Isolation and Characterization of a Streptococcus thermophilus Plasmid Closely Related to the pMV158 Family

Plasmid 45, 171–183 (2001) doi:10.1006/plas.2001.1517, available online at http://www.academicpress.com on

Isolation and Characterization of a Streptococcus thermophilus Plasmid Closely Related to the pMV158 Family Nathalie Turgeon and Sylvain Moineau1 Department of Biochemistry and Microbiology, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P4 Received October 9, 2000; revised January 12, 2001 Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids. Fifteen S. thermophilus plasmids were divided into four groups using DNA homology. Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S. thermophilus plasmids. Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D). Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode. The only plasmid of group C, named pSMQ172, was further characterized. This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance. Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis. These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci. © 2001 Academic Press Key Words: Streptococcus; plasmid; rolling-circle; single-stranded DNA; pMV158; bacteriophage; lactic acid bacteria.

Streptococcus thermophilus is a gram-positive bacterium used to manufacture yogurt and specialty cheeses. These fermented dairy products are produced at record level in many countries, and consequently, the research interest in this lactic acid bacterium (LAB)2 has increased sharply in recent years. Plasmids play a critical role in LAB2 such as Lactococcus, since they carry important industrial phenotypes, including carbohydrate fermentation, bacteriocin and exopolysaccharide production, and antiphage mechanisms. However, plasmids are rarely found in S. thermophilus strains and very few encode useful phenotypes. Indeed, studies have consistently reported that less than 20% of S. thermophilus strains carry one or two small plasmids (Girard et al., 1987; Herman and Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF295100. 1 To whom correspondence should be addressed. Fax: 418-656-2861. E-mail: [email protected]. 2 Abbreviations used: LAB, lactic acid bacteria; RC, rolling-circle mechanism; sso , single-stranded origin. 171

McKay, 1985; Janzen et al., 1992; Somkuti and Steinberg, 1986). Two previous studies have shown that S. thermophilus plasmids may be divided into five distinct DNA homology groups (Janzen et al., 1992; Somkuti and Steinberg, 1986). Only five plasmids have been previously sequenced and they range in size from 2093 to 6499 bp (Hashiba et al., 1993; Janzen et al., 1992; O’Sullivan et al., 1999; Solaiman and Somkuti, 1998; Somkuti et al., 1998). They belong to the same DNA homology group, as they are all members of the pC194/pUB110 family (del Solar et al., 1998; Khan, 1997). Although the presence of single-stranded DNA (ssDNA) intermediates has never been demonstrated with any of these five plasmids, they appear to replicate via a rolling-circle mechanism (RC) as suggested by their homology to known RC plasmids. Some of them were also used to construct cloning vectors for the genetic modification of S. thermophilus strains (Solaiman and Somkuti, 1993, 1998; Somkuti et al., 1995). Thus, despite an apparent diversity of the S. thermophilus plasmids, the molecular charac0147-619X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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terization has only been determined for similar plasmids. In this study, the plasmid content of several S. thermophilus strains was examined. Plasmids were then separated into groups based on DNA homology. Assays for detection of ssDNA were performed to elucidate the mode of replication of each group. A unique plasmid was investigated in order to increase the general knowledge of S. thermophilus plasmids. Sequence analysis revealed that this plasmid was closely related to the pMV158 family. It is the first report of a S. thermophilus plasmid linked to plasmids of pathogenic streptococci. MATERIALS AND METHODS Bacterial strains, plasmids, and media. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5a was grown at 37°C in Luria broth (Sambrook et al., 1989). S. thermophilus strains were grown at 30°C at 42°C in Elliker broth (Difco Laboratories, Detroit, MI) supplemented with 1% beef extract (Difco) and 1.9% b-glycerophosphate (Sigma–Aldrich, Oakville, Ontario, Canada) (Le Marrec et al., 1997). L. lactis was grown at 30°C in M17 (Difco Laboratories) supplemented with 0.5% glucose (GM17). S. salivarius was grown at 37°C in Hogg-Jago glucose broth supplemented with 0.4 M sorbitol (HJGS) (Buckley et al., 1999). When appropriate, antibiotics were added as follows: For E. coli, 50 mg of ampicillin per milliliter and 20 mg chloramphenicol per milliliter; for S. thermophilus, S. salivarius, and L. lactis, 5 mg of chloramphenicol per milliliter. All antibiotics were purchased from Sigma–Aldrich. DNA manipulation. Plasmid DNA was purified from E. coli with a Qiagen plasmid maxi kit (Qiagen, Chatsworth, CA) or as described by Birnboim and Doly (1979). Plasmid DNA was purified from S. thermophilus as described by O’Sullivan and Klaenhammer (1993) followed by a continuous CsCl gradient. Total DNA of S. thermophilus was obtained as described by Noirot-Gros and Ehrlich (1994), except that 30 mg/ml of lysozyme was used for cell lysis. Restriction and modification enzymes were used according to the manufacturer’s recommenda-

