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
Received 8 October 2004; returned 24 November 2004; revised 26 November 2004; accepted 27 November 2004
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Abstract |
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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
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Introduction |
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In this study, we analysed a 12 kb tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium to assess the molecular basis of these resistances.
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Materials and methods |
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Results and discussion |
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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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The Tn1721-homologous part in pFPTB1 (positions 496510 179) revealed 99% identity to the sequence located between bases 591311 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 (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 24610 457 and 11 37112 612 in pFPTB1), both of which shared 98% identity with the corresponding parts of the nucleotide sequence of plasmid pJHCMW1 (positions 30853296 and 5311770, 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 88211 374 in pFPTB1) and RNA I (positions 11 77811 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 1014) and immediately downstream of the Tn1721-homologous part (positions 10 18010 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-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 Tn3Tn1721 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 Tn3Tn1721 element can be considered as some kind of evidence that a transposition eventeither regular or one-endedhas occurred.
Although the processes that led to the formation of the Tn3Tn1721 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
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.
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Acknowledgements |
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