Plasmid (2004) 51 :24-36.
Characterization of a theta replicating plasmid from
Nathalie Turgeon1, Michel Frenette1 and Sylvain Moineau1
1Département de biochimie et de microbiologie, Faculté des sciences et de génie, Groupe de
recherche en Écologie buccale (GREB), Faculté de médecine dentaire, Université Laval,
Québec, Canada G1K 7P4
Corresponding author. Fax: +418-656-2861. E-mail address: firstname.lastname@example.org
Abbreviations used: LAB, lactic acid bacteria; RC, rollingcircle replication; ssDNA, single-
stranded DNA; ssi, singlestranded initiating signal; sso, single-stranded replication origin.
Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology,
into at least four groups (A to D). Here, we report the characterization of plasmids of group B
and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains
4,449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of
the pGI3 family. The plasmid pSMQ308 (group B) contains 8,144 bp and six ORFs. Two
ORFs likely encode a primase/helicase and an integrase. Northern experiments demonstrate
that these two orfs are transcribed within the three strains containing plasmids of group B.
Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta
mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode
in S. thermophilus. Finally, a classification of S. thermophilus plasmids into six groups based
on their mode of replication is proposed.
Streptococcus thermophilus is a Gram-positive lactic acid bacterium (LAB) used to transform
milk into several fermented dairy products (i.e., yogurt, cheeses, etc.). The increasing
consumer demands for these products in recent years have given rise to active research
activities on this economically relevant LAB. These fundamental and applied studies have
been aimed at increasing our understanding of this microorganism in order to select better
strains for various food applications as well as to improve specialized strains through genetic
Plasmids play a central role in these studies, as they are the primary vehicles used to
manipulate targeted DNA sequences. However, these extrachromosomal genetic elements are
infrequently observed in S. thermophilus strains. In fact, the few studies that addressed this
issue reported between 20 to 30% of S. thermophilus strains carrying one or two small-sized
plasmids (Herman and McKay 1985, Somkuti and Steinberg 1986, Girard, Lautier et al. 1987,
Janzen, Kleinschmidt et al. 1992, Turgeon and Moineau 2001). These plasmids encode very
few identified phenotypes, such as restriction and modifications systems, as well as small heat
shock proteins (Somkuti, Solaiman et al. 1998, O'Sullivan, van Sinderen et al. 1999, Geis, El
Demerdash et al. 2003). S. thermophilus plasmids were previously classified into four or five
groups based on DNA homology (Somkuti and Steinberg 1986, Janzen, Kleinschmidt et al.
1992, Turgeon and Moineau 2001, Geis, El Demerdash et al. 2003). To date, 18 S.
thermophilus plasmids have been sequenced and their size varies from 2,1-kbp to 10-kbp.
DNA sequence analysis of the replication region indicates that 13 of the 18 sequenced S.
thermophilus plasmids belong to the pC194/pUB110 family of rolling-circle replicating
plasmids (RC). S. thermophilus plasmid pSMQ172 also replicates via a RC mode, but is
highly related to the pMV158 family (Turgeon and Moineau 2001), while the mode of
replication of the four others has yet to be analyzed in details.
Contrary to other LAB (i.e., Lactococcus lactis), limited cloning tools are available for the
genetic modification of S. thermophilus. Some of the broad-range cloning vectors (pNZ123,
pMIG, pSA3, pTRKH2, and pTRKL2) constructed for other bacterial genera are functional in
S. thermophilus (Dao and Ferretti 1985, de Vos 1987, O'Sullivan and Klaenhammer 1993,
Wells, Wilson et al. 1993). Few shuttle vectors based on cryptic plasmids of S. thermophilus
have also been developed (Solaiman and Somkuti 1993, Solaiman and Somkuti 1997,
Solaiman and Somkuti 1997, Solaiman and Somkuti 1998, Somkuti and Steinberg 1999, Su,
Jury et al. 2002). All these vectors are based on one of the three following replication
mechanisms: pC194, pMV158, and pAMβ1 (Khan 1997, del Solar, Giraldo et al. 1998, Khan
2000). Given that plasmids of the same replication family are usually incompatible, this lack
of diversity may pose a problem in many genetic studies. These vectors are also excluded from
food applications, because they contain antibiotic resistance genes or exogenous DNA or both
(Boucher, Parrot et al. 2002).