tions (Boehringer Mannheim, Laval, Québec, Canada). Competent E. coli cells were prepared and transformed with the Gene Pulser II apparatus as described by the manufacturer (Bio-Rad Laboratories, La Jolla, CA). The methods for preparing competent cells and the electrotransformation of L. lactis and S. thermophilus have been described elsewhere (Holo and Nes, 1989). Competent S. salivarius cells were prepared as described by Buckley et al. (1999). Southern hybridization. DNA was transferred to positively charged nylon membranes (Boehringer-Mannheim) by capillary blotting as described by Sambrook et al. (1989). Plasmids and PCR products were labeled randomly with the DIG High Prime kit (BoehringerMannheim). Prehybridization, hybridization, posthybridization washes, and detection were performed as suggested in the DIG System User’s Guide for Filter Hybridization. DIG Easy Hyb buffer and CSPD were utilized for hybridization steps and chemiluminescent detection, respectively. ssDNA detection. Single-stranded DNA was detected in total DNA samples of S. thermophilus as described by Noirot-Gros and Ehrlich (1994) in the presence or absence of S1 nuclease treatment as well as with or without DNA denaturation prior to gel blotting. DNA sequencing and analysis. The 16S rRNA of S. thermophilus SMQ-172 was amplified by PCR with primers ssu27 (5⬘AGAGTTTGATCMTGGCTCAG-3⬘) and ssu1492⬘ (5⬘-TACGGYTACCTTGTTACGACTT3⬘) before sequencing. Plasmid restriction fragments were cloned in pBluescript II KS and sequenced using universal primers (forward and reverse). The sequence was completed by primer walking using synthetic oligonucleotide primers (Gibco/BRL, Burlington, Ontario, Canada). DNA was sequenced on both strands with an Applied Biosystems 373A automated DNA sequencer (Applied Biosystems, Foster City, CA). The assembly and sequence analysis were performed with the Wisconsin Package version 9.0 (Genetics Computer Group, Madison, WI) (Devereux et al., 1984). The open reading frames were compared with databases (GenBank, EMBL, SwissProt, PIR, PDB,

