMP 1-SS

1.3

7.5

19.9

.. .. .. a~~~~.

v

1.6

6.2

22.5

5.2

4.1

10.3 >28.1

0.2

0.96

3.5

7.7

0.23 0.34

1.6

9.6 a22.6

2.6

6.3

8.0

ND >20.6

ND

ND

9.0

ND

28.3

6.8 25.5

5.9

ND

7.1

ND

ND

4.5

ND

30.3

ND

ND

15.2

ND 326.4

6.6 15.0

FIG. 6. Loss frequencies of pKMP1 and its deletion derivatives from L. lactis LM0230 and L. cremoris EB5 during growth at 25, 37, and 39°C without Em. Except for pKMP1, the solid single lines represent regions remaining after deletion. The open block on the map indicates the pSK11L replication region. Open bars below the map indicate regions whose deletion decreased stability. Solid bars indicate regions whose deletion increased stability. The speckled bar indicates the region whose deletion apparently abolished the temperature-sensitive maintenance phenotype. The plasmid loss rates per generation represent averages from at least two independent experiments. ND, not determined. Restriction site abbreviations are the same as in Fig. 2.

regions 2b (pKMP1-N), 2d (pKMP1-SP), and 6 (pKMP1-X), which had no effect on stability in LM0230, caused greater than 10-fold increases in plasmid loss in EB5. Deletion of region 2c (pKMP1-B) caused decreased plasmid loss in EB5, while deletion of this region had no effect in LM0230. However, regions which behaved similarly in EB5 and LM0230 were also identified. Deletion of regions 1 (pKMP1-A) and 3 (pKMP1-BS) destabilized pKMP1 in both LM0230 and EB5, while deletion of region 2a (pKMP1-E) stabilized the plasmid in both strains. Deletion of region 6 and regions 3, 4, and 5 together resulted in a plasmid (pKMP1-SBH) as unstable as the minimum replicon (pKMP1-SS), despite the presence of stabilizing region 1. pKMP1-SPH and pKMP1-C, which individually lack LM0230 stabilization region 5 and temperature-sensitive maintenance region 4, respectively, were not tested in EB5. Temperature-sensitive maintenance of pKMP1 and its deletion derivatives in EB5 was determined by comparing the loss frequencies at 25, 32, and 37°C (EB5 does not grow at 39°C) (Fig. 6). No obvious difference between plasmid loss rates was observed for growth at 25 and 32°C (data not shown). However, all plasmids except pKMP1-N, which lacks region 2b, showed an increase (average, 2.6-fold) in loss frequency at 37°C compared with that at 25C. There-

fore, pKMP1 and its derivatives appeared to produce a temperature-sensitive maintenance phenotype in both LM0230 and EB5. However, since deletion of no particular region consistently correlated with loss of temperaturesensitive maintenance, genes affecting this phenotype in L. cremoris EB5 could not be localized. The temperaturesensitive maintenance phenotype does not seem to be as strongly expressed in EB5, since most plasmids were significantly more stable in EB5 than in LM0230 at 37°C and the relative decrease in plasmid stability at 37°C was greater in LM0230. DISCUSSION We describe cloning of the replication region, potential plasmid maintenance functions, and temperature-sensitive maintenance region of pSK11L (10). The replication region of pSK11L was localized to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this region revealed an ORF encoding the putative Rep protein and an upstream noncoding ori region containing direct and inverted repeats. Stability assays of pKMP1 and its deletion derivatives showed that, like pSK11L, pKMP1 and most of its derivatives were significantly less stable in L. lactis LM0230 than in L.

7580

HORNG ET AL.

