TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens

Jessica A Wisniewski, Daouda A K Traore, Trudi L Bannam, Dena Lyras, James C Whisstock, Julian I Rood

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) have been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP), encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.
Original languageEnglish
Pages (from-to)884-896
Number of pages13
JournalMolecular Microbiology
Volume99
Issue number5
DOIs
Publication statusPublished - 1 Mar 2016

Cite this

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title = "TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens",
abstract = "Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) have been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP), encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.",
author = "Wisniewski, {Jessica A} and Traore, {Daouda A K} and Bannam, {Trudi L} and Dena Lyras and Whisstock, {James C} and Rood, {Julian I}",
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TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens. / Wisniewski, Jessica A; Traore, Daouda A K; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I.

In: Molecular Microbiology, Vol. 99, No. 5, 01.03.2016, p. 884-896.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens

AU - Wisniewski, Jessica A

AU - Traore, Daouda A K

AU - Bannam, Trudi L

AU - Lyras, Dena

AU - Whisstock, James C

AU - Rood, Julian I

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N2 - Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) have been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP), encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

AB - Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) have been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP), encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

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