Streptococcus thermophilus Plasmids

DDBJ, and PRF) using Blast version 2.0.4 (Altschul et al., 1997). Phage resistance assays. Phages DT1 and Q1 were used as representatives of the two main S. thermophilus phage groups (Le Marrec et al., 1997; Moineau et al., 1995; Tremblay and Moineau, 1999). For phage propagation and assay, calcium chloride was added to the medium to a final concentration of 10 mM as described previously (Moineau et al., 1995). RESULTS AND DISCUSSION Species identification and plasmid content. Twenty-two S. thermophilus strains were obtained from various collections. Thirteen strains are currently used in large-scale industrial milk fermentations and 9 strains were previously isolated from artisanal cheeses. Species identification was confirmed by sugar fermentation profiles on API 50CH. All S. thermophilus strains produced acid from glucose, lactose, and sucrose. Few strains were also able to utilize mannose and fructose. These S. thermophilus strains were analyzed for their plasmid content. Nine strains were plasmid-free, 11 strains contained one plasmid, and 2 strains (SMQ-173 and SMQ-312) carried two plasmids (Fig. 1E). Overall close to 60% of the strains tested possessed plasmids, a result that is much higher than reported in previous studies in which fewer than 20% possessed plasmids (Girard et al., 1987; Herman and McKay, 1985; Janzen et al., 1992; Somkuti and Steinberg, 1986). Interestingly, most industrial strains were plasmid-free, whereas all the strains isolated from artisanal cheeses carried plasmids (Table 1). This observation suggests that plasmids may exert a negative effect during the selection of industrial S. thermophilus strains. The presence of a plasmid within a cell may increase the metabolic load and elicit a detrimental effect on cell growth or acid production, both key factors in large-scale milk fermentation. Alternatively, less stable plasmids or plasmids that do not confer a selective advantage may be rapidly lost in the dairy environment. These results also suggest that novel plasmid-encoded activities should be sought mainly within wild-type strains.

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Identification of four plasmid groups. DNA–DNA hybridization experiments were used to separate the 15 plasmids into four groups (Fig. 1, Table 2). The first probe was made of a PCR product obtained with the primers RCNT1/RCNT2 and pSMQ310 as the template. The two primers (RCNT1, 5⬘-CTAGGTCTCACCATCATC-3⬘; RCNT2, 5⬘-CGAGA CTGGCGAGGACG-3⬘) were designed from a conserved region between the repB gene of the previously sequenced plasmids pST1 (Janzen et al., 1992) and pST1-no29 (Hashiba et al., 1993). Ten plasmids hybridized with the first probe and were included within group A (Fig. 1A, Table 2). A second probe consisted of an XbaI-restricted plasmid (pSMQ308) that did not hybridize with the previous probe. Three plasmids hybridized with the second probe and were classified into the group B (Fig. 1B, Table 2). Groups C (pSMQ172) and D (pSMQ173b) contained only one plasmid, as they hybridized only with themselves (Fig. 1C and Fig. 1D, Table 2). These results concur with two previous investigations that distributed 9 and 13 S. thermophilus plasmids within five DNA groups (Janzen et al., 1992; Somkuti and Steinberg, 1986). Group A, which shares DNA homology with the five previously sequenced plasmids, appears to be the most dominant type in S. thermophilus. Finally, plasmids of group A are compatible with plasmids of groups B and D since the strain SMQ312 carried one plasmid from group A and one from group B, whereas SMQ-173 harbored one plasmid from group A and one from group D. Detection of ssDNA. DNA–DNA hybridizations with or without denaturation as well as the S1 nuclease treatment revealed the presence of ssDNA for plasmids of groups A, C, and D (Fig. 2). For pSMQ172 (group C), many forms of the plasmid were detected. The presence of ssDNA intermediates confirmed that these plasmids replicate via a rolling-circle mode. The replication mode of group B plasmids remains unclear due to the absence of detectable ssDNA. These results are the first experimental evidence of the mode of replication of plasmids in S. thermophilus and they showed that most of the plasmids in this species replicate via a RC mode.

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FIG. 1. Plasmid profiles of S. thermophilus strains and plasmid grouping based on DNA–DNA hybridizations. Lanes 1, 11, 12, and 17, supercoiled DNA markers (Gibco/BRL); lane 2, SMQ-308; lane 3, SMQ-309; lane 4, SMQ-310; lane 5, SMQ-311; lane 6, SMQ-312; lane 7, SMQ-315; lane 8, SMQ-316; lane 9, SMQ-318; lane 10, SMQ-319; lane 13, SMQ-171; lane 14, SMQ-172; lane 15, SMQ-173; lane 16, SMQ-307. (A) pSMQ310 probe, (B) pSMQ308 probe, (C) pSMQ172 probe, (D) pSMQ173b probe, and (E) agarose gel.

