Characterization of a theta-replicating plasmid from Streptococcus thermophilus

Département de biochimie et de microbiologie, Faculté des sciences et de génie, Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec, Canada G1K 7P4.
Plasmid (Impact Factor: 1.58). 02/2004; 51(1):24-36. DOI: 10.1016/j.plasmid.2003.09.004
Source: PubMed
ABSTRACT
Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-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 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot 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 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.

Full-text

Available from: Nathalie Turgeon, Jun 19, 2014
Plasmid (2004) 51 :24-36.
Characterization of a theta replicating plasmid from
Streptococcus thermophilus.
Nathalie Turgeon
1
, Michel Frenette
1
and Sylvain Moineau
1
1
partement 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: sylvain.moineau@bcm.ulaval.ca
(S. Moineau).
Abbreviations used: LAB, lactic acid bacteria; RC, rollingcircle replication; ssDNA, single-
stranded DNA; ssi, singlestranded initiating signal; sso, single-stranded replication origin.
doi:10.1016/j.plasmid.2003.09.004
http://www.sciencedirect.com/science/article/pii/S0147619X03000805
Page 1
2
2
ABSTRACT
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.
Page 2
3
3
INTRODUCTION
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
modification.
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
Page 3
4
4
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
proposed.
Page 4
5
5
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
Page 5
6
6
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 cm
2
. CDP-Star was utilized for
chemiluminescent detection.
Accession number. The DNA sequences of pSMQ173b and pSMQ308 are available under
the GenBank accession numbers AY312235 and AY312234, respectively.
Page 6
7
7
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 (
62
TTAATA-16-TATAAT), orf3 (
1351
TTGAAC-
19-TAAAAT), orf4 (
1609
TTGAAC-20-TATAAT), and on the complementary strand upstream
of orf5 (
4443
TTGACT-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
Page 7
8
8
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 (
1333
TTGGAA-13-TATACT), orf3 (
4152
TTGATT-17-TATAAT), and orf6 (
6633
TTAAAC-
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
Page 8
9
9
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.
2002).
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
binding proteins.
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
(Figure 3.2E).
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
of orf6.
Page 9
10
10
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,
Page 10
11
11
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.
Page 11
12
12
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
Page 12
13
13
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.
Page 13
14
14
ACKNOWLEDGEMENTS
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
FQRNT.
Page 14
15
15
REFERENCES
Abremski, K. and R. Hoess (1984). "Bacteriophage P1 site-specific recombination.
Purification and properties of the Cre recombinase protein." J. Biol. Chem. 259(3):
1509-1514.
Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman
(1997). "Gapped BLAST and PSI-BLAST, a new generation of protein database
search programs." Nucleic Acids Res. 25: 3389-3402.
Azcarate-Peril, M. A. and R. R. Raya (2002). "Sequence analysis of pLBB1, a cryptic plasmid
from Lactobacillus delbrueckii subsp. bulgaricus." Can. J. Microbiol. 48(2): 105-112.
Begg, K. J., S. J. Dewar and W. D. Donachie (1995). "A new Escherichia coli cell division
gene, ftsK." J. Bacteriol. 177(21): 6211-6222.
Birnboim, H. C. and J. Doly (1979). "A rapid alkaline extraction procedure for screening
recombinant plasmid DNA." Nucleic Acids Res. 7: 1513-1523.
Boucher, I., M. Parrot, H. Gaudreau, C. P. Champagne, C. Vadeboncoeur and S. Moineau
(2002). "Novel food-grade plasmid vector based on melibiose fermentation for the
genetic engineering of Lactococcus lactis." Appl. Environ. Microbiol. 48(12): 6152-
6161.
Bourniquel, A. A., M. G. Casey, B. Mollet and R. D. Pridmore (2002). "DNA sequence and
functional analysis of Lactobacillus delbrueckii subsp. lactis plasmids pN42 and
pJBL2." Plasmid 47(2): 153-157.
Brewer, B. J. and W. L. Fangman (1987). "The localization of replication origins on ARS
plasmids in S. cerevisiae." Cell 51: 463-471.
