DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli

International Journal of Medical Microbiology 302 (2012) 4–9

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Short Communication

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli Péter Z. Fekete a,1 , Elzbieta Brzuszkiewicz b,e,1 , Gabriele Blum-Oehler c,∗ , Ferenc Olasz d , Mónika Szabó d , Gerhard Gottschalk b , Jörg Hacker e , Béla Nagy a a

Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, University of Göttingen, Germany c Graduate School of Life Sciences, University of Würzburg, Germany d Agricultural Biotechnology Center, Gödöllo, ˝ Hungary e German National Academy of Sciences Leopoldina, Halle, Germany b

a r t i c l e

i n f o

Article history: Received 14 April 2009 Received in revised form 29 June 2011 Accepted 31 July 2011 Keywords: Enterotoxigenic Escherichia coli (ETEC) Virulence and antibiotic resistance plasmid Sequence analysis Toxin-specific locus (TSL)

a b s t r a c t In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. © 2011 Published by Elsevier GmbH.

Introduction Diarrhoeal diseases frequently develop as a result of infection by enterotoxigenic Escherichia coli (ETEC). ETEC bacteria cause severe watery diarrhea and death of newborn calves and of suckling and weaned pigs (reviewed in Nagy and Fekete, 2005). In humans, ETEC bacteria are recognized as one of the most frequent causative agents of (sometimes fatal) childhood diarrhea in the developing countries, and of traveller’s diarrhea worldwide (Qadri et al., 2005). Due to this social and economic significance and due to similarities between the pathogenesis of ETEC infections of animals and man, ETEC bacteria and their plasmids have been the subject of intensive studies in human and veterinary medicine, and several human ETEC virulence plasmids have been fully or partially sequenced (reviewed in Johnson and Nolan, 2009), but no completed animal ETEC virulence plasmid sequences are available at the moment. ETEC bacteria of weaned pigs adhere to the microvilli of epithelial cells in the small intestine through their fimbrial adhesins

∗ Corresponding author. E-mail address: [email protected] (G. Blum-Oehler). 1 These two authors contributed equally to this paper. 1438-4221/$ – see front matter © 2011 Published by Elsevier GmbH. doi:10.1016/j.ijmm.2011.07.003

(F4, F18). Subsequently, they produce enterotoxins (STa, STb and/or LT), thereby stimulating secretion of water and electrolytes into the intestinal lumen (reviewed in Gyles, 1994; Nagy and Fekete, 2005). Production of these toxic and adhesive virulence factors is mostly determined by virulence plasmids encoding the above mentioned adhesive and enterotoxic virulence determinants (reviewed in Johnson and Nolan, 2009). Thus heat-stable enterotoxin STa is carried by transposon Tn1681 (So and McCarthy, 1980), and STb was described as part of Tn4521 (Lee et al., 1983, 1985). STb and F18 are characteristic plasmid-mediated virulence factors of porcine postweaning ETEC. One prototype strain of F18 fimbria-producing ETEC is Ec2173 (O147:K+, NM, STa, STb, Hy, F18ac) isolated in Hungary from a fatal case of porcine post-weaning diarrhea (PWD) (Nagy et al., 1990; Rippinger et al., 1995). Ec2173 harbours two large virulence plasmids: one (pF18) is approximately 200 kb in size with a RepFIc replicon, coding for F18ac and haemolysin (Fekete et al., 2002; Olasz et al., 2005). The other plasmid (pTC) was originally estimated to be a 120 kb plasmid carrying heat-stable toxin genes (estA, estB) and a tetracycline resistance class B determinant (tetB) according to the published nomenclature (Levy et al., 1989, 1999). The plasmid pTC is also characterised by a ColE1 type replicon (Olasz et al., 2005). Our preliminary studies have proved the pathogenetic significance and

