Salmonella enterica subsp. enterica serovar Enteritidis harbours ...

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Salmonella enterica subsp. enterica serovar Enteritidis harbours ColE1, ColE2, and rollingcircle-like replicating plasmids Daniela Gregorova, Jitka Matiasovicova, Alena Sebkova, Marcela Faldynova, and Ivan Rychlik

Abstract: Using DNA hybridization, at least three distinct groups of low molecular mass plasmids were identified in Salmonella enterica subsp. enterica serovar Enteritidis. After sequencing representative plasmids from each group, we concluded that they belonged to ColE1, ColE2, and rolling-circle-like replicating plasmids. Plasmid pK (4245 bp) is a representative of widely distributed ColE1 plasmids. Plasmid pP (4301 bp) is homologous to ColE2 plasmids and was present predominantly in single-stranded DNA form. The smallest plasmids pJ (2096 bp) and pB (1983 bp) were classified as rolling-circle-like replicating plasmids. Both encoded only a single protein essential for their own replication, and they must have existed in an unusual molecular structure, as (i) they were capable of hybridization without denaturation, (ii) their DNA could be linearized with S1 nuclease, and (iii) even after such treatment, the ability to hybridize without denaturation did not disappear. Key words: Salmonella enterica subsp. enterica serovar Enteritidis, ColE1, ColE2, RCR, plasmid, rolling-circle replication. Résumé : Nous avons identifié par hybridation de l’ADN au moins trois groupes distincts de plasmides de masses moléculaires réduites chez Salmonella enterica subsp. enterica serovar Enteritidis. À la suite du séquençage de plasmides représentatifs de chaque groupe, nous avons déduit qu’ils appartenaient aux plasmides se répliquant par ColE1, ColE2 et par cercle roulant. Le plasmide pK (4245 pb) est un représentant des plasmides communs ColE1. Le plasmide pP (4301 pb) est homologue aux plasmides ColE2 et se retrouvait principalement sous forme monocaténaire. Les plus petits plasmides pJ (2096 pb) et pB (1983 pb) ont été classés dans les plasmides à réplication par cercle roulant. Ces derniers codaient une seule protéine essentielle à leur propre réplication. Ils devaient présenter une structure singulière puisque (i) ils étaient capables d’hybridation sans dénaturation, (ii) leur ADN pouvait être linéarisé par la nucléase S1 et (iii) même après ce traitement, leur capacité à s’hybrider sans dénaturation n’a pas été enrayée. Mots clés : Salmonella enterica subsp. enterica serovar Enteritidis, ColE1, ColE2, RCR, plasmides, réplication par cercle roulant. [Traduit par la Rédaction]

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Introduction Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is the most frequent causative agent of human salmonellosis in Europe (Rabsch et al. 2001), and therefore its epidemiology is a subject of particular interest. Numerous methods have been used to investigate the spread of the infection during outbreaks. One method is the plasmid profile analysis, which utilizes the presence or absence of different plasmids in bacterial cells (Dorn et al. 1993; Millemann et al. 1995). Using the plasmid profile analysis for differentiation of Salmonella Enteritidis (Rychlik et al. Received 25 July 2003. Revision received 1 December 2003. Accepted 8 December 2003. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 16 March 2004. D. Gregorova, J. Matiasovicova, A. Sebkova, M. Faldynova, and I. Rychlik.1 Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic. 1

Corresponding author (e-mail: [email protected]).

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1998), we identified nearly 30 different low molecular mass plasmids and later characterized them in more detail (Rychlik et al. 2001; Gregorova et al. 2002). We have found that the plasmid designated as pI codes for an unusual enzyme retron reverse transcriptase and influences phage resistance (Rychlik et al. 2001). Another plasmid pC we have determined to encode a functional restriction and modification system (Gregorova et al. 2002). Despite this, only limited information is available on the small plasmids present in Salmonella spp. and on and biological functions for which they are responsible. In Salmonella Enteritidis, plasmid pFM366 encodes a nonfunctional restriction–modification system (Ibanez and Rotger 1993). In related S. enterica subsp. enterica serovar Typhimurium, only a few plasmids have been characterized in detail by DNA sequencing, and their biological roles remain unclear (Bernardi and Bernardi 1984; Astill et al. 1993). In the rather rare serovar S. enterica subsp. enterica serovar Borreze, a plasmid was shown to encode the O:54 antigen (Keenleyside and Whitfield 1995). A small plasmid was described in the vaccine for S. enterica subsp. enterica serovar Choleraesuis strain (Liu et al. 2002). Finally, in S. enterica