cremoris EB5 at all of the temperatures tested. Unlike that of pSK11L, maintenance of pKMP1 and its deletion derivatives appeared to be temperature sensitive in both hosts, although the relative decrease in stability at 37°C was greater in LM0230. Plasmid stability decreased further in LM0230 at 39°C. A region seemingly responsible for the temperaturesensitive maintenance phenotype in LM0230 was localized to a 1.2-kbp ClaI-HindIII fragment distinct from the minimum replicon. No region potentially responsible for the temperature-sensitive maintenance phenotype in EB5 was identified. Comparison of plasmid stabilities at 25°C resulted in identification of three regions (Fig. 6, regions 1, 3, and 5) whose deletion decreased plasmid stability and one region (Fig. 6, region 2a) whose deletion increased plasmid stability in LM0230. Comparison of plasmid stabilities at 25°C in EB5 showed that two of the stability regions identified in LM0230 (Fig. 6, regions 1 and 3) affected stability similarly in EB5. Additional regions on pKMP1 whose deletion had no effect on plasmid stability in LM0230 but affected stability in EB5 were identified (Fig. 6, regions 2b, 2c, 2d, and 6). The ability to ferment lactose has been reported to be extremely stable in some L. cremoris strains, including SK11 (8, 10). Feirtag et al. (10) have shown that for SK11, this stability is due to encoding of the lactose genes on an unusually stable plasmid, pSK11L. As pSK11L did not appear on agarose gels to exhibit a high copy number which

could account for stable inheritance (data not shown), it seemed likely that pSK11L, like most low-copy-number plasmids, encodes several systems to safeguard stable plasmid maintenance in its native L. cremoris strains (24). The instability of the minimum replicon (pKMP1-SS) in L. cremoris EB5 supports this hypothesis. However, the instability of pSK11L in L. lactis LM0230 suggests that these stability regions do not function or function poorly in L. lactis LM0230. This hypothesis is consistent with our identification of regions (2b, 2c, 2d, and 6 [Fig. 6]) whose deletion decreased plasmid stability in L. cremoris EB5 but had no effect on plasmid stability in L. lactis LM0230. Our results suggest that L. lactis LM0230 lacks essential host-derived plasmid maintenance factors. Alternatively, some of the pSK11L stability-affecting regions may have evolved to the point of being unable to interact efficiently with corresponding L. lactis LM0230 host factors. Further study will be needed before any conclusions can be drawn about the nature of the stability-affecting regions or how many stability-affecting functions are present on pKMP1 or pSK11L. However, it is necessary to keep in mind that pKMP1 does not appear to be as stable as pSK11L in L. cremoris EB5 (10). Although this discrepancy may be due to the different assays used to measure stability (determination of the number of Lac- cells in single colonies for pSK11L versus determination of the number of Ems cells after growth for approximately 20 generations in broth for pKMP1), it is also likely that pKMP1 does not contain all or contains truncated versions of stability determinants present on pSK11L. For example, we found that pSK11L contains two insertion elements, both of which are present on the lactococcal chromosome (28) and could provide homology for plasmid integration. Such integration could contribute to the stability of pSK11L and to the reported linkage of the lactose operon to the chromosome in L. cremoris strains. Therefore, studies based solely on pKMP1 stability may provide an incomplete model for pSK11L stability. pKMP1 displayed temperature-sensitive maintenance in both L. lactis LM0230 and L. cremoris EB5. The plasmid loss rate was greater in L. lactis LM0230, perhaps because

