Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium

Frédérique Pasquali1, Corinna Kehrenberg2, Gerardo Manfreda1 and Stefan Schwarz2,*

1 Department of Food Science, Alma Mater Studiorum, University of Bologna, via San Giacomo 9, 40126 Bologna, Italy; 2 Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany


* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; Email: stefan.schwarz{at}fal.de

Received 8 October 2004; returned 24 November 2004; revised 26 November 2004; accepted 27 November 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The complete nucleotide sequence of the 12 656 bp plasmid pFPTB1 from Salmonella enterica subsp. enterica serovar Typhimurium, which mediates resistance to tetracyclines and ampicillin, was determined. The plasmid was analysed for potential reading frames and structural features indicative of transposons and transposon relics.

Methods: Plasmid pFPTB1 was transformed into Escherichia coli JM109, overlapping restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was carried out to confirm the resistance phenotype mediated by this plasmid.

Results: Plasmid pFPTB1 contains a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, a novel gene for ampicillin resistance blaTEM-135, and the right terminal repeat. Immediately downstream, the terminal 5215 bp at the right end of a Tn1721-like transposon, including the right terminal repeat, a truncated transposase gene, as well as the genes tet(A) and tetR for tetracycline resistance, were detected. A 5 bp direct repeat, TAAAA, was seen immediately upstream of the Tn3 part and immediately downstream of the Tn1721 part. Plasmid pFPTB1 also carries a replication region similar to that of the Klebsiella pneumoniae plasmid pJHCMW1.

Conclusion: Plasmid pFPTB1 is one of the few completely sequenced plasmids from S. Typhimurium and harbours a novel transposon-like structure consisting of a Tn3-related part containing the blaTEM-135 gene for ampicillin resistance and a Tn1721-related part containing the tetR-tet(A) genes for tetracycline resistance.

Keywords: transposon Tn1721 , transposon Tn3 , recombination , resistance gene transfer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The presence of antibiotic resistance genes on transposable elements is a matter of concern with regard to the dissemination of the resistance genes via horizontal gene transfer. In particular, genes coding for resistances to tetracyclines or ß-lactams in Gram-negative bacteria have been associated with transposons. The tetracycline resistance gene of hybridization class A [tet(A)] which codes for a membrane-associated efflux protein, is associated with the 11.1 kb non-conjugative transposon Tn1721.1 Complete or truncated variants of Tn1721 have been detected on conjugative or mobilizable plasmids in a wide variety of Gram-negative bacteria obtained from humans,2,3 animals,46 plants,7 or environmental6,7 sources worldwide. Genes coding for ß-lactamases of the TEM-type are among the most prevalent ß-lactam resistance genes in Gram-negative bacteria.811 Particular blaTEM genes code for extended spectrum ß-lactamases (ESBLs) or inhibitor-resistant ß-lactamases. Up to now, more than 130 TEM variants have been identified (www.lahey.org/studies; last accessed 16 September 2004). The blaTEM-1 gene has been detected on transposon Tn3 which is also located either on conjugative and non-conjugative plasmids, or on the chromosome.8 Tn3 and Tn1721 are members of the Tn3 family of transposons and exhibit similar mechanisms of replicative transposition.12 Moreover, both transposons have terminal inverted repeats of 35–38 bp and produce characteristic 5 bp direct repeats at their integration sites.12 Both Tn1721-associated tet(A) genes and Tn3-associated TEM-type bla genes, have been detected in various Salmonella enterica serovars.4,5,9 A recent study also identified both genes in the same multiresistant isolates of S. Typhimurium var. Copenhagen phage types DT002, DT003 and DT104.11