doi: 10.1139/W03-113

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Fig. 1. Gel electrophoresis of the most frequent plasmids present in Salmonella enterica subsp. enterica serovar Enteritidis (A), and hybridization results when plasmid pC (B), plasmid pP (C), or plasmid pJ (D) were used as probes. Lanes: M, λ/HindIII molecular weight standard; 1, pP; 2, pN and pO; 3, pM; 4, pL; 5, pK; 6, pC; 7, pD; 8, pS; 9, pI and pJ; 10, pH and pF and pU; 11, pA and pH and pB; 12, pH; 13, pE; 14, pE and pG; 15, pG.

subsp. enterica serovar Typhi D4, a multicopy plasmid has been identified encoding a restriction–modification system (Miyahara et al. 1997). From the point of view of plasmid replication, plasmids are classified into several groups. In Gram-negative bacteria, Col-like plasmids replicating by a theta-form mechanism have been described the most frequently (Bernardi and Bernardi 1984; Mruk et al. 2001; Fu et al. 1995). Rollingcircle replicating plasmids have been found less frequently in Gram-negative bacteria (Yasukawa et al. 1991) but frequently in Gram-positive bacteria (Khan 1997). Having a well-established system for plasmid identification (Rychlik et al. 1998) and having characterized the first two plasmids in detail (Rychlik et al. 2001; Gregorova et al. 2002), we were interested in a more general characterization of plasmids occurring in Salmonella Enteritidis. We therefore characterized plasmids by DNA–DNA hybridization, sequenced at least one representative plasmid from each group of plasmids, and examined the predicted modes of replication.

Materials and methods Bacterial strains, their storage and propagation Over 1000 of the field strains of Salmonella Enteritidis were initially characterized by plasmid profiling (Rychlik et

al. 1998). Out of these strains, 15 strains representing the most frequent plasmid profiles were selected. The strains were stored at –70 °C in Luria–Bertani broth (Difco Laboratories, Detroit, Mich., U.S.A.) supplemented with 15% glycerol. During laboratory passages, the strains were grown in Luria–Bertani broth or agar at 37 °C. Recombinant DNA techniques Unless otherwise stated, all the plasmids were purified by a QIAprep Miniprep kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions, with only one exception (in contrast to maunfacturer’s recommendations): the strains were grown on Luria–Bertani agar plates, and approximately 4 cm2 of the dense culture was scraped by loop and resuspended directly in the initial P1 buffer. After the purification, the plasmids were electrophoresed in 0.8% agarose, visualized by ethidium bromide staining, and vacuum blotted on the Hybond N nylon membrane (Amersham Biosciences UK Limited, Buckingshire, U.K.). The whole plasmids labelled by an ECL Direct Labelling kit (Amersham) were used as hybridization probes. An initial plasmid to be used as a probe was randomly selected. After signal development, the bound probe was stripped off by incubation of the membrane in 0.4 mol/L NaOH at 45 °C for 30 min. Plasmid DNA probes were selected, from among those giving a negative reaction, for subsequent re-hybridization of © 2004 NRC Canada



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Fig. 2. Genetic maps of plasmids pK, pP, pJ, and pB. DR, direct repeat; IR, inverted repeat.