16S rRNA of SMQ-172. The unique plasmid of group C (pSMQ172) was further characterized to increase the basic knowledge of S. thermophilus plasmids. Before this plasmid was sequenced, the 16S rRNA of SMQ-172 was sequenced. The sequence between position 27 and 1492 showed the highest identity (98%) with 16S rRNA of S. thermophilus (data not shown) (Ludwig et al., 1992). DNA sequence and analysis of pSMQ172. The plasmid pSMQ172 was sequenced and found to contain 4230 bp. The GC content was 38%, a value that is in accordance with the S. thermophilus genome (39%). Several open reading frames (orfs) were identified on the pSMQ172 sequence. Only four orfs with more than 40 codons and preceded by a Shine–Dalgarno (SD) sequence were analyzed further (Table 3). Putative consensus promoters were recognized upstream of orf1 (TTGGCG-18TAAAAT), orf3 (TTACGA-17-TATACT), orf4

(TTCATT-16-TAAAAT), and within the noncoding strand of orf2 (TTGCTT-17-TATAAT). DNA homologies were detected with several rolling-circle plasmids of gram-positive bacteria (Table 3), including Lactobacillus acidophilus (Sano et al., 1997), Lactobacillus curvatus (Klein et al., 1993), Lactobacillus helveticus (Pridmore et al., 1994), Lactobacillus hilgardii (Josson et al., 1990), Lactobacillus plantarum (Bates and Gilbert, 1989; Cocconcelli et al., 1996), Lactococcus lactis (Leenhouts et al., 1991; Perreten et al., 1997; Prevots et al., 1998; Xu et al., 1991), Streptococcus agalactiae (Lacks et al., 1986; van der Lelie et al., 1989), Streptococcus bovis (Nakamura et al., 2000), Streptococcus ferus (Leblanc et al., 1993), and Streptococcus suis (Takamatsu et al., 2000). Overall, pSMQ172 shares a high degree of homology with the rolling-circle plasmids pSSU1 of S. suis (Fig. 3B) and pMV158 of S. agalactiae (Fig. 3A).

175

Streptococcus thermophilus Plasmids

TABLE 1 Bacterial Strains and Plasmids Used in This Study Bacteria or Plasmids S. thermophilus SMQ-119 SMQ-171 SMQ-172 SMQ-173 SMQ-307 SMQ-308 SMQ-309 SMQ-310 SMQ-311 SMQ-312 SMQ-315 SMQ-316 SMQ-318 SMQ-319 SMQ-495 SMQ-645 SMQ-646 L.lactis SMQ-647 E. coli SMQ-648 S. salivarius SMQ-649 Plasmids pBS pSMQ171 pSMQ172 pSMQ172cat pSMQ173a pSMQ173b pSMQ307 pSMQ308 pSMQ309 pSMQ310 pSMQ311 pSMQ312a pSMQ312b pSMQ315 pSMQ316 pSMQ318 pSMQ319

Relevant characteristics

Source

Industrial strain used in yogurt production; host for phage Q1 Industrial strain used in cheese production Industrial strain used in cheese production Industrial strain used in cheese production Industrial strain used in cheese production Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Strain isolated from artisanal cheese Industrial strain used in cheese production, host for phage DTI SMQ-495 (pSMQ172cat), Cmr SMQ-119 (pSMQ172cat), Cmr

Moineau et al. (1995) Moineau et al. (1995) Moineau et al. (1995) Moineau et al. (1995) This study QI QI QI QI QI QI QI QI QI This study This study This study

MG1363 (pSMQ172cat), Cmr

This study

DH5a (pSMQ172cat), Cmr

This study

ATCC 25975 (pSMQ172cat), Cmr

This study

Cloning vector for sequencing, Apr, 2.9 kb Resident plasmid of SMQ-171, 2 kb Resident plasmid of SMQ-172, 4.2 kb pSMQ172, cat gene of pC194 (Horinouchi et al., 1982), 5.7 kb Resident plasmid of SMQ-173, 3 kb Resident plasmid of SMQ-173, 5 kb Resident plasmid of SMQ-307, 3.5 kb Resident plasmid of SMQ-308, 8 kb Resident plasmid of SMQ-309, 3.5 kb Resident plasmid of SMQ-310, 6 kb Resident plasmid of SMQ-311, 3 kb Resident plasmid of SMQ-312, 5.5 kb Resident plasmid of SMQ-312, 7 kb Resident plasmid of SMQ-315, 4 kb Resident plasmid of SMQ-316, 7 kb Resident plasmid of SMQ-318, 3 kb Resident plasmid of SMQ-319, 3 kb