In this study, we analysed a 12 kb tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium to assess the molecular basis of these resistances.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Plasmid pFPTB1 was found in a Salmonella Typhimurium phage type U302 strain isolated from a rabbit at a slaughterhouse in Italy. Plasmid DNA was prepared by alkaline lysis and subsequent purification via affinity chromatography columns (Qiagen, Hilden, Germany). In vitro susceptibility testing was carried out by disc diffusion according to the German DIN standard 58940.13 Plasmid pFPTB1 was transformed into E. coli JM109 and transformants were selected on Luria-Bertani (LB) agar supplemented with 20 mg/L of tetracycline. PCR analysis for the most commonly found tetracycline and ampicillin resistance genes in Enterobacteriaceae was carried out as previously described.11 After restriction mapping, PvuII and EcoRV fragments of pFPTB1 were cloned separately into pCR-Blunt II-TOPO (Invitrogen, Groningen, The Netherlands) and transformed into E. coli TOP10. In addition, BamHI–ClaI fragments were cloned into pBluescriptII SK+ (Stratagene, Amsterdam, The Netherlands) and the recombinant plasmids were transformed into E. coli JM109. Initial sequence analyses were conducted with the M13 reverse and forward primers. For determination of the complete sequence, primer walking was carried out with oligonucleotide primers (MWG, Ebersberg, Germany) designed from sequences previously obtained with the M13 reverse and forward primers. Sequence analysis was carried out with the BLAST programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; last accessed 24 September 2004) as well as with the ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; last accessed 24 September 2004). The nucleotide sequence of pFPTB1 has been deposited in the European Molecular Biology Laboratory (EMBL) database under accession number AJ634602.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Structure and organization of plasmid pFPTB1

Plasmid pFPTB1 has a total size of 12 656 bp. In vitro susceptibility testing of E. coli JM109:pFPTB1 transformants revealed that this plasmid mediates resistance to tetracyclines and ampicillin. PCR analysis identified the corresponding resistance genes as tet(A) and blaTEM (data not shown). Restriction mapping of pFPTB1 showed a high degree of similarity between parts of pFPTB1 and the blaTEM-carrying transposon Tn3 (accession no. V00613) and a part of the tet(A)-carrying transposon Tn1721 (accession no. X61367) (Figure 1). Sequence analysis confirmed the presence of a complete copy of a Tn3-like transposon and a truncated copy of a Tn1721-like transposon on plasmid pFPTB1.



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Figure 1. Comparative analysis of Tn3 (accession no. V00613), Tn1721 (accession no. X61367) and pFPTB1 (accession no. AJ634602). Restriction endonucleases are abbreviated as follows: B (BamHI), Bg (BglII), C (ClaI), D (DraI), Ev (EcoRV), E (EcoRI), P (PstI), Pv (PvuII), and S (SmaI). A distance scale in kb is presented below each map. The genes tnpA, {Delta}tnpA, tnpR, mcp, blaTEM-1, blaTEM-135, tet(A), tetR, ORF294 are presented as arrows with the arrowhead indicating their direction of transcription. The black and black/white striped boxes represent the inverted 38 bp repeats of Tn1721 and Tn3, respectively. The 5 bp direct repeats at the boundaries of the Tn3{Delta}Tn1721 element in pFPTB1 are shown in boxes below the map of pFPTB1. The grey boxes in pFPTB1 represent the two sequences homologous to plasmid pJHCMW1 (accession no. AF479774). The grey shaded regions indicate similarity between pFPTB1 and either Tn3 or Tn1721.

 
The Tn3-homologous part in pFPTB1 (positions 15–4964) showed 97% identity to the complete sequence of the 4957 bp transposon Tn3. This 4950 bp segment of pFPTB1 comprised the blaTEM gene coding for a ß-lactamase, the genes tnpA and tnpR whose products are essential for transposition of Tn3 as well as the left 38 bp terminal inverted repeat of Tn3. The right terminal inverted repeat consisted of only 37 bp due to the lack of a single ‘T’ between positions 4939 and 4941. The blaTEM gene of pFPTB1 codes for a structurally unique 286 amino acid protein, designated TEM-135, which differs from the TEM-1 ß-lactamase (accession no. P00810, AAR25033 by a single amino acid substitution at position 180 (M->T in TEM-135). Single amino acid substitutions were also seen between TEM-135 and the enzymes TEM-106 (accession no. AAM52207 at position 102 (K->E in TEM-135) and TEM-126 (accession no. AAT45742 at position 177 (E->D in TEM-135). Since the pFPTB1 transformant was resistant to ampicillin, but susceptible to ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, cefazolin, cefuroxime, cefotaxime, ceftazidime, imipenem and meropenem, these substitutions are believed to have no influence on the substrate spectrum and the sensitivity of TEM-135 to ß-lactamase inhibitors. Moreover, the TEM-135-associated resistance phenotype does not correspond to an ESBL phenotype.