Previously, we classified S. thermophilus plasmids into four DNA homology groups (Turgeon
and Moineau 2001). Single-stranded DNA was detected within strains containing plasmids of
groups A (pC194 family), C (pMV158 family), and D, indicating that they replicate via a
rolling-circle mode. Here, we present the analysis of plasmids that belong to the DNA
homology groups B and D as well as the identification of their mode of replication. A
classification of S. thermophilus plasmids based on their replication mechanism is also
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study
are listed in Table 3.1. Escherichia coli DH5α was grown at 37°C in Luria broth (Sambrook
and Russell 2001). S. thermophilus strains were grown at 42°C in M17 broth (Quélab,
Montréal, Québec, Canada) supplemented with 0.5% lactose. When appropriate, 100 µg per
ml of ampicillin (Sigma-Aldrich, Oakville, Ontario, Canada) were added to E. coli culture.
DNA and RNA manipulations. Plasmid DNA was purified from E. coli with Qiagen plasmid
maxi kit (Qiagen, Chatsworth, CA) or as described by Birnboim and Doly (Birnboim and Doly
1979). Plasmid DNA was isolated from S. thermophilus as described previously (O'Sullivan
and Klaenhammer 1993) and then purified using a continuous CsCl gradient. Total DNA of S.
thermophilus was extracted as described elsewhere (Bruand, Ehrlich et al. 1991), except that
60 mg/ml of lysozyme were used for cell lysis. RNA was obtained from S. thermophilus with
the Qiagen RNeasy kit, following the incubation of cells at 37°C for 10 min in the presence of
lysozyme (60 mg/ml). Restriction and modification enzymes were used according to the
manufacturer’s recommendations (Roche Diagnostics, Laval, Québec, Canada). Competent E.
coli cells were prepared and transformed with the Gene Pulser II as described by the
manufacturer (Bio-Rad Laboratories, La Jolla, CA).
DNA sequencing and analysis. Plasmid restriction fragments were cloned into pBluescript II
KS and sequenced using universal primers (forward and reverse). The sequence was
completed by primer walking using synthetic oligonucleotide primers (Invitrogen Life
Technologies, Burlington, Ontario, Canada). DNA sequencing was carried out by the DNA
sequencing service at Université Laval using an ABI Prism 3100 apparatus. The assembly and
sequence analysis were performed with the Wisconsin Package Version 10.2 (Genetics
Computer Group, Madison, WI) (Devereux, Haeberli et al. 1984). The open reading frames
were compared with databases (GenBank, EMBL, SwissProt, PIR, PDB, DDBJ, and PRF)
using Blast version 2.0.6 (Altschul, Madden et al. 1997).
Two-dimensional agarose gel electrophoresis. Theta-replication intermediates were detected
from total DNA of S. thermophilus SMQ-308 using the method of neutral/neutral 2D agarose
gel electrophoresis described by Brewer et al. (Brewer, Sena et al. 1988). Ten micrograms of
total DNA were digested with HindIII before electrophoresis. The plasmid pSMQ308 bears
two HindIII restriction sites at coordinates 5913 and 6309. The digested DNA was loaded on a
20 cm X 20 cm X 0.6 cm agarose gel with 6.7 mm X 2 mm combs and separated by
electrophoresis in a 2.2-L submersed chamber containing 0.5X TBE buffer. The first
dimension was run in a 0.4% agarose gel at 1 V/cm for 42 h. The lane of interest was excised
and turned 90°. The second dimension was performed at 4°C in a 1% agarose gel of the same
size, in presence of ethidium bromide (0.3 µg/ml) at 5 V/cm for 14 h with recirculating buffer.