Brewer, B. J., E. P. Sena and W. L. Fangman (1988). "Analysis of replication intermediates by
two-dimensional agarose gel electrophoresis." Cancer Cells 6: 229-234.
Bruand, C., S. D. Ehrlich and L. Jannière (1991). "Unidirectional theta replication of the
structurally stable Enterococcus faecalis plasmid pAMb1." EMBO J. 10(8): 2171-
2177.
Dao, M. L. and J. J. Ferretti (1985). "Streptococcus-Escherichia coli shuttle vector pSA3 and
its use in the cloning of streptococcal genes." Appl. Environ. Microbiol. 49(1): 115-
119.
de Vos, W. M. (1987). "Gene cloning and expression in lactic streptococci." FEMS Microbiol.
Rev. 46: 281-295.
del Solar, G., R. Giraldo, M. J. Ruiz-Echevarria, M. Espinosa and R. Diaz-Orejas (1998).
"Replication and control of circular bacterial plasmids." Microbiol. Mol. Biol. Rev.
62(2): 434-464.
Devereux, J., P. Haeberli and O. Smithies (1984). "A comprehensive set of sequence analysis
programs for the VAX." Nucleic Acids Res. 12: 387-395.
Ehrlich, S. D., C. Bruand, S. Sozhamannan, P. Dabert, M. F. Gros, L. Jannière and A. Gruss
(1991). "Plasmid replication and structural stability in Bacillus subtilis." Res.
Microbiol. 142(7-8): 869-873.
Emond, E., R. Lavallee, G. Drolet, S. Moineau and G. LaPointe (2001). "Molecular
characterization of a theta replication plasmid and its use for development of a two-
component food-grade cloning system for Lactococcus lactis." Appl. Environ.
Microbiol. 67(4): 1700-1709.
Page 15
16
16
Esposito, D. and J. J. Scocca (1997). "The integrase family of tyrosine recombinase: evolution
of a conserved active site domain." Nucleic Acids Res. 25(18): 3605-3614.
Frère, J., M. Novel and G. Novel (1993). "Molecular analysis of the Lactococcus lactis subsp.
lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22." Mol.
Microbiol. 10(5): 1113-1124.
Geis, A., H. A. M. El Demerdash and K. J. Heller (2003). "Sequence analysis and
characterization of plasmids from Streptococcus thermophilus." Plasmid 50: 53-69.
Girard, F., M. Lautier and G. Novel (1987). "DNA:DNA homology between plasmids from
Streptococcus thermophilus." Lait 67: 537-544.
Hashiba, H., R. Takiguchi, J. Kenichi, A. Kenji and T. Hirota (1993). "Identification of the
replication region of Streptococcus thermophilus No. 29 plasmid pST1." Biosci.
Biotechnol. Biochem. 57: 1646-1649.
Hayes, F. and D. J. Sherratt (1997). "Recombinase binding specificity at the chromosome
dimer resolution site dif of Escherichia coli." J. Mol. Biol. 266(3): 525-537.
Herman, R. E. and L. L. McKay (1985). "Isolation and partial characterisation of plasmid
DNA from Streptococcus thermophilus." Appl. Environ. Microbiol. 50: 1103-1106.
Ilyina, T. V., A. E. Gorbalenya and E. V. Koonin (1992). "Organization and evolution of
bacterial and bacteriophage primase-helicase systems." J. Mol. Evol. 34(4): 351-357.
Jannière, L., C. Bruand and S. D. Ehrlich (1990). "Structurally stable Bacillus subtilis cloning
vectors." Gene 87(1): 53-61.
Janzen, T., J. Kleinschmidt, H. Neve and A. Geis (1992). "Sequencing and characterization of
pST1, a cryptic plasmid from Streptococcus thermophilus." FEMS Microbiol. Lett.
95: 175-180.
Khan, S. A. (1997). "Rolling-circle replication of bacterial plasmids." Microbiol. Mol. Biol.
Rev. 61(4): 442-455.
Khan, S. A. (2000). "Plasmid rolling-circle replication: recent developments." Mol. Microbiol.
37(3): 477-484.
Kiewiet, R., S. Bron, K. de Jonge, G. Venema and J. F. M. L. Seegers (1993). "Theta
replication of the lactococcal plasmid pWV02." Mol. Microbiol. 10(2): 319-327.