Strategene This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

Note. Apr, ampicillin resistance; cat, chloramphenicol acetyl transferase; Cmr, chloramphenicol resistance; QI, Quest International (Rochester, MN).

Functions associated with distinct DNA regions. Putative functions were assigned to three of the four ORFs present in pSMQ172 and to noncoding regions. ORF1 shared homology with proteins involved in plasmid copy number control (Cop) (Table 3). The product of orf1

shared 93% identity with the CopG protein of pMV158 (Table 3). A ribbon–helix–helix motif was also identified on ORF1 (Gomis-Rüth et al., 1998). ORF2 was similar to several replication initiator proteins (Rep) (Table 3). The two conserved motifs of the Rep (HUH and catalytic

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TABLE 2 Distribution of Plasmids in Four Groups Group A

Group B

Group C

Group D

pSMQ171 pSMQ173a pSMQ307 pSMQ309 pSMQ310 pSMQ311 pSMQ312a pSMQ315 pSMQ318 pSMQ319

pSMQ308 pSMQ312b pSMQ316

pSMQ172

pSMQ173b

site) were found on ORF2 (del Solar et al., 1998). The critical tyrosine residue of the catalytic site was also present on ORF2. Another highly conserved region in the pMV158 plasmid family was identified upstream of the orf1 gene of pSMQ172. This area contained a putative double-stranded origin (dso) (Fig. 4). A short DNA sequence repeated three times was recognized 82 bp downstream of the putative nick site and may represent the Rep binding site. As with pMV158, a singlestranded origin (ssoA) was observed downstream of orf3. The pSMQ172 ssoA had a 146bp stem–loop structure that shares 70% identity with the pMV158 ssoA (Fig. 5). A recombination site (RSB) was also noticed in the ssoA region of pSMQ172. However, the region did not contain the usual six conserved nucleotides (Cs6) found in this sso family (del Solar et al., 1998). The Cs-6 region is involved in the termination of primer RNA synthesis (Kramer et al., 1997). A putative antisense RNA was also detected between orf1 and orf2. This potential RNA displayed 71% identity with RNA II of pLS1 (Fig. 6) (del Solar and Espinosa, 1992). In addition, the promoter and the terminator of this antisense RNA were well conserved. In pLS1, this antisense is involved in the downregulation of the plasmid copy number. ORF3 was associated with various proteins involved in plasmid mobilization (Mob) (Table 3). A putative oriT was also uncovered upstream of the orf3. It was at the same location and it

shared common elements (the RSA recombination site, two inverted repeats, and the nick site) with the oriT identified on pMV158 (Fig. 7). The RSA, the IR2, and the nick site are especially well conserved in both oriT. Finally, ORF4 had no homology with known protein in databases except for a deduced protein (ORF6) of unknown function found on a plasmid of S. suis (Table 3). Host range of pSMQ172. Since pSMQ172 does not contain a selective marker, the chloramphenicol acetyltransferase gene (cat) of the plasmid pC194 was cloned into pSMQ172 between the ssoA and the orf4, at the unique AatII site. The recombinant plasmid pSMQ172cat was successfully introduced into S. ther-

FIG. 2. Detection of ssDNA by Southern hybridizations. Total DNA from S. thermophilus SMQ-307 (A), SMQ-308 (B), SMQ-172 (C), and SMQ-173 (D) were electrophoresed on a 0.8% agarose gel containing ethidium bromide (0.5 ␮g per milliliter), with (⫹) or without (⫺) prior S1 nuclease treatment. DNA was denatured (left) or not (right) prior to transfer on nylon membrane. Membranes were hybridized with respective DIG-labeled probes: pSMQ307 (A), pSMQ308 (B), pSMQ172 (C), and pSMQ173b (D).

