Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments

Vicki Adams, Radhika Bantwal, Lauren Stevenson, Kit-Ling Jackie Cheung, Milena M Awad, Adam Joel Nicholson, Glen P Carter, Kate E Mackin, Julian I Rood, Dena Lyras

Research output: Contribution to journalArticleResearchpeer-review

6 Citations (Scopus)

Abstract

TnpX is a site-specific recombinase responsible for excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes, however, a limited number are available for use in the clostridia. The ability of TnpX to recognise and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a double plc virR C. perfringens mutant, by allowing the removal and subsequent re-use of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host, C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.
Original languageEnglish
Pages (from-to)3597-3603
Number of pages7
JournalApplied and Environmental Microbiology
Volume80
Issue number12
DOIs
Publication statusPublished - 2014

Cite this

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title = "Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments",
abstract = "TnpX is a site-specific recombinase responsible for excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes, however, a limited number are available for use in the clostridia. The ability of TnpX to recognise and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a double plc virR C. perfringens mutant, by allowing the removal and subsequent re-use of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host, C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.",
author = "Vicki Adams and Radhika Bantwal and Lauren Stevenson and Cheung, {Kit-Ling Jackie} and Awad, {Milena M} and Nicholson, {Adam Joel} and Carter, {Glen P} and Mackin, {Kate E} and Rood, {Julian I} and Dena Lyras",
year = "2014",
doi = "10.1128/AEM.04285-13",
language = "English",
volume = "80",
pages = "3597--3603",
journal = "Applied and Environmental Microbiology",
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Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments. / Adams, Vicki; Bantwal, Radhika; Stevenson, Lauren; Cheung, Kit-Ling Jackie; Awad, Milena M; Nicholson, Adam Joel; Carter, Glen P; Mackin, Kate E; Rood, Julian I; Lyras, Dena.

In: Applied and Environmental Microbiology, Vol. 80, No. 12, 2014, p. 3597-3603.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Utility of the clostridial site-specific recombinase TnpX to clone toxic-product-encoding genes and selectively remove genomic DNA fragments

AU - Adams, Vicki

AU - Bantwal, Radhika

AU - Stevenson, Lauren

AU - Cheung, Kit-Ling Jackie

AU - Awad, Milena M

AU - Nicholson, Adam Joel

AU - Carter, Glen P

AU - Mackin, Kate E

AU - Rood, Julian I

AU - Lyras, Dena

PY - 2014

Y1 - 2014

N2 - TnpX is a site-specific recombinase responsible for excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes, however, a limited number are available for use in the clostridia. The ability of TnpX to recognise and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a double plc virR C. perfringens mutant, by allowing the removal and subsequent re-use of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host, C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

AB - TnpX is a site-specific recombinase responsible for excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes, however, a limited number are available for use in the clostridia. The ability of TnpX to recognise and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a double plc virR C. perfringens mutant, by allowing the removal and subsequent re-use of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host, C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

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