Martin-Parras, L., P. Hernandez, M. L. Martinez-Robles and J. B. Schvartzman (1992).
"Initiation of DNA replication in ColE1 plasmids containing multiple potential
origins of replication." J. Biol. Chem. 267(31): 22496-22505.
Moineau, S., S. A. Walker, B. J. Holler, E. R. Vedamuthu and P. A. Vandenbergh (1995).
"Expression of a Lactococcus lactis phage resistance mechanism by Streptococcus
thermophilus." Appl. Environ. Microbiol. 61: 2461-2466.
O'Sullivan, D. J. and T. R. Klaenhammer (1993). "High- and low-copy-number Lactococcus
shuttle cloning vectors with features for clone screening." Gene 137(2): 227-231.
O'Sullivan, D. J. and T. R. Klaenhammer (1993). "Rapid mini-prep isolation of high quality
plasmid DNA from Lactococcus lactis and Lactobacillus spp." Appl. Environ.
Microbiol. 59: 2730-2733.
O'Sullivan, T. F., D. van Sinderen and G. F. Fitzgerald (1999). "Structural and functional
analysis of pCI65st, a 6.5 kb plasmid from Streptococcus thermophilus NDI-6."
Microbiology 145: 127-134.
Osborn, M. "Database of plasmid replicons (DPR)." [En ligne].
http://www.essex.ac.uk/bs/staff/osborn/DPR_home.htm (Page consultée le 12 juin
2003).
Page 16
17
17
Sakai, H. and T. Komano (1996). "DNA replication of IncQ broad-host-range plasmids in
gram-negative bacteria." Biosci. Biotechnol. Biochem. 60(3): 377-382.
Sambrook, J. and D. W. Russell (2001). Molecular cloning: a laboratory manual. Cold Spring
Harbor, N.Y., Cold Spring Harbor Laboratory Press.
Santamaria, D., E. Viguera, M. L. Martinez-Robles, O. Hyrien, P. Hernandez, D. B. Krimer
and J. B. Schvartzman (2000). "Bi-directional replication and random termination."
Nucleic Acids Res. 28(10): 2099-2107.
Schouler, C., M. Gautier, S. D. Ehrlich and M. C. Chopin (1998). "Combinational variation of
restriction modification specificities in Lactococcus lactis." Mol. Microbiol. 28(1):
169-178.
Seegers, J. F., D. van Sinderen and G. F. Fitzgerald (2000). "Molecular characterization of the
lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I
restriction/modification system in a single lactococcal strain." Microbiology 146(( Pt
2)): 435-443.
Solaiman, D. K. Y. and G. A. Somkuti (1993). "Shuttle vectors developed from Streptococcus
thermophilus native plasmid." Plasmid 30: 67-78.
Solaiman, D. K. Y. and G. A. Somkuti (1997). "Characterization of pER371-based
Streptococcus thermophilus- Escherichia coli shuttle vectors." Biotechnol. Lett. 19:
595-598.
Solaiman, D. K. Y. and G. A. Somkuti (1997). "Construction of a green-fluorescent protein-
based, insertion-inactivation shuttle vector for lactic acid bacteria and Escherichia
coli." Biotechnol. Lett. 19(12): 1175-1179.
Solaiman, D. K. Y. and G. A. Somkuti (1998). "Characterization of a novel Streptococcus
thermophilus rolling-circle plasmid used for vector construction." Appl. Microbiol.
Biotechnol. 50(2): 174-180.
Solow, B. T. and G. A. Somkuti (2000). "Comparison of low-molecular-weight heat stress
proteins encoded on plasmids in different strains of Streptococcus thermophilus."
Curr. Microbiol. 41(3): 177-181.
Somkuti, G. A., D. K. Y. Solaiman and D. H. Steinberg (1998). "Structural and functional
properties of the hsp16.4-bearing plasmid pER341 in Streptococcus thermophilus."
Plasmid 40(1): 61-72.
Somkuti, G. A. and D. H. Steinberg (1986). "Distribution and analysis of plasmids in
Streptococcus thermophilus." J. Ind. Microbiol. 1: 157-163.