J. BACTERIOL.

the plasmid was initially more unstable in this host. It was also found that while the data on stability at 25°C were fairly consistent between independent assays of the same plasmid, the results of assays at 37 and 39°C were more variable (data not shown). In L. lactis LM0230, this may be related to the ability of pKMP1 to integrate into the host chromosome (10) and may reflect a difference in the prevalence of integrants in different assays. Chromosomal integration may also explain the lack of temperature sensitivity of pKMP1-C maintenance. It has recently been observed (15) that the proportion of cells containing the integrated form of the plasmid is higher for pKMP1-C than for pKMP1 or any of the other deletion derivatives. Therefore, the region deleted in pKMP1-C may indirectly affect the temperature-sensitive maintenance phenotype by increasing the frequency of plasmid integration which appears to occur by homologous recombination (27) or because deletion of this region destabilizes the plasmid to such an extent that there is strong selective pressure for cells containing integrated plasmids. The fact that flanking regions 3 and 5 (Fig. 6) affect stability supports the latter alternative. In either case, when the plasmid is in the integrated state the temperature-sensitive maintenance function would not be observed. Therefore, region 4 may not be the cause of the temperature-sensitive maintenance phenotype of pKMP1 in L. lactis LM0230. Instead, the temperature-sensitive maintenance phenotype may be a function of the replication region itself. This report also presents a restriction map of pSK11L and the positions of genes encoding several traits known to be associated with this plasmid. The replication origin appeared to be flanked on either side by stability regions. The lactose operon was separated from the replication origin on one side by a copy of ISSJ and on the other by a sequence which had intermediate homology to ISSJ under stringent conditions in Southern hybridizations. This sequence may represent a degenerate ISSJ element. Such a structure suggests that the lactose operon was originally part of a transposable element whose ends were ISSJ elements that transposed to a smaller plasmid. A similar structure of two ISSJ elements surrounding a lactose operon has been reported on the lactose plasmid of L. lactis ML3 (29). Our results support the idea that ISSJ played a significant role in the acquisition by lactococci of genes necessary for maximum growth and acid production in milk. The insertion elements may also play a role in the biology of pSK11L. ISSJ has previously been shown to mediate cointegration with conjugal plasmids or chromosomal conjugal elements in other lactococcal strains (2, 12). Yu et al. (35) reported isolation of a lactose plasmid (pDI-21) from L. cremoris H2 by conjugal transfer to a plasmid-free L. lactis strain. Interestingly, the order and distribution of restriction sites in pDI-21 are very similar to those of the restriction sites in pSK11L up to the region that contains ISSJ. pSK11L is a nonconjugative plasmid (25). It seems likely that pDI-21 is a cointegrate plasmid created by transposition of ISSJ from the L. cremoris H2 lactose plasmid to a conjugative plasmid present in the same host. This has been observed for conjugal mobilization of the lactose plasmid in ML3 (2, 12). To our knowledge, the pSK11L replicon is the only sequenced replicon from a lactococcal plasmid with a known biological function. All previously sequenced lactococcal replicons were from small cryptic plasmids (7, 14, 17). The replication and stabilization functions of pSK11L are interesting to study for a number of reasons. (i) pSK11L is extremely stable in L. cremoris strains (8, 10) but unstable in L. lactis strains (10). Regions surrounding the pSK11L

VOL. 173, 1991

LACTOSE PLASMID pSK11L FROM L. LACTIS SUBSP. CREMORIS

replication region which have differing effects on stability in each of these hosts have been identified. Such a phenomenon has never before been reported for an L. cremoris plasmid in L. lactis strains or vice versa. pSK11L may, therefore, provide a unique opportunity for examination of differences between interacting host and plasmid replication functions in two closely related subspecies. (ii) pSK11L may also offer a model system to study the evolutionary transition of a plasmid to a restricted host range. (iii) pSK11L represents the first reported example of a lactococcal plasmid whose stability does not appear to be determined entirely by the minimum replicon. Thus, pSK11L will allow identification of plasmid stabilization functions in lactococci. Isolation of such functions is currently in progress and could enable engineering of stable plasmids for use in commercial dairy starter strains. ACKNOWLEDGMENTS This work was supported in part by the Minnesota-South Dakota Dairy Foods Research Center, the Kraft General Foods Chair in Food Science granted to L.L.M., and a doctoral dissertation fellowship granted to K.M.P. REFERENCES 1. Anderson, D. G., and L. L. McKay. 1983. A simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549-552. 2. Anderson, D. G., and L. L. McKay. 1984. Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3. J. Bacteriol. 158:954-962. 3. Baldwin, K. A., and L. L. McKay. Unpublished data. 4. Chang, A. C. Y., and S. N. Cohen. 1978. Constructioii and characterization of amplifiable multicopy DNA cloning vehicles derived from the P1SA cryptic miniplasmid. J. Bacteriol. 134: 1141-1156. 5. Chen, E. Y., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencirdg plasmid DNA. DNA 4:165-170. 6. de BruiJn, F. J., and J. R. Lupski. 1984. The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids-a review. Gene 27:131-149. 7. de Vos, W. M. 1986. Gene cloning in lactic streptococci. Neth. Milk Dairy J. 40:141-154. 8. de Vos, W. M., and F. L. Davies. 1984. Plasmid DNA in lactic streptococci: bacteriophage and proteinase plasmid in Streptococcus cremoris SK11, p. 201-206. In Third European Congress on Biotechnology. Verlag Chemie, Deerfield Beach, Fla. 9. Efstathiou, J. D., and L. L. McKay. 1977. Inorganic salts resistance associated with a lactose-fermenting plasmid in Streptococcus lactis. J. Bacteriol. 130:257-265. 10. Feirtag, J. M., J. P. Petzel, E. Pasalodos, K. A. Baldwin, and L. L. McKay. 1991. Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lctis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 57:539-548. 11. Garvie, E. I. 1984. Taxonomy and identification of bacteria important in cheese and fermented dairy products, p. 35-66. In F. L. Davies and B. A. Law (ed.), Advances in the microbiology and biochemistry of cheese and fermented milk. Elsevier Applied Science Publishers, New York, N.Y. 12. Gasson, M. J. 1990. In vivo genetic systems in lactic acid bacteria FEMS Microbiol. Rev. 87:43-60. 13. Hayes, F., C. Daly, and G. F. Fitzgerald. 1990. Identification of the minimal replicon of Lactococcus lactis subsp. lactis UC317