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wide distribution of pTC as a self conjugative model ETEC plasmid, with major virulence determinants causing PWD of swine (Fekete et al., 2003; Olasz et al., 2005). Our earlier studies also indicated that pTC contains an approximately 40 kb DNA fragment, as a putative pathogenicity island (PAI) with a 10 kb toxin-specific locus (TSL) carrying sta and stb, and we found that the TSL is characterised by a so far unknown genetic background for stb (“pTC-like” stb) (Fekete et al., 2003). This putative PAI with the TSL has been mobilized into plasmid pACYC177 resulting in a transposition product (pAKR2) with the original colE1 origin of replication of pTC but without Tet B (Fekete et al., 2003; Suppl. Fig. 1). Here we hypothesized that this transposition product may have attributes differing from classical PAI and may represent the plasmid backbone of pTC. Considering the pathogenetic significance of pTC, and the fact that so far there have been no complete plasmid sequences available for animal ETEC, in this study we aimed to determine the complete nucleotide sequence of pTC and describe the genetic and functional properties of this self-conjugative model ETEC plasmid containing determinants of pathogenicity and antimicrobial resistance. Materials and methods Bacterial strains, plasmids and DNA techniques Plasmid pTC (Sta, Stb, Tet B; Olasz et al., 2005) was isolated from EC2173 (Nagy et al., 1990) and served as a ‘transposition donor’ in a previous experiment (Fekete et al., 2003). Plasmid pAKR2 is the transposition fusion product of pTC and pACYC177 (AmpR , KmR ) (Fekete et al., 2003; Suppl. Fig. 1). General DNA techniques were performed according to Maniatis et al. (1989). Sequencing and comparative studies The sequence of pTC was assembled in two sequencing phases: first the pTC core backbone (transposed into the pACYC177 plasmid resulting in pAKR2) was sequenced; in the second phase the Tn10 transposon (encoding the tetracycline resistance) was amplified by long-distance PCR resulting in the complete Tn10 tetracycline transposon, and this amplicon was sequenced. In the first phase the plasmid pAKR2 was randomly fragmented using the nebulizer technique and size-fractioned by sucrose density gradient in the range of 1.0–4.0 kb. Purified, end-repaired fragments were subcloned into the cloning vector pCR4Topo (Invitrogen, Karlsruhe, Germany) forming a random library. Sequencing data were collected on an ABI 3730xl sequencer. The plasmid was sequenced with a 9-fold coverage; 1914 sequence runs were assembled and the resulting contigs were ordered. The remaining gaps were closed using conventional PCR-based methods. In the second step the Tn10 region of pTC was amplified in a long-distance PCR using primers specific for the IS10 flanking the Tn10 transposon and sequenced. The orientation of the transposon was determined by PCR amplifications and sequencing using primers specific for the flanking of IS10 elements and by restriction cleavage with MluI endonuclease (data not shown). Sequence editing was performed using the GAP4 application of the Staden software package. Annotation was done using the ERGO (Integrated Genomics, Inc., Chicago, IL) software package. The predictions were verified and modified manually by searching derived protein sequences against public nucleotide or protein domain databases such as GenBank. For comparative analysis, the complete sequence of pTC was analysed using the NCBI BLAST search engine on the NCBI nucleotide collection (nr/nt) taking NR1 and pC15-1a as examples of E. coli resistance plasmids. The complete nucleotide

5

sequence of the pTC plasmid has been deposited in the GenBank database (accession no. CP000913). Results and discussion As a first step, the sequence of the pTC plasmid was assembled from the sequence data of pAKR2 (Fekete et al., 2003) containing the transposed pTC core backbone and the sequence of the pTCderived Tn10 transposon (encoding tetracycline resistance). This latter segment was amplified by PCR and sequenced separately. To verify the similarity between pTC and pAKR2, the BamHI, EcoRI, SalI and XhoI restriction patterns of the two plasmids were compared and PCR analyses were performed as well (data not shown). The results completely corresponded to the derived pTC sequence obtained from pAKR2 and Tn10 sequences (Suppl. Fig. 2). The characteristic 9 bp target duplication (tacttaaca) generated by IS10 was identified on pAKR2 as well, indicating that IS10 elements are bordering the pTC core region and Tn10. In the following sections the sequence derived and assembled from pAKR2 and from the separately sequenced Tn10 amplicon is described as the whole pTC sequence, as follows: The overall sequence of pTC is 91,019 bp (Fig. 1) containing altogether 112 ORFs (encoding STa and STb enterotoxins, 7 ORFs of the Tet B region of Tn10, 26 verified or putative mobile elements homologous to bacteriophages and insertion sequences (IS), 22 plasmid maintenance (stability and replication) proteins, 31 proteins of the plasmid transfer (tra) locus, 9 proteins of the origin of replication, and 15 hypothetical proteins (Table 1 and Fig. 1). The overall G+C content of pTC is 50.23%, from which one of the dissimilar segments was the region Tn10 with a G+C content of 39.95%. The five main regions of pTC were identified based on their functional properties: (i) the plasmid maintenance/stability region is responsible for the stability of the plasmid, (ii) the TSL (toxin specific locus) conferring virulence attributes to the plasmid by encoding the sta and stb toxin genes, (iii) the Tn10 transposon, encoding the tetracycline resistance genes, (iv) the tra (plasmid transfer) region responsible for spread of pTC, and (v) the colE1-like origin of replication. The borders of the plasmid transfer, Tn10 and origin of replication regions were identified based on earlier sequence data and on our present sequence studies. The assumed boundaries of plasmid maintenance and TSL regions were determined by homologies to other sequences in databases. In these cases it was supposed that the region started and finished with the beginning and end of DNA homology, respectively. The five main regions of pTC The plasmid maintenance/stability region The plasmid maintenance/stability region could be divided into three, well defined sub-regions (designated I–III) separated by independent insertion of TSL and Tn10 and contained several genes with unknown functions. The TSL region is flanked by two direct duplications at bp 784–1467 and bp 18,308–18,992, respectively (97% similarity in BLAST2 comparison). The repetition contained copies of the yacAB genes [encoding a plasmid stabilization system protein belonging to the RelE/ParE family (Johnson et al., 2005)] on both sides which are most likely responsible for the extreme stability of pTC (Olasz et al., 2005). The sub-region I (positions 784–1467) between the origin of replication and the TSL contained the ORFs for yacA and yacB. However, the third gene of the yac operon (yacC) is missing, probably as a consequence of the TSL insertion. In addition, the duplication of yacA and yacB genes (18,308–18,992) surrounding the TSL serves as the starting point of the maintenance sub-region II (18,308–34,773). This subregion showed good similarity between position 21,637–26,926