TABLE 3 General Features of the Four orfs of Plasmid pSMQ172 and Comparisons with Databases Start

Stop

%G⫹C

Size (aa)

MW (kDa)

1

262

399

32

45

5.0

2

460

1131

37

223

3

1496

2995

39

4

3891

3439

38

SD sequence AAAGGAGGTGA 16S rRNA

Function or similarity

Percentage identity (aa)

Accession Number

TTGAGAGGTAAttcATG

S. suis pSSU1 CopG S. agalactiae pMV158 CopG S. bovis pSB02 Cop L. lactis pFX2 CopX L. lactis pWV01 ORFC Lb. plantarum pSC22 CopA Lb. acidophilus pLA106 Rep

95 (43/45) 93 (42/45) 42 (18/42) 42 (17/40) 42 (17/40) 42 (17/40) 42 (15/35)

AB019522 X15669 AB021465 X54310 X56954 X95843 D88438

25.8

TACTTGGGTTAgttataccgtATG

Lb. curvatus pLC2 Rep S. suis pSSU1 CopG Lb. helveticus pLH2 ORF1 L. lactis pPF107-3 ORF1 S. bovis pSB02 Rep S. agalactiae pMV158 RepB L. lactis pWV01 RepA Lb. plantarum pSC22 RepA L. lactis pFX2 RepX

72 (161/223) 71 (160/223) 69 (152/219) 69 (153/219) 65 (135/207) 64 (132/205) 49 (111/225) 48 (110/225) 46 (105/226)

Z14234 AB019522 X81981 Y12675 AB021465 X15669 X56954 X95843 X54310

499

58.1

TGAGGAGGAAAagcaATG

S. suis pSSU1 Mob S. agalactiae pMV158 MobM S. ferus pVA3-1 Mob L. lactis pK214 Mob Lb. hilgardii pLAB1000 Mob Lb. plantarum pLB4 PreA

97 (486/499) 66 (338/505) 68 (302/440) 37 (745/385) 43 (141/324) 43 (141/324)

AB019522 X15669 L23803 X92946 M55222 M33531

150

16

AACGGAGGTAAaatattATG

S. suis pSSU1 ORF6

68 (103/150)

AB019522

Streptococcus thermophilus Plasmids

ORF

Note. SD, underlined are conserved nucleotides compared to the consensus Shine–Dalgarno sequence; aa, amino acids; Lb. acidophilus, Lactobacillus acidophilus; Lb. curvatus, Lactobacillus curvatus; Lb. helveticus, Lactobacillus helveticus; Lb. hilgardii, Lactobacillus hilgardii; Lb. plantarum, Lactobacillus plantarum; L. lactis, Lactococcus lactis; S. agalactiae, Streptococcus agalactiae; S. bovis, Streptococcus bovis; S. ferus, Streptococcus ferus.

177

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FIG. 3. Comparative DNA sequence analysis of pSMQ172 with the rolling-circle plasmid pMV158 from Streptococcus agalactiae (A) and pSSU1 from Streptococcus suis (B). The predicted orfs are identified in boxes. The length of the box is proportional to the length of the predicted ORF. The colored bars give the percentages of nucleotide identity between both sequences as defined in the color code at the top of the figure. The vertical lines indicate the transition zones between the two plasmids. The figure was created with the SIM Alignment tool and Lalnview (Huang and Miller, 1991; http://www.expasy.hcuge.ch).