Somkuti, G. A. and D. H. Steinberg (1999). "Promoter activity of the pER341-borne ST(Phsp)
in heterologous gene expression in Escherichia coli and Streptococcus thermophilus."
FEMS Microbiol. Lett. 179(2): 431-436.
Su, P., K. Jury, G. E. Allison, W. Y. Wong, W. S. Kim, C. Q. Liu, T. Vancov and N. W. Dunn
(2002). "Cloning vectors for Streptococcus thermophilus derived from a native
plasmid." FEMS Microbiol. Lett. 216(1): 43-47.
Turgeon, N. and S. Moineau (2001). "Isolation and characterization of a Streptococcus
thermophilus plasmid closely related to the pMV158 family." Plasmid 45: 171-183.
Viguera, E., A. Rodriguez, P. Hernandez, D. B. Krimer, O. Trellez and J. B. Schvartzman
(1998). "A computer model for the analysis of DNA replication intermediates by two-
dimensional agarose gel electrophoresis." Gene 217(1-2): 41-49.
Walker, J. E., M. Saraste, M. J. Runswick and N. J. Gay (1982). "Distantly related sequences
in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-
requiring enzymes and a common nucleotide binding fold." EMBO J. 1(8): 945-951.
Page 17
18
18
Wells, J. M., P. W. Wilson and R. W. Le Page (1993). "Improved cloning vectors and
transformation procedure for Lactococcus lactis." J. Appl. Bacteriol. 74(6): 629-636.
Zhou, X., P. W. Caufield, Y. Li and F. Qi (2001). "Complete nucleotide sequence and
characterization of pUA140, a cryptic plasmid from Streptococcus mutans." Plasmid
46(2): 77-85.
Page 18
19
19
Table 1 Bacterial strains and plasmids used in this study.
Bacteria
or plasmids
Source
Bacteria
S. thermophilus
SMQ-173
(Moineau, Walker
et al. 1995)
SMQ-308
(Turgeon and
Moineau 2001)
SMQ-312
(Turgeon and
Moineau 2001)
SMQ-316
(Turgeon and
Moineau 2001)
E. coli
DH5α
Stratagene
Plasmids
pBS KS+
Stratagene
pSMQ173b
(Turgeon and
Moineau 2001)
pSMQ308
(Turgeon and
Moineau 2001)
pSMQ312b
(Turgeon and
Moineau 2001)
pSMQ316
(Turgeon and
Moineau 2001)
Ap
r
, ampicillin resistance
Page 19
20
Table 2 General features of the five orfs of plasmid pSMQ173b and comparison with databases.
ORF
Start
Stop
pI
Size
aa
MW
kDa
SD sequence
AAAGGAGGTGA 16S rRNA
Function or similarity
Percentage
identity (aa)
Accession
number
1
123
443
4.3
106
12.7
AAAGGAGGATTtacattATG
S. thermophilus, pER1-2 ORF1
99 (105/106)
AJ242478.2
2
941
1348
9.1
135
15.2
AAAGGACATTTtttatATG
S. thermophilus, pER1-2 ORF2
99 (131/132)
AJ242478.2
3
1473
2579
9.0
368
42.5
ATAGG.GGTGTtagaATG
S. thermophilus, pER1-2 ORF3
B. subtilis, ydcR
S. agalactiae, SAG1988
N. meningitidis, phage rep protein
E. faecalis, Tn916 ORF20
St. aureus, SAV0398
97 (191/195)
25 (73/285)
25 (72/288)
24 (60/248)
22 (73/324)
22 (69/302)
AJ242478.2
AB001488.1
NC_004116.1
AL162754.2
U09422
AP003359
4
2581
3927
9.4
448
49.5
AAAGAAGGTGAatactaatATG
S. thermophilus, pER1-2 ORF4
C. acetobutylicum, Ftsk-like
B. subtilis, SpoIIIE
E. faecalis, Tn916 ORF21
L. lactis IL1403, FtsK
99 (447/448)
28 (64/227)
25 (61/241)
24 (77/313)
23 (61/257)
AJ242478.2
AE007695.1
Z99119
U09422
AE006396.1
5
4361
3921
9.0
146
16.8
AAAAGAGACATatATG
S. thermophilus pER1-2 ORF5
Pseudoalteromonas sp. pPS1M3 Rep
Se. ruminantium pJW1 RepA
98 (100/102)
46 (18/39)
34 (34/100)
AJ242478.2
AB022096.1
AF141869.1
SD, underlined are conserved nucleotides as compared to the consensus Shine-Dalgarno sequence; aa, amino acids; B. subtilis, Bacillus subtilis; C.