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Fig. 1. Circular physical map of the pTC plasmid. pTC is composed of five main distinct regions: (i) the plasmid maintenance region (divided into three sub-regions designated I-III), (ii) the TSL (encoding the sta and stb toxin genes), (iii) a Tn10 transposon, encoding tetracycline resistance genes tetR(B), tetA(B) and tetC(B), (iv) the tra (plasmid transfer) region, and (v) the origin of replication (ori). Identified ORFs depicted by boxes, coloured according to their functional categories: black, origin of replication; blue, plasmid maintenance genes; violet, IS elements and transposase genes; red, heat-stable enterotoxin genes; orange, genes of antibiotic (tetracycline) resistance, green, genes of the plasmid transfer (tra) region; the genes encoding ORFs of hypothetical proteins are pale grey. The dark grey ring indicates the G+C content of the corresponding region. The innermost ring is a base pair scale.

to Shigella flexneri 301 virulence plasmids pCP301 (AF386526.1), pWR100 (AL391753.1) and pWR501 (AF348706.1). The ORFs TC1 0032 and TC1 0033 represented plasmid stability proteins StbA and StbB which are similar to the stability cassette of plasmid pCTX-M3 (NC 004464.2). The maintenance sub-region III (positions 43,935–52,152) was highly similar to the corresponding sequence of NR1 (DQ364638.1). The genes in this region can also contribute to the stability/maintenance of the plasmid: i.e. TC1 0059 and TC1 0060 correspond to psiB and psiA genes, possibly inhibiting the generation of a signal to induce SOS genes (Bagdasarian et al., 1986); TC1 0061, an flmC analogous putative gene (F-plasmid maintenance protein C) whose function is probably analogous to the “mok” and “hok” system, playing a central role in the plasmid maintenance due to a membrane-associated lethal protein - antisense RNA system (Gerdes et al., 1986). Toxin-specific locus (TSL) As stated earlier (Fekete et al., 2003) the region corresponding to the enterotoxigenicity of strain EC2173 is the large

toxin-specific locus (TSL). Based on our present finding, it is located between bp 1468 and 18,307. In contrast to our earlier findings (Fekete et al., 2003) is a 16,839 bp fragment, and it contains three copies of the IS91 element instead of two in the 9571 bp ‘inter-toxin’ region between the sta and stb genes. TSL as a whole is flanked by a truncated IS679 (1468–3367 bp) and an IS21 (16,641–18,307 bp) element, and it contains 24 ORFs: the two heat-stable enterotoxin genes sta and stb, 4 encoding hypothetical proteins, and 18 encoding known or recently identified transposases (IS Finder; http://www-is.biotoul.fr/). The sta (bp 14,840–15,058) and stb (bp 5054–5269) genes encoding the heat-stable enterotoxins are relatively closely linked. The sta gene shows 100% (216/216) nucleotide identity with the canonical sta gene (So and McCarthy, 1980). On the complementary strand the 213 bp ORF (5054–5269 bp) shows 99% (211/213) similarity to the stb gene described earlier (Lee et al., 1983). One point mutation resulting in a His–Asn change at the 12th amino acid position of the STb toxin has been identified, contrasting to our earlier finding (Fekete et al., 2003) and confirming recent observations (Taillon et al., 2008).