Southern and Northern hybridizations. Nucleic acids were transferred to positively charged
nylon membranes (Roche Diagnostics) by capillary blotting (Sambrook and Russell 2001).
Probes were labeled during the PCR reaction using DIG-dNTP according to the
manufacturer’s recommendations (Roche Diagnostics). Prehybridization, hybridization, post-
hybridization washes, and detection were performed in sealed bags as suggested in the DIG
System User’s Guide for Filter Hybridization. The amount of DIG Easy Hyb buffer used for
the prehybridization and hybridization steps was 20 ml per 100 cm2. CDP-Star was utilized for
Accession number. The DNA sequences of pSMQ173b and pSMQ308 are available under
the GenBank accession numbers AY312235 and AY312234, respectively.
RESULTS AND DISCUSSION
DNA sequence analysis of pSMQ173b. The S. thermophilus plasmid pSMQ173b belongs to
the homology group D (Turgeon and Moineau 2001). It has a size of 4,449 bp and a G+C
content of 37%, which is in accordance with the G+C content of the S. thermophilus genome
(37%). Five open reading frames (orfs) with more than 50 codons and preceded by a Shine-
Dalgarno (SD) sequence were found and analyzed further (Table 3.2). Putative consensus
promoters were recognized upstream of orf1 (62TTAATA-16-TATAAT), orf3 (1351TTGAAC-
19-TAAAAT), orf4 (1609TTGAAC-20-TATAAT), and on the complementary strand upstream
of orf5 (4443TTGACT-18-TAAAAT). A putative terminator was identified downstream of orf5
on the complementary strand (coordinates 3929 to 3980, ΔG = –9.7 kcal/mol). Overall, the
DNA sequence of pSMQ173b is 99% identical to the recently published S. thermophilus
plasmid pER1-2 (Geis, El Demerdash et al. 2003).
A putative replication origin was uncovered upstream of the orf2. This region shares several
features of replication origin, such as inverted (coordinates 607 to 627) and direct (coordinates
847 to 893) repeats. The inverted repeats contain a stem-loop structure similar to the pGI3 dso
family. A putative single strand replication origin (ssoA-type) was also found within this
region (coordinates 448 to 574). This sso contains an imperfect stem loop structure, and has
five of the six conserved nucleotides (aAGCGA) found within the ssoA-type loop (Khan 1997,
del Solar, Giraldo et al. 1998, Khan 2000).
Presumptive functions were assigned to three of the five ORFs present on pSMQ173b. ORF3
was linked to a replication initiator protein of the pGI3 family of RC plasmid. ORF3 also
displays homology with several transposon proteins and phage replication proteins. ORF4
possessed a FtsK domain with a putative ATP binding P-loop motif (Walker, Saraste et al.
1982). Proteins of the FtsK/SpoIIIE family encoded by conjugative plasmids and transposons
are required for DNA transfer (Begg, Dewar et al. 1995). Interestingly, ORF5 also shares
homologies to proteins involved in plasmid replication, but of the pSN2 family of RC
plasmids (Table 3.2) (Khan 1997). Thus, it seems likely that ORF3, ORF4, and ORF5 are
necessary for the replication or stability of pSMQ173b. However, Geis et al. (Geis, El
Demerdash et al. 2003) have shown that a mini-replicon containing only the ORFs 2 and 3 of
pER1-2 can be established. It was also previously demonstrated that pSMQ173b replicates via
a rolling circle mode (Turgeon and Moineau 2001). Consequently, these data strongly suggest
that pER1-2 and pSMQ173b replicate via a RC mechanism of the pGI3 family.