The Tn1721-homologous part in pFPTB1 (positions 4965–10 179) revealed 99% identity to the sequence located between bases 5913–11 139 in the Tn1721 sequence. This 5215 bp segment of pFPBT1 includes the genes tet(A) and tetR coding for a class A tetracycline efflux protein of 399 amino acids and a repressor protein of 216 amino acids, the ORF294 coding for a hypothetical transmembrane protein of 294 amino acids, a truncated tnpA gene ({Delta}tnpA) of Tn1721, and the 38 bp right terminal inverted repeat of Tn1721. The remaining parts of Tn1721, shown in Figure 1, were absent in pFPTB1. The deduced amino acid sequence of ORF294 proved to be indistinguishable from that of the PecM protein (accession no. AAR25036 which is involved in the regulation of virulence factor synthesis in Erwinia chrysanthemi.

Comparative analysis of the sequences downstream of the truncated Tn1721 in pFPTB1 revealed two stretches of 212 bp and 1242 bp (positions 10 246–10 457 and 11 371–12 612 in pFPTB1), both of which shared 98% identity with the corresponding parts of the nucleotide sequence of plasmid pJHCMW1 (positions 3085–3296 and 531–1770, respectively) (accession no. AF479774). In pJHCMW1, the 1242 bp segment has been shown experimentally to contain the region responsible for plasmid replication.14 Two RNA molecules have been identified in pJHCMW1 with RNA II acting as the primer for initiation of replication and RNA I representing the antisense RNA molecule that controls initiation of replication by binding to RNA II and preventing primer formation.14 Almost identical sequences for RNA II (positions 11 882–11 374 in pFPTB1) and RNA I (positions 11 778–11 879 in pFPTB1) have been found in the nucleotide sequence of pFPTB1. Thus, replication of plasmid pFPTB1 is also believed to be mediated and regulated by RNA molecules.

Analysis of the physically linked transposon segments in pFPTB1

Transposons of the Tn3 family, such as Tn1721 and Tn3, do not show extended insertion site specificity, but appear to prefer A + T-rich sequences.12 When integrating into a new vector molecule, these transposons produce characteristic 5 bp direct repeats at the insertion site.12 The 5 bp direct repeat, TAAAA, was detected immediately upstream of the Tn3-homologous part (positions 10–14) and immediately downstream of the Tn1721-homologous part (positions 10 180–10 184) whereas no direct repeats were seen, either at the junction between Tn3-homologous and -non-homologous, or at those between Tn1721-homologous and -non-homologous sequences in pFPTB1. This observation suggests that the entire Tn3-{Delta}Tn1721 segment might have integrated into the pFPTB1 basic replicon as one unit.

During replicative transposition of Tn3-like transposons, binding of the transposase to the 38 bp terminal inverted repeats is essential for the formation of a cointegrate intermediate between the donor and the recipient replicon.12 Previous studies have shown that the terminal inverted repeats vary among the different members of the Tn3 family and that the transposases of the Tn3 family bind more or less specifically to the different terminal inverted repeats. It has been shown that the Tn3 transposase does not mediate regular transposition of transposons that carry mixed Tn1000/Tn3 termini although these termini are closely related.12 Thus, it is questionable whether the Tn3 transposase may allow transposition of the Tn3{Delta}Tn1721 structure seen on plasmid pFPTB1 in which the Tn3/Tn1721 termini are only distantly related.

In addition to regular transposition, so-called ‘one-ended transposition’ has also been observed among Tn3-like transposons. In this case, a single transposon terminus may be sufficient to generate transposase-dependent cointegrate formations between donor and recipient molecules.12 In these cases, which occur at distinctly lower frequencies, the typical 5 bp direct repeats may also be observed at the integration site.12 Thus, the presence of the 5 bp direct repeats at the boundaries of the Tn3{Delta}Tn1721 element can be considered as some kind of evidence that a transposition event—either regular or one-ended—has occurred.

Although the processes that led to the formation of the Tn3{Delta}Tn1721 structure in pFPTB1 are unknown, the finding of such a structure is an interesting observation since it underlines that cointegrates are not only formed between resistance plasmids, but also between resistance mediating transposons. If such a Tn3{Delta}Tn1721 structure is disseminated as a unit, simultaneous spread of resistance to the two most frequently used classes of antimicrobial agents in veterinary medicine, penicillins and tetracyclines, will occur.


    Acknowledgements
 
We wish to thank Vera Nöding for excellent technical assistance as well as Franz-Josef Schmitz and Jasmina Petridou for confirming the resistance phenotype.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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