the membrane to classify all plasmids into individual hybridization groups (Fig. 1). All DNA modifying enzymes (restriction endonucleases, S1 nuclease) were used with buffers, following the manufacturer’s recommendations. Whenever multiple DNA modifying enzymes were used in succession, the DNA was purified using a QIAquick Gel Extraction kit (Qiagen) in between each manipulation. Plasmids digested with restriction endonuclease were electrophoresed in 1.5% agarose gel. Plasmid DNA sequencing, sequence assembly, and homology searches Selected plasmids were sequenced using an ABI310 Genetic Analyzer (Applied Biosystems, Foster City, Calif., U.S.A.) following the strategy described previously (Rychlik et al. 2001). The nucleotide sequences of the plasmids have been deposited in GenBank under accession Nos. AY079200 (pK), AY079199 (pP), AF268389 (pJ), and AY178821 (pB). Characterization of replication mode in selected plasmids Sequence analysis revealed the presence of rolling-circle plasmids, which replicate via single-stranded (ss) DNA intermediates. To detect such single-stranded intermediates, purified plasmid DNA was treated with S1 nuclease (20 U S1 nuclease for 30 min at 23 °C; 1 U ≈ 16.67 nkat). After inactivation of the S1 nuclease at 65 °C for 15 min, treated and nontreated (control) plasmid DNA was electrophoresed in 0.8% agarose gel, depurinated with 10 mmol/L HCl for 10 min, denaturated with 0.2 mol/L NaOH for 20 min, and vacuum blotted. Alternatively, the gels were vacuum blotted without depurination and denaturation to detect naturally occurring ssDNA.

Results DNA hybridization For initial hybridization, the plasmid designated as pC was selected as a probe. Most plasmids hybridized except for five, namely plasmids pP (lane 1), pD (lane 7), pJ (the smallest plasmid in lane 9), and pA and pB (the largest and smallest plasmids in lane 11) (Figs. 1A and 1B). From them, plasmid pP was used as a probe in the second round of hybridization (Figs. 1A and 1C). After the second round of hybridization, two plasmids remained, which did not react. One of these plasmids designated as pJ was finally labelled and shown to react with itself and plasmid pB (Figs. 1A and 1D). We therefore concluded that there are at least three groups of completely different plasmids present in Salmonella Enteritidis.

Plasmid sequencing and GenBank comparison One or two representative plasmids of each group were characterized by DNA sequencing to examine what types of plasmids the individual hybridization groups comprised, to determine the reasons for the cross-hybridization within individual groups, and to identify specific genes encoded by the plasmids. Representative plasmid(s) of each group were characterized by DNA sequencing. Plasmid pK (4245 bp), representing the most frequent plasmid group, encoded three open reading frames (ORFs) (Fig. 2). For the first two ORFs, no function could be predicted based on the BLAST comparison. The last ORF was homologous to mbeA (Boyd et al. 1989; Varsaki et al. 2003). Plasmid pK’s closest homologue, among already sequenced S. enterica plasmids, was plasmid pBert from S. enterica subsp. enterica serovar Berta (GenBank acc. No. AF025795). Plasmid pK was also closely related to plasmids pSW100 and pSW200 of Erwinia stewartii (Fu et al. 1995, 1998). Based on the sequence comparison, the homologous sequences were always localized in genes coding for RNA I and RNA II molecules (RNA I gene, 74–4213 bp; RNA II gene, 4213–475 bp) and in genes essential for mobilization for conjugative transfer. Therefore, the plasmid pK and all the plasmids of this hybridization group were predicted to belong to the ColE1 type of plasmids. Plasmid pP (4301 bp) encoded three ORFs (Fig. 2). The first ORF coded for a protein of which 250 N-terminal amino acids were 61% identical and 71% similar to relaxase encoded by pSC101 of Salmonella Typhimurium (Bernardi and Bernardi 1984) and of which 100 carboxy-terminal amino acids were 58% identical to and 72% similar to relaxase of R721 shufflon of Escherichia coli (Kim and Komano 1992). For the second ORF encoded by plasmid pP, no function could be predicted based on BLAST comparison. The last ORF coded for a protein that was similar to the Rep proteins of ColE2 plasmids. The Rep protein of plasmid pP was homologous to the Rep protein of plasmid ColE2imm-K317 (88% identical and 89% similar) or to the Rep protein of plasmid ColE2-GEI602 (85% identical and 88% similar) (Hiraga et al. 1994). Plasmid pJ (2096 bp) encoded only a single ORF (Fig. 2). The protein encoded by this ORF showed extensive homology to Rep proteins of plasmids replicating via the rolling-circle mechanism. Its closest homologue was plasmid pKYM from Shigella sonnei (Yasukawa et al. 1991). The pJ Rep protein was 68% identical and 80% similar to the pKYM-encoded Rep protein. In the position 1330–1442 bp of plasmid pJ, eight direct repeats of TGT GGG sequence were identified. Plasmid pB was the smallest plasmid (Fig. 2). Its nucleotide © 2004 NRC Canada