DNA sequence analysis of pSMQ308. The S. thermophilus plasmid pSMQ308 is a member
of the group B DNA homology (Turgeon and Moineau 2001). It contains 8,144 bp with a G+C
content of 38%. Nucleotide sequence analysis of pSMQ308 revealed a certain number of orfs,
but only six were studied further (Table 3.3). Putative promoters were recognized upstream of
orf1 (1333TTGGAA-13-TATACT), orf3 (4152TTGATT-17-TATAAT), and orf6 (6633TTAAAC-
17-TATAAT). Furthermore, several putative factor-independent transcriptional terminators
were found downstream of orf2 (coordinates 4057 to 4103, ΔG = –13.6 kcal/mol) and orf6
(coordinates 8023 to 8068, ΔG = –11.9 kcal/mol; coordinates 362 to 407, ΔG = –11.8
kcal/mol; coordinates 1096 to 1147, ΔG = –7.9 kcal/mol).
The region upstream of orf1 shares several features with the origin of theta-replicating
plasmids. This non-coding DNA sequence contains an iteron-like structure with four 22-bp
repeats (coordinates 1144 to 1233). A short sequence of 9-bp is also repeated three times
(coordinates 1288 to 1312) within an A+T rich segment (coordinates 1277 to 1316). Two
inverted repeats of 20-bp and 24-bp were also found upstream of orf1 (coordinates 38 to 57
and 104 to 127, respectively). However, this region upstream of orf1 does not contain known
ssi signals and dnaA box found in several origins of replication (Sakai and Komano 1996, del
Solar, Giraldo et al. 1998). Interaction between the host DnaA and the plasmid Rep protein at
the origin facilitates the loading of DnaB and DnaC, which are essential for initiation of
replication of various theta plasmids (del Solar, Giraldo et al. 1998). The ssi signals are the
sites of specific primers synthesis by plasmid primase in replication origin of several plasmids,
like ColE2 and RSF1010 (Sakai and Komano 1996, del Solar, Giraldo et al. 1998).
Putative functions were assigned to three of the six ORFs. ORF1 shares homologies with
primase/helicase usually encoded on plasmids as well as with chromosomally encoded
helicases (Table 3.3). Most of the motifs commonly found within primase (motifs 1 to 6) and
helicase (motifs 1 to 4) are present in ORF1 (Figure 3.1), including the Walker motifs A and B
(Walker, Saraste et al. 1982, Ilyina, Gorbalenya et al. 1992). Primase and helicase are
essentials for the replication of the E. coli plasmid RSF1010 (Sakai and Komano 1996) as well
as for bacteriophage genome (Ilyina, Gorbalenya et al. 1992). The primase/helicase of
Lactobacillus plasmid pN42 is also implicated in plasmid replication (Bourniquel, Casey et al.
ORF3 is similar to several integrases of the tyrosine recombinase family (Table 3.3).
Moreover, conserved motifs (boxes A, B and C) of this protein family were found within
ORF3 (Table 3.4). Integrases of the tyrosine recombinase family are involved in plasmid copy
number control and dimer resolution (Abremski and Hoess 1984, Hayes and Sherratt 1997).
Finally, ORF4 has a high pI and contains an helix-turn-helix motif found in many DNA
Gene expression of pSMQ308 and related plasmids. The transcriptional unit of theta
replication modules often contains several orfs (Schouler, Gautier et al. 1998, Seegers, van
Sinderen et al. 2000, Emond, Lavallee et al. 2001, Boucher, Parrot et al. 2002). To determine
whether orfs of pSMQ308 are transcribed as mono- or polycistronic mRNA, Northern blot
transcriptional analysis was performed on exponentially-grown cells of S. thermophilus SMQ-
308. Four probes were used to cover most of the pSMQ308 plasmid. Hybridizations with
probe A, which was specific to orf1, revealed one transcript of 3 kb (Figure 3.2A). The size of
this transcript corresponds to the expected length of an orf1-orf2 mRNA initiated from the
promoter upstream of orf1 and stopped at a terminator-like structure downstream from orf2
A large transcript of 4.5 kb was detected in S. thermophilus SMQ-308 with probes B (orf3), C
(orf4 and orf5), and D (orf6). This transcript size indicates that orf3, orf4, orf5, and orf6 are
cotranscribed within this strain (Figure 3.2B, 3.2C, 3.2D, and 3.2E). This polycistronic mRNA
would start at the promoter recognized upstream of orf3 and end at one of the terminators
identified downstream of orf6. A smaller transcript of 3 kb was also obtained with probe C,
which corresponds to an orf3-orf4-orf5 mRNA (Figure 3.2C). Finally, a 0.5-kb transcript was
also detected in SMQ-308 with probe D. This transcript may correspond to an internal region
Similar transcriptional analyses were performed on two other S. thermophilus strains (SMQ-
312 and SMQ-316) harboring plasmids of group B (Turgeon and Moineau 2001). The
plasmids pSMQ312b and pSMQ316 have a size of approximately 7-kb. The 3-kb transcript
(probe A) was detected in the two strains indicating that the polycistronic orf1-orf2 mRNA is
conserved in the three plasmids of group B (data not shown). A 1.5-kb mRNA was detected
with the probe B indicating that the integrase gene is also transcribed in these two strains, but
on a smaller transcript (data not shown). These data suggest that pSMQ308, pSMQ312b, and
pSMQ316 replicate by a similar mechanism involving orf1, orf2, and orf3.
