The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

H M Wang, M C M Smith, P Mullany

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd) attL, attR, attTn, and attB(Bs3) with relative binding affinities attTn approximate to attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.

Original languageEnglish
Pages (from-to)4871-4878
Number of pages8
JournalJournal of Bacteriology
Volume188
DOIs
Publication statusPublished - 2006

Keywords

  • LARGE RESOLVASE
  • BACILLUS-SUBTILIS
  • ENTEROCOCCUS-FAECALIS
  • RECOMBINATION SYSTEM
  • BXB1 INTEGRATION
  • EXCISION
  • TNPX
  • ELEMENT
  • TN4451
  • TN916

Cite this

The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site. / Wang, H M ; Smith, M C M ; Mullany, P .

In: Journal of Bacteriology, Vol. 188, 2006, p. 4871-4878.

Research output: Contribution to journalArticle

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title = "The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site",
abstract = "Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100{\%} of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd) attL, attR, attTn, and attB(Bs3) with relative binding affinities attTn approximate to attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.",
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T1 - The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

AU - Wang, H M

AU - Smith, M C M

AU - Mullany, P

PY - 2006

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N2 - Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd) attL, attR, attTn, and attB(Bs3) with relative binding affinities attTn approximate to attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.

AB - Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd) attL, attR, attTn, and attB(Bs3) with relative binding affinities attTn approximate to attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.

KW - LARGE RESOLVASE

KW - BACILLUS-SUBTILIS

KW - ENTEROCOCCUS-FAECALIS

KW - RECOMBINATION SYSTEM

KW - BXB1 INTEGRATION

KW - EXCISION

KW - TNPX

KW - ELEMENT

KW - TN4451

KW - TN916

U2 - 10.1128/JB.00210-06

DO - 10.1128/JB.00210-06

M3 - Article

VL - 188

SP - 4871

EP - 4878

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

ER -