acetobutylicum, Clostridium acetobutylicum; E. faecalis, Enterococcus faecalis; L. lactis, Lactococcus lactis; N. meningitidis, Neisseria
meningitidis; S. agalactiae, Streptococcus agalactiae; S. thermophilus, Streptococcus thermophilus; Se. ruminantium, Selenomonas ruminantium;
St. aureus, Staphylococcus aureus.
Page 20
21
Table 3 General features of the six orfs of plasmid pSMQ308 and comparison with databases.
ORF
Start
Stop
pI
Size
aa
MW
kDa
SD sequence
AAAGGAGGTGA 16S rRNA
Function or similarity
Percentage
identity (aa)
Accession
number
1
1429
3507
5.3
692
76.9
AAAGGAGATTTtgacaATG
Lb. delbrueckii pLBB1, primase/helicase
Lb. delbrueckii pN42, DNA binding protein
Lb. delbrueckii pJBL2, replication protein
Lb. delbrueckii pLL1212, primase/helicase
L. lactis IL1403, DNA helicase
B. subtilis, replicative DNA helicase
B. halodurans, replicative DNA helicase
31 (173/542)
31 (154/483)
31 (153/483)
30 (146/472)
27 (91/335)
26 (118/451)
26 (113/425)
AF236060
AJ421627.1
AJ421486.1
AF109691
AE006308.1
NC_000964.1
AP001520
2
3524
4069
9.6
181
20.6
GTAGGAGCGTTcttatATG
Unknown
3
4220
5146
9.5
308
35.5
AAAGGAGCGCGtagtATG
Lb. delbrueckii pWS58, integrase
T. tengcongensis, integrase
C. tetani, integrase/recombinase
Anabaena sp., integrase/recombinase
P. abyssi, integrase/recombinase xerD
Lp. interrogans, integrase xerC
Synechocystis sp., integrase xerC
35 (110/310)
32 (90/313)
31 (97/318)
30 (87/274)
30 (83/275)
29 (82/282)
28 (83/296)
Z50864.1
NC_003869.1
AE015940.1
AP003605.1
NC_003413.1
NC_004342.1
D90899.1
4
5190
5372
9.6
60
7.0
AAAGGCGGTTAataATG
HTH motif
5
5513
6946
4.9
478
56.0
AGAGGGGTTTAgaATG
Unknown
6
6939
7988
9.6
349
39.4
AAAGAGGGGGTagaagATG
Unknown
SD, underlined are conserved nucleotides as compared to the consensus Shine-Dalgarno sequence; aa, amino acids; B. halodurans, Bacillus
halodurans; B. subtilis, Bacillus subtilis; C. tetani, Clostridium tetani;L. lactis, Lactococcus lactis; Lb. delbrueckii, Lactobacillus delbrueckii; Lp.
interrogan, Leptospira interrogan; P. abyssi, Pyrococcus abyssi; T. tengcongensis, Thermoanaeobacter tengcongensis.
Page 21
22
Table 4 Comparative sequence analysis between the ORF3 of pSMQ308 and conserved motifs of tyrosine recombinases.
Box A
Box B
Box C
Consensus
A
S
UUxUUxxTGURXGEUxxLxxxDUD
S S
A A
HxLRHTxATxLxxxG
UxxxLGHxxxxxTxxYxH
pSMQ308
ILSLMVTgGLRTiEVSRADVGDLr
HSLRHTAiTLaLLAG
VQQFarHANLNTTMIYNH
Capital letters denote amino acids that respect the consensus (Esposito and Scocca 1997). In the consensus sequence, U denotes
hydrophobic amino acid, x represents any amino acid, capital letters indicate conserved position in more than 50% of the tyrosine
recombinase proteins and obligate conserved positions are underlined.