Replication mechanism of pSMQ308. In a previous study, no ssDNA was detected within S.
thermophilus SMQ-308, suggesting that pSMQ308 does not replicate via a rolling circle
mechanism (Turgeon and Moineau 2001). According to the DNA sequence analysis,
pSMQ308 may replicate via a theta mode. To confirm this hypothesis, 2D agarose gel
electrophoresis was performed. This method has been used in the last decade to detect the
presence of theta-shape replication intermediates (Brewer and Fangman 1987). Following the
DNA isolation, the replication intermediates are linearized with restriction enzymes prior to
electrophoresis. In the first dimension, intermediates are separated on the basis of their mass
using low voltage and low agarose density. In the second dimension, DNA molecules are
separated according to their shape with high voltage and high agarose gel density. Finally,
replication intermediates are detected by Southern hybridizations.
During the initial stages of replication, plasmids differ in size and shape from the molecules
that are produced at the end of their replication cycle. Consequently, a pool of molecules at
different stages of replication will give a specific pattern of curves on the gel. These various
replication intermediates can be observed using digestions at restriction sites located at
different positions from the origin. When the restriction site is localized on the opposite side of
a replication origin, where a bidirectional replication begins, theta shape replication
intermediates are obtained (Figure 3.3A). These intermediates give the classic bubble shape
curve and demonstrate that the plasmid replicates via a theta mechanism (Figure 3.3). A
computer program was used to predict the 2D gel pattern obtain with the HindIII fragment of
pSMQ308 with a bidirectional replication from the putative origin (Figure 3.3B) (Viguera,
Rodriguez et al. 1998). The corresponding pattern of bubble shape molecules was observed
with pSMQ308 HindIII digestion (Figure 3.3C).
Two other curves were also observed in the 2D agarose gel. One curve was characteristic of
intermediates initiated outside the restriction fragment studied, and also of replication forks
already beyond this DNA region. The other curve may represent molecules in which plasmid
replication ended at random (Santamaria, Viguera et al. 2000). Similar plasmid replicating
patterns were observed with the lactococcal theta-replicating plasmid pWV02 (Kiewiet, Bron
et al. 1993). Different hypotheses may explain these patterns. The plasmid replication may
start at many locations, as observed in many eukaryotic systems (Santamaria, Viguera et al.
2000). It may also suggest that the velocity of the replication forks is not the same in both
directions or that the two forks did not start at the same time or both (Santamaria, Viguera et
al. 2000). Furthermore, one or both of these curves may be caused by the progression of the
replication fork along plasmid multimers (Bruand, Ehrlich et al. 1991, Martin-Parras,
Hernandez et al. 1992, Frère, Novel et al. 1993).
Nonetheless, the presence of bubble replication intermediates indicated that pSMQ308
replicates by a theta mechanism. To our knowledge, this is the first demonstration of a S.
thermophilus plasmid that replicates via a theta mode. Plasmids encoding similar
primase/helicase (Table 3.3) have been previously reported in Lactobacillus delbrueckii,
although their mode of replication was not investigated. These plasmids could also replicate
via a theta mechanism (Azcarate-Peril and Raya 2002, Bourniquel, Casey et al. 2002).