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Table 1 List of open reading frames (ORFs) of pTC plasmid. # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

ORF TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1

0001 0002 0003 0004 0005 0006 0007 0008 0009 0010 0011 0012 0013 0014 0015 0016 0017 0018 0019 0020 0021 0022 0023 0024 0025 0026 0027 0028 0029 0030 0031 0032 0033 0034 0035 0036 0037 0038 0039 0040 0041 0042 0043 0044 0045 0046 0047 0048 0049 0050 0051 0052 0053 0054 0055 0056 0057 0058 0059 0060 0061 0062 0063 0064 0065 0066 0067 0068 0069 0070 0071 0072 0073 0074 0075

Start

End

397 948 1214 1564 1766 3691 4388 5054 5475 5795 6309 6536 7723 8144 8640 9527 10231 11418 12060 13247 13714 14117 14840 15248 16087 16600 17034 18473 18742 19284 20560 21627 22022 23396 24319 24706 25592 26110 26964 27299 27771 28733 29701 31109 31758 32482 33100 33400 34891 36465 38114 38511 38821 39485 40142 41508 42615 43945 44586 45020 45961 46510 47160 47527 49019 49805 50212 51330 52153 52832 53640 53900 54274 54607 55214

783 1217 1501 1746 3337 4098 4666 5269 5750 6172 6599 7723 8088 8833 9530 9853 11418 11783 13247 13612 14043 14491 15058 16090 16593 17034 18206 18742 19020 20471 21471 22022 23026 24322 24630 25389 26059 26886 27218 27724 28193 29155 31062 31672 32219 33048 33339 34821 36099 37670 38434 38813 39507 40111 41395 42164 43823 44520 45023 45739 46173 47160 47507 49098 49504 50101 51033 51932 52638 53503 53867 54259 54585 55173 55888

Strand

c c c

c c c c c c c c

c c c c

c c

c

c

c

c c c c c c

c

c

Function of encoded protein

Region

Hypothetical replication protein, similar to protein RepA4 Conserved hypothetical protein YacA Conserved hypothetical protein YacB Transposase IS679orfB transposase IS679orfC Transposase homologous to IS1400 orfB truncated Transposase homologous to IS1400 orfA, transposase 6 Heat-stable enterotoxin STb (enterotoxin B) Insertion element IS1 protein InsA Insertion element IS1 protein InsB Putative transposase tnpA (IS91) Transposase IS91 TnpA truncated Putative IS91 ORF2 truncated Truncated insertion sequence IS629 Putative transposase OrfB of insertion sequence IS629 Putative transposase OrfA of insertion sequence IS629 Putative IS91 ORF1 Putative IS91 ORF2 Putative IS91 ORF1 Putative IS91 ORF2 Putative transposase OrfA Putative transposase and inactivated derivatives Heat-stable enterotoxin I (STa) Putative transposase Putative transposase ATP-binding protein for insertion sequence IS21 Transposase for insertion sequence element IS21 Conserved hypothetical protein YacA2 Plasmid stabilisation system YacB protein Hypothetical protein Hypothetical protein Plasmid stable inheritance protein StbB Plasmid stable inheritance protein StbA Conserved hypothetical protein: similar to YccB Conserved hypothetical protein putative site-specific DNA-methyltransferase Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein Putative antirestriction protein KlcA Conserved hypothetical protein Hypothetical protein Conserved hypothetical protein YdbA Conserved hypothetical protein Conserved hypothetical protein Single-stranded DNA binding protein ssb Conserved hypothetical protein Putative ParB-like nuclease Transposase of Tn10 Conserved hypothetical protein YdjA protein Conserved hypothetical protein Transcription regulatory protein Tetracycline repressor protein TetR(B) Tetracycline antiporter protein TetA(B) tetracycline transcriptional regulator TetC(B) Transposase of Tn10 Conserved hypothetical protein Plasmid SOS inhibition protein PsiB plasmid SOS inhibition protein PsiA Putative protein FlmC homologue (F-plasmid maintenance protein C) transposase IS679 orfA IS element transposase IS679 orfB IS element transposase IS679 orfC Conserved hypothetical protein Conserved hypothetical protein YeiA Conserved hypothetical protein Putative lytic transglycosylase TraM protein TraJ protein TraY protein TraA fimbrial protein (pilin) TraL protein TraE protein TraK lipoprotein precursor

ori Maintenance I Maintenance I TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL TSL Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Maintenance II Tn10 Tn10 Tn10 Tn10 Tn10 Tn10 Tn10 Tn10 Tn10 Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III Maintenance III tra tra tra tra tra tra tra