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sequence was 1983 bp long; plasmid pB was essentially identical to pJ with only one exception: the direct repeat region present in plasmid pJ was absent in plasmid pB. Instead, in plasmid pB, 84-bp-long inverted repeats were identified. Based on the sequence similarities, both plasmids pJ and pB belonged to the pC194/pUB110 rolling-circle replicating plasmid family (Yasukawa and Masamune 1997). Detection of ssDNA During rolling-circle replication, temporarily formed ssDNA intermediates are generated (Noirot-Gros and Ehrlich 1994). To detect such intermediates during replication of pB and pJ, plasmid DNA preparations were treated with S1 nuclease and run on agarose gel together with nontreated plasmids. Two parallel gels were run and blotted, with or without denaturation, with NaOH. Plasmids pK and pP were included as controls. As expected, no ssDNA intermediates were present in the bacterial strain harboring plasmid pK (Fig. 3). Plasmid pP preparations gave a strong hybridization signal, indicating the presence of ssDNA. The detected molecule allowed for hybridization without denaturation and disappeared after the S1 nuclease treatment. The signal derived from the ssDNA was stronger than that derived from the double-stranded plasmid DNA (Fig. 3). Plasmid pJ allowed for hybridization without denaturation, presenting a relatively weak signal (Fig. 3). Comparing the results of hybridization with that of the gel stained with ethidium bromide, we concluded that the signal originated either from the ssDNA replication intermediate, which comigrated together with a covalently closed circular (CCC) plasmid, or from the CCC molecule itself. S1 nuclease treatment resulted in a disappearance of this signal and also in a linearization of the CCC plasmid DNA. Interestingly, even after such treatment, which should degrade all ssDNA sequences, the linearized plasmid was capable of hybridization without denaturation (Fig. 3). To avoid any denaturation of plasmid DNA during purification, this experiment was repeated also with plasmid DNA purified without alkaline denaturation using a DNeasy Tissue kit (Qiagen), and exactly the same results were observed (not shown). To determine the sequences responsible for S1 nuclease sensitivity, plasmid pJ DNA was treated with S1 nuclease followed by digestion with selected restriction endonucleases. The fragments obtained were compared with restriction fragments of the S1 nuclease nontreated plasmid. If S1 nuclease was active only in a specific position, there should be a difference between the treated and nontreated plasmids in a single particular band. However, the restriction profiles of both the S1 nuclease treated and nontreated plasmid preparations were identical (Fig. 4). In the next experiment, we attempted to determine the sequence responsible for hybridization. To detect it, plasmid DNA was digested with restriction endonucleases, electrophoresed in agarose gel, and vacuum blotted without denaturation. The blot was hybridized with the whole plasmid pJ labelled as a probe, and we found that all the restriction fragments were capable of hybridization (Fig. 5). Exactly the same results were obtained with plasmid pB. Both the experiments supported the hypothesis that S1-nuclease-sensitive and hybridization-capable sequences are not located within a

Can. J. Microbiol. Vol. 50, 2004 Fig. 3. Characterization of replication intermediates. DNA of plasmids pK, pC, and pJ were treated (+) or nontreated (–) with S1 nuclease, and electrophoresed and blotted with (D) or without (ND) denaturation. The blots were hybridized with probes derived from a particular plasmid sequence. No single-stranded DNA was observed in plasmid pK. In plasmid pP, high levels of plasmid single-stranded DNA were observed. The nature of the DNA was confirmed by both its sensitivity to S1 nuclease and by its ability to hybridize without denaturation. Plasmid pJ was capable of hybridization without denaturation, no matter if it was treated or nontreated with S1 nuclease.