Classification of S. thermophilus plasmids. The report of the nucleotide sequence of
pSMQ173b and pSMQ308 now brings to 20 the total of completely sequenced S. thermophilus
plasmids (Table 3.5). Janzen et al. (1992) have previously proposed to classify S.
thermophilus plasmids within five DNA homology groups (I to V). Recently, Geis et al.
(2003) sequenced at least one plasmid of each of these groups and proposed to reduce the
number of DNA homology groups to four. We also reported a classification of S. thermophilus
plasmids into four DNA groups (Turgeon and Moineau 2001). All the necessary information is
now available to establish links between these two classifications.
Comparative sequence analysis of the 20 S. thermophilus plasmids promptly revealed that two
of our DNA homology groups are not present in the plasmid collection of Geis et al. (2003)
and vice versa. Consequently, at least six DNA homology groups of plasmids are found in S.
thermophilus strains. The mechanism of replication and incompatibility was then used to
classify these S. thermophilus plasmids.
Thirteen S. thermophilus plasmids share significant homologies with plasmids of the rolling-
circle pC194/pUB110 family (Table 3.5). This set of plasmids includes the DNA homology
group A (Turgeon and Moineau 2001) as well as the groups I and IV of Janzen et al. (Janzen,
Kleinschmidt et al. 1992). The three highly related S. thermophilus plasmids pER1-2, pSt22-2
(group II, Janzen, Kleinschmidt et al. 1992, Geis, El Demerdash et al. 2003), and pSMQ173b
(group D, this study) were classified into the pGI3 family of RC plasmids (Osborn) (Table
3.5). The plasmid pSMQ172 (group C, Turgeon and Moineau 2001) is unique among the S.
thermophilus plasmids analyzed thus far and is closely related to the RC plasmids of the
pMV158 family (Table 3.5). The Rep protein of plasmid pST0 (group V, Janzen,
Kleinschmidt et al. 1992, Geis, El Demerdash et al. 2003) shares similarities with the Rep
protein of the S. mutans plasmid pUA140, which belongs to the pT181 family of the RC
plasmids (Zhou, Caufield et al. 2001) (Table 3.5). The plasmid pSt106 (group III, Janzen,
Kleinschmidt et al. 1992, Geis, El Demerdash et al. 2003) does not share similarity with any
existing family of replicons. While waiting for more information on its mechanism of
replication, it was placed in a distinct group (Table 3.5). Finally, pSMQ308 (group B) was
classified into a primase/helicase theta family (Table 3.5).
Cloning vectors using the replicons from these six S. thermophilus plasmid groups are likely
to be compatible as they use different replication machineries. Besides, RC plasmids of the
pC194/pUB110 and pGI3 families have already been found in S. thermophilus strains, namely
SMQ-173, St22, and ER1 (Turgeon and Moineau 2001, Geis, El Demerdash et al. 2003).
Similarly, a RC plasmid of the pC194/pUB110 and a theta plasmid were identified in S.
thermophilus SMQ-312 (Turgeon and Moineau 2001, this study).
This proposal should simplify the analysis of additional S. thermophilus plasmids as well as
the development of compatible cloning vectors. Vectors based on theta-type replicon are
generally favored, because they are more stable and have a limited host range as opposed to
RC plasmids (Jannière, Bruand et al. 1990, Ehrlich, Bruand et al. 1991). This classification is
also likely to be extended as more studies on plasmid replication are conducted.
We are very grateful to J.B. Schvartzman for the gift of the computer program for 2D gel
analysis. We also acknowledge P.H. Roy for his help in the sequence analysis. This work was
funded by research grants from the Natural Sciences and Engineering Research Council of
Canada (NSERC) and the Fonds Québécois pour la Recherche sur la Nature et les
Technologies (FQRNT). N.T. is a recipient of graduate scholarships from NSERC and
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