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Table 1 (Continued) #

ORF

76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1 TC1

0076 0077 0078 0079 0080 0081 0082 0083 0084 0085 0086 0087 0088 0089 0090 0091 0092 0093 0094 0095 0096 0097 0098 0099 0100 0101 0102 0103 0104 0105 0106 0107 0108 0109 0110 0111 0112

Start

End

55888 57356 57921 58199 58849 59138 59709 60268 60741 63368 63751 64380 65396 65716 66351 68445 69235 69698 69999 70519 70874 71223 72521 75359 75890 76761 77615 79849 85139 85944 86907 87596 87978 88807 89637 89998 90196

57339 57895 58202 58714 59070 59476 59942 60615 63371 63754 64383 65372 65707 66354 68201 69221 69579 69982 70514 70791 71164 72524 75343 75844 76624 77564 79849 85119 85885 86804 87467 87808 88514 89397 89891 90183 91019

Strand

Pathogenetic significance of TSL characterised by the “pTC-like” stb was indicated by the loss of in vivo enterotoxigenicity of pTCcured derivatives of the ETEC strain Ec2173 in our previous study (Olasz et al., 2005). Furthermore, TSL has been detected in the majority of porcine ETEC F18+ isolates of Hungarian, Austrian and US origin tested (Fekete et al., 2003; Olasz et al., 2005).

Tn10 transposon encoding tetracycline resistance Sequencing of the long-distance PCR amplicon of Tn10 revealed that a 9146 bp sequence between positions 34,773 and 43,935 determines a Tn10 composite transposon carrying the tetracycline resistance genes tetR(B), tetA(B) and tetC(B) located on the 3 end of the Tn10, with two almost identical (98%) IS10 elements at the ends. In comparison to other parts of the plasmid this region has a very low G+C content (39.95%), suggesting an autonomous mobilization ability. During the transposition events between pTC and pACYC177 the IS10 elements might have mediated the ‘inverse’ transposition of the pTC core region (maintenance and transfer regions, origin of replication and TSL) resulting in plasmid pAKR2 (Fekete et al., 2003). Based on this fact, it seems that the IS10 elements play a role not only in the genetic transfer of antimicrobial resistance determinant Tet B but also in transfer of virulence determinants (STa, STb).

Plasmid transfer (tra) region The 33,729 bp stretch of DNA between bp 52,153 and 85,882 represents the largest distinct region responsible for the plasmid transfer function of pTC. This tra region contains 35 ORFs (TC1 0069 to TC1 0104) and is highly similar (up to 98% similarity) to known transfer regions of other plasmids of the NR1 family, raising the

Function of encoded protein

Region

TraB protein TraP protein TrbD protein TraV protein precursor TraR protein Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein TraC protein TrbI protein TraW protein precursor TraU protein precursor conserved hypothetical protein periplasmic protein TrbC TraN protein precursor TraF protein precursor TrbA protein TraQ protein TrbB protein precursor TrbJ protein TrbF protein TraH protein precursor TraG protein Surface exclusion inner membrane protein TraS TraT complement resistance protein precursor Conserved hypothetical protein TraD protein TraI protein (DNA helicase I/relaxase) TraX protein Hypothetical alpha/beta hydrolase - RNA-binding protein Fertility inhibition protein (conjugal transfer repressor FinO) Hypothetical protein Hypothetical nuclease precursor Negative regulator of repA1 expression Replication regulatory protein RepA2 (CopB protein) Protein CopA/IncA, protein RepA3 Replication initiation protein RepA1

tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra tra ori ori ori ori ori ori ori ori

possibility that those self-transmissible plasmids are of common origin.

Origin of replication The origin and the replication genes (ori) are located on the fragment between bp 85,944 and 783 representing a ColE1 type replicon (Olasz et al., 2005). This region is highly (99%) similar to the origin of replication of the self-conjugative NR1 plasmid, as an example, which is an autonomously replicating plasmid closely related to the F plasmid.