7581

plasmid pCI305. Appl. Environ. Microbiol. 56:202-209. 14. Hayes, F., P. Vos, G. F. Fitzgerald, W. M. de Vos, and C. Daly. 1991. Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305. Plasmid 25:1626. 15. Horng, J. S., K. M. Polzin, and L. L. McKay. Unpublished data. 16. Imanaka, T., T. Ano, M. Fugii, and S. Aiba. 1984. Two replication determinants of an antibiotic-resistance plasmid, pTB19, from a thermophilic bacillus. J. Gen. Microbiol. 130: 1399-1408. 17. Leenhouts, K. 1990. Ph.D. Thesis. University of Groningen, Groningen, The Netherlands. 18. Lin, L.-S., and R. J. Meyer. 1986. Directly repeated, 20-bp sequence of plasmid R1162 DNA is required for replication expression of incompatibility and copy-number control. Plasmid 15:35-47. 19. Lin, L.-S., and R. J. Meyer. 1987. DNA synthesis is initiated within the origin of replication of plasmid R1162. Nucleic Acids Res. 15:8319-8333. 20. Ludwig, W., E. Sewaldt, R. Kilpper-Balz, K. H. Schleifer, L. Magrum, C. R. Woese, G. E. Fox, and E. Stackebrandt. 1985. The phylogenetic position of Streptococcus and Enterococcus. J. Gen. Microbiol. 131:543-551. 21. Macrina, F. L., R. P. Jones, J. A. Tobian, D. L. Hartley, D. B. Clewell, and K. R. Jones. 1983. Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning. Gene 25: 145-150. 22. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Meissner, P. S., W. P. Sisk, and M. L. Berman. 1987. Bacteriophage X cloning system for the construction of directional cDNA libraries. Proc. Natl. Acad. Sci. USA 84:4171-4178. 24. Nordstrom, K. 1989. Mechanisms that contribute to the stable segregation of plasmids. Annu. Rev. Genet. 23:37-69. 25. Pasalodos, E., and L. L. McKay. Unpublished data. 26. Persson, C., and K. Nordstrom. 1986. Control of replication of the broad host range plasmid RSF1010: the incompatibility determinant consists of direEtly repeated DNA sequences. Mol. Gen. Genet. 203:189-192. 27. Petzel, J. P., and L. L. McKay. Unpublished data. 28. Polzin, K. M., and L. L. McKay. 1991. Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci. AppI. Environ. Microbiol. 57:734-743. 29. Polzin, K. M., and M. Shimizu-Kadota. 1987. Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3. J. Bacteriol. 169:5481-5488. 30. Roberts, R. C., R. Burioni, and D. R. Helinski. 1990. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J. Bacteriol. 172:6204-6216. 31. Rodriquez, R. L., and R. C. Tait. 1983. Recombinant DNA techniques: an introduction. Addison-Wesley Publishing Co., Reading, Mass. 32. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 33. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813. 34. Winberg, G., and M. L. Hammerskjola. 1980. DNA from agarose gels using DEAE-paper: applications to restriction site mapping of adenovirus type 16 DNA. Nucleic Acids Res. 8:253-264. 35. Yu, P.-L., R. D. Appleby, G. G. Pritchard, and G. E. Y. Limsowtin. 1989. Restriction mapping and localization of the lactose-metabolizing genes of Streptococcus cremoris pDI-21. Appl. Microbiol. Biotechnol. 30:71-74.