Fig. 4. Identification of the S1-nuclease-sensitive site in plasmid pJ. Plasmid pJ was treated with S1 nuclease, followed by digestion with restriction endonucleases MseI (lane 1), HaeIII (lane 2), and DdeI (lane 3). Lanes 4–6 contained natural plasmid pJ digested with the same restriction endonucleases (lane 4, MseI; lane 5, HaeIII; lane 6, DdeI). Lane 7, S1-nuclease-treated plasmid pJ. Lane 8, nontreated plasmid pJ. Lanes M, 200-bp ladder.

specific locus but instead are distributed evenly across the whole plasmid sequence.

Discussion Our hybridization studies showed that there are at least three different types of plasmids present in Salmonella © 2004 NRC Canada



Gregorova et al. Fig. 5. Identification of the part of plasmid pJ capable of hybridization without denaturation. Plasmid pJ was digested with restriction endonucleases TaqI (lane 1), MseI (lane 2), HaeIII (lane 3), and DdeI (lane 4), and was electrophoresed and blotted without denaturation (A). The blots were hybridized with the whole plasmid pJ used as a probe (B). Lane M, 200-bp ladder.

Enteritidis. In the dominant group of ColE1-like plasmids, by sequence comparison of plasmid pK and previously sequenced plasmids pI and pC (Rychlik et al. 2001; Gregorova et al. 2002), we confirmed that the sequences responsible for the cross hybridization were located in the parts of plasmids involved in the regulation of plasmid replication, and the same could be concluded also for the rolling-circle-like replicating plasmids pJ and pB. To our knowledge, ColE2-type replicons (pP) or rolling-circle-like replicating plasmids (pJ, pB) have not been identified in Salmonella spp. so far. One reason for this may be that all the sequenced ColE1 Salmonella spp. plasmids contained genes that enable them to be mobilized and transferred to the host by conjugation, while no such genes were detected in either pP or pJ and pB. It is therefore likely that ColE1 plasmids were acquired by Salmonella spp. from other bacteria. Because the virulence plasmid present in some Salmonella serovars is considered to be non-self-transmissible or self-transmissible at very low frequency (Jones et al. 1982; Ou et al. 1990; Ahmer et al. 1999) and because S. enterica only sporadically harbours any other conjugative plasmids (Taylor and Levine 1980; Sanderson et al. 1981; Poppe et al. 1996), it can be considered the terminal host of such plasmids. When we experimentally investigated replication modes of selected plasmids, the expected results were observed in plasmid pK. Partially surprising results were observed in the ColE2-like plasmid pP, as it was predominantly present as a ssDNA molecule. It has been described before that ColE2 plasmids follow a unidirectional mode of replication, and how the second strand is being synthesized is not clear (Takechi and Itoh 1995). Consistent with this, our finding

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documents that the second strand synthesis is being completed only in a few of the plasmid pP molecules. The most unusual behavior was observed in rolling-circle replicating plasmids pJ and pB. Although these plasmids were capable of hybridization without denaturation and the signal disappeared after S1 nuclease treatment, the reaction was slightly atypical because this signal was identical to the localization of CCC plasmid DNA on the agarose gel. We propose that the CCC form itself was capable of weak hybridization because of the unusual structures discussed below. Another unusual feature of these two plasmids was that the CCC form of the plasmid molecule could be linearized with S1 nuclease. And most surprisingly, even after such treatment, the plasmids were still capable of hybridization without denaturation (Fig. 3). Initially, we expected that the S1-nuclease-sensitive region and the site capable of hybridization were located either around the origin of replication or at the region with the direct or inverted repeat motifs that might be involved in the regulation of plasmid replication. However, subsequent experiments showed that neither the S1nuclease-sensitive site nor the site responsible for hybridization without denaturation were located at any specific part of the plasmids. We therefore speculate that during each replication cycle of plasmid pJ or pB, the synthesis of DNA proceeds several base pairs farther, forming a short triplestranded DNA structure that can be used for the next round of replication. Such a hypothesis is at least partially supported by the findings of Rasooly and Novick (1993) who described such extension of replication in rolling-circle replicating plasmids of Staphylococcus aureus, although in this case the extended sequence was bound to the Rep protein and was released from the plasmid upon the termination of its replication.

Acknowledgements We would like to thank Michaela Dekanova for the technical assistance and Ayanna Jefferson for the manuscript revision. This work has been supported by a grant from the Czech Ministry of Agriculture QC0195 and M03-99-01.

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