Possible origin of pTC DNA sequence comparisons of the plasmids NR1 (DQ364638.1) and pC15-1a (AY458016.1) showed a high degree of similarity (98% or more) to pTC and represented different types of pTC relatives (Suppl. Fig. 3). The Tn10 transposon was found exactly in the same location both in NR1 and pTC but in an inverse orientation as suggested by the reverse direction of the bordering IS10 elements, and proved by restriction cleavage with MluI endonuclease (data not shown). The Tn10 transposon was absent from pC15-1a. Overall, the first 24 kb (including the TSL encoding the two enterotoxins) seemed to be characteristic of plasmid pTC, it was not found in plasmids NR1 or pC15-1a (Suppl. Fig. 3). It seems that the tra region, together with the plasmid replication and maintenance regions represented their common backbone containing clusters of other genes (drug resistance and pathogenicity determinants) acquired by horizontal transfer. Other E. coli plasmids [for example: pO26L (FJ449539.1), pO86A1 (AB255435.1), pAPEC-O2-R (AY214164.3) and pUTI89 (CP000244.1)] are also similar to pTC in the tra and

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plasmid maintenance subregions II and III, suggesting that these plasmids might have a common ancestor. The fact that the first approximately 24 kb segment (positions 921–24,366) of pTC does not fit the corresponding sequences of these plasmids does not contradict this concept. The nonhomologous part could be divided into two regions: one was the TSL, while the other (positions 21,637–26,926) showed significant homology to the Shigella flexneri 301 virulence plasmids pCP301 (AF386526.1), pWR100 (AL391753.1) and pWR501 (AF348706.1), raising the possibility that this sub-region might have originated from Shigella plasmids or their relatives. Based on the above described homologies a starting point of this theoretical scenario could have been an ancient plasmid highly similar to NR1 and pC15-1a (and to several other plasmids) subjected to multiple rearrangements. The first event in these developments might have been the insertion of Tn10, separating the ancestors of pTC and NR1 from that of pC15-1a. This hypothesis is supported by the fact that Tn10 is present both in NR1 and pTC exactly at the same position but absent from pC15-1a. As a possible further development the TSL and IS679 might have become inserted into the plasmid backbone of pTC. IS679 (pTC positions: 46,425–49,128) is present in pTC but missing from both NR1 and pC15-1a, indicating that the insertion probably took place after the segregation of pTC and NR1. It is also interesting to note that TSL is surrounded by sequences which are characteristic of Shigella plasmids such as pCP301 and others mentioned above. Furthermore, TSL is bordered by the duplication of the yacA and yacB genes, suggesting that a putative Shigella virulence plasmid harbouring TSL might have been inserted into the ancestor of pTC (probably by homologous recombination). The origin of this putative Shigella plasmid remains unclear. Recently only one full sequence of ETEC plasmids has been available in databases. The ETEC plasmid pEntH10407 (Ochi et al., 2009) shows significant similarities to the plasmid backbone of pTC (origin of replication, maintenance subregions II and III and parts of transfer region), however the transfer region in pEntH10407 contains a large deletion (genes between traC and TraT are missing). Both pTC and pEntH10407 harbours the sta gene in their pathogenicity islet, but the stb gene is missing from pEntH10407 and, on the contrary, the LT toxin gene is absent from pTC. Comparing the TSL region of pTC to that of pEntH10407 it turned out, that there are no significant similarities between the two islets: homologies were only found to the sta gene and to IS elements IS1 and IS679. The boundaries of the whole islets and the toxin genes differ, moreover, they are located on different positions of the plasmid. These data strongly suggest that the human and animal ETEC plasmids have different evolutionary origins. Based on the above findings it can be concluded that all the complex rearrangements resulted in a unique feature of pTC. This large stretch of pTC-DNA has many characteristics of a PAI: while it harbours a set of genes in the TSL region necessary for pathogenicity, it also shows the characteristics of a transposon because almost the whole pTC could be integrated into a new DNA species (Fekete et al., 2003). Above all, pTC is a self-transmissible plasmid securing the rapid spread of the virulence genes in the presence of tetracycline. Considering all these features, pTC can be interpreted as a composite plasmid representing a selection advantage in the propagation and spread of pathogenicity and antimicrobial determinants. The differing characteristics of pTC from those of the above human ETEC plasmid pEntH10407 are in harmony with our studies on 58 human ETEC strains from Egypt, Mexico and Thailand without detection of TSL (results not shown) providing further evidence for a difference between human and porcine ETEC plasmid populations and indicating low or no zoonotic potential of pTC.

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