Page 22
Table 5 Classification of S. thermophilus plasmids.
Plasmid
Size
(kb)
Gene encoding
putative function
Accession
number
References
pC194/pUB110 rolling-circle family
pST1
2.1
-
X65856
(Janzen, Kleinschmidt et al. 1992)
pST1
2.7
-
S67974
(Hashiba, Takiguchi et al. 1993)
pER371
2.7
-
AF022180
(Solaiman and Somkuti 1998)
pER341
2.8
hsp
AF019139
(Somkuti, Solaiman et al. 1998)
pSt04
3.1
hsp
AJ242477
(Geis, El Demerdash et al. 2003)
pER1-1
3.4
hsp
AJ242476
(Geis, El Demerdash et al. 2003)
pJ34
3.4
-
AJ242475
(Geis, El Demerdash et al. 2003)
pND103
3.5
hsp
-
(Su, Jury et al. 2002)
pER36
3.7
hsp
AF177168
(Solow and Somkuti 2000)
pER16
4.5
HsdS (R/M type I)
hsp
AF177166
(Solow and Somkuti 2000)
pCI65st
6.5
Enolase
HsdS (R/M type I)
hsp
AF027167
(O'Sullivan, van Sinderen et al.
1999)
pSt08
7.5
R/M type II
AJ239049
(Geis, El Demerdash et al. 2003)
pER35
10
R/M type I
hsp
AF177167
(Solow and Somkuti 2000)
pGI3 rolling-circle family
pER1-2
4.4
-
AJ242478
(Geis, El Demerdash et al. 2003)
pSMQ173b
4.4
-
AY312235
This study
pSt22-2
4.4
-
-
(Geis, El Demerdash et al. 2003)
pMV158 rolling-circle family
pSMQ172
4.2
mob
AF295100
(Turgeon and Moineau 2001)
pT181 rolling-circle family
pSt0
8.1
R/M type II
AJ242480
(Geis, El Demerdash et al. 2003)
Primase/helicase theta family
pSMQ308
8.1
integrase
AY312234
This study
pST106 like
pSt106
5.3
primase
resolvase
AJ242479
(Geis, El Demerdash et al. 2003)
Page 23
Figure 1 Comparative sequence analysis between the ORF1 of pSMQ308 and the primase/helicase (gp4) of bacteriophage T7.
Asterisks indicate conserved amino acids. The six distinct conserved motifs of primases and the four conserved motifs of helicases are
boxed (Ilyina, Gorbalenya et al. 1992). Amino acids conserved in most of the primases and helicases are indicated in boxes.
Page 24
Figure 2 Transcriptional analysis of pSMQ308. The 0.3 kb to 9.5 kb RNA ladder is from
Invitrogen Life Technologies. Panel A, experiment performed with probe A
specific to orf1 (coordinates 1372-3522); panel B, experiment carried out with
probe B specific to orf3 (coordinates 4688-5157); panel C, experiment done with
probe C specific to orf4 and orf5 (coordinates 5241-6504); panel D, experiment
completed with probe D specific to orf6 (coordinates 7198-8023). Panel E, Map of
pSMQ308. Thick arrows on the map designate orfs (the direction of transcription
is indicated). Small arrows symbolize the promoters. Terminators are indicated by
stem-loop structures. Thick lines above the map represent transcripts with respect
of the promoters and terminators found in the sequence analysis. Thick lines below
the map illustrate the probes used in the experiments.
3 kb
0.5 kb
3 kb
4.5 kb
4.5 kb
4.5 kb
Page 25
26
Figure 3 Replication intermediates analysis of pSMQ308. Panel A, representation of replication intermediates of a 7.5-kb HindIII
fragment from pSMQ308 (coordinates 6309-5913). Panel B, schematic representation of 2D gel patterns of the same
HindIII fragment made with a computer program (ori coordinates 1300) (Viguera, Rodriguez et al. 1998). Panel C, 2D
agarose gel electrophoresis of HindIII digested pSMQ308. The membrane was hybridized with probe B (coordinates
4688-5157).
Page 26
Show more