Streptococcus thermophilus Plasmids

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FIG. 4. Comparison between dso of four plasmids of the pMV158 family. Capital letters denote conserved nucleotides. Nick sites are identified by arrows. dso of pMV158, pE194, and pFX2 have been previously characterized. For a review, see del Solar et al. (1998) and Khan (1997).

mophilus SMQ-119 and SMQ-495, S. salivarius ATCC 25975, L. lactis MG1363, and E. coli DH5␣, indicating that pSMQ172 replicates in both gram-positive and gram-negative bacteria. It is worth mentioning that the copy number of pSMQ172cat was reduced in E. coli and L. lactis. Lacks et al. (1986) previously showed that chloramphenicol-resistant derivatives of pMV158 exhibited a low copy number in RecA⫺ mutants of E. coli. Phage resistance. In an attempt to identify a function to ORF4, the phage-sensitive S. thermophilus strains SMQ-119 and SMQ-495, transformed with pSMQ172cat, were tested for phage resistance. S. thermophilus phages are currently classified into two groups, based on the number of major structural proteins (MSP) and the mode of DNA packaging (Le Marrec et al., 1997). SMQ-119 is sensitive to phage Q1, a member of the phage group that has three MSP and a DNA packaging scheme that proceeds via a headful mechanism (Le Marrec et al., 1997). SMQ-495 is sensitive to phage DT1, a member of the phage group that possesses two MSP and cohesive genome extremities (Tremblay and Moineau, 1999). Both transformed strains (SMQ-645 and SMQ-646) were still phage sensitive, indicating that orf4 does not encode a phage resistance mechanism. A link between pSMQ172 and plasmids of pathogenic streptococci. The ecological niche of S. thermophilus is not well established but it has been isolated from raw milk. S. agalactiae is a group B Streptococcus with pathogenicity to humans and animals (Kawamura et al., 1995). It is also one of the leading causes of bovine mastitis (Keefe, 1997). S. suis has been implicated

in a wide range of diseases in farm animals as well as in humans (Staats et al., 1997). It is tempting to speculate that horizontal transfer occurred between the three species. The major difference between pSMQ172 and pMV158 was the absence of the tetracycline resistance gene on pSMQ172 (Fig. 3A). Also missing on pSMQ172 was an ssoU located between tet and oriT on pMV158. The ssoU is believed to be involved in the conversion of singlestranded DNA into double-stranded DNA after the transfer from one strain to another (del Solar et al., 1993). The absence of the ssoU and the tet suggests that a deletion may have occurred in pSMQ172. Alternatively, the ssoU–tet cassette could have been acquired by pMV158. Many industrial and academic research programs worldwide are currently isolating LAB from various nonindustrial sources (raw milk, artisanal fermented products, or plant material) to find novel biochemical activities and applications. With the recent discovery of a natural L. lactis plasmid carrying antibiotic resistance genes (Perreten et al., 1997) and now a natural S. thermophilus plasmid related to pathogenic streptococci, one could wonder about the longterm risks associated with the isolation of LAB from nature. In any case, it illustrates the importance of the molecular characterization of LAB strains and their plasmids. ACKNOWLEDGMENTS We thank the members of the plasmid group of the Canadian Network on Lactic Acid Bacteria for helpful discussion. We are very grateful to L.-A. Lortie and M. Frenette for the gift of competent cells of S. salivarius and to M. Dup-

180 TURGEON AND MOINEAU

FIG. 5. Comparison between pSMQ172 and pLS1 ssoA structures. The pLS1 ssoA structure has been previously proposed (del Solar et al., 1993). RSB is a recombination site. Cs-6 is a region of six conserved nucleotides in the pMV158-ssoA group (del Solar et al., 1998). Asterisk delineates identical nucleotides.

Streptococcus thermophilus Plasmids

FIG. 6. Comparative sequence analysis between pSMQ172 putative antisense RNA and pLS1 RNA II. Arrows indicate inverted repeats of putative transcription terminators. pLS1 promoter and terminator have been previously proposed (del Solar and Espinosa, 1992; Lacks et al., 1986). Asterisk delineates identical nucleotides.

181

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FIG. 7. Comparative sequence analysis between oriT of pSMQ172 and pMV158. The two inverted repeats (IR, arrows) and the conserved recombination site (RSA) are indicated, as well as the nick site (䉲) of MobM. Features of the plasmid pMV158 oriT were previously described (Grohmann et al., 1999). Asterisk delineates identical nucleotides.

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