The ClosTron: a universal gene knock-out system for the genus Clostridium

Heap, John T.; Pennington, Oliver J.; Cartman, Stephen T.; Carter, Glen P.; Minton, Nigel P.

doi:10.1016/j.mimet.2007.05.021

Cited by 663Bacterial Genetics and BiotechnologyCRISPR and Genetic EngineeringClostridium difficile and Clostridium perfringens research

The ClosTron: a universal gene knock-out system for the genus Clostridium

Heap, John T., Pennington, Oliver J., Cartman, Stephen T., Carter, Glen P., Minton, Nigel P.·2007·Journal of Microbiological Methods

DOI 10.1016/j.mimet.2007.05.021

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Abstract

Progress in exploiting clostridial genome information has been severely impeded by a general lack of effective methods for the directed inactivation of specific genes. Those few mutants that have been generated have been almost exclusively derived by single crossover integration of a replication-deficient or defective plasmid by homologous recombination. The mutants created are therefore unstable. Here we have adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. Integrants are readily selected on the basis of acquisition of resistance to erythromycin, and are generated from start to finish in as little as 10 to 14 days. Unlike single crossover plasmid integrants, the mutants are extremely stable. The system has been used to make 6 mutants of Clostridium acetobutylicum and 5 of Clostridium difficile, exceeding the number of published mutants ever generated in these species. Genes have also been inactivated for the first time in Clostridium botulinum and Clostridium sporogenes, suggesting the system will be universally applicable to the genus. The procedure is highly efficient and reproducible, and should revolutionize functional genomic studies in clostridia.

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Heap et al. adapted mobile group II introns for use in a wide spectrum of clostridia, and the technology is termed ClosTron.
“Heap et al. (2007) adapted such mobile elements for use in a wide spectrum of clostridia, and the technology is termed “ClosTron.””
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Heap et al., 2007
The ClosTron technique is reproducible and has been widely applied to various clostridia including acetogen C. autoethanogenum.
“Thanks to these properties, this technique is reproducible and has been widely applied to various clostridia (Heap et al., 2007, 2010; Camiade et al., 2010) including acetogen C. autoethanogenum (Mock et al., 2015; Marcellin et al., 2016).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Heap et al., 2007
By incorporating a clostridial retro-transposition activating marker based on the ermB gene of the Enterococcus faecalis plasmid pAMβ1, positive selection for the desired integration can be made by the bacteria's acquisition of erythromycin resistance.
“By incorporating a clostridial retro-transposition activating marker (RAM) based on the ermB gene of the Enterococcus faecalis plasmid pAMβ1, positive selection for the desired integration can be conveniently made by the bacteria's acquisition of erythromycin resistance (Heap et al., 2007, 2010; Kuehne and Minton, 2012).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Heap et al., 2007

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Authors: Heap, J. T., Pennington, O. J., Cartman, S. T., Carter, G. P., & Minton, N. P.
Affiliations: University of Nottingham (GB)
Corresponding author: Minton, Nigel P.
ORCID: Heap, John T.
Carter, Glen P.
Minton, Nigel P.
Venue: Journal of Microbiological Methods
Publisher: Elsevier BV
Published: 2007-09
Volume: 70
Issue: 3
Pages: 452-464
ISSN: 0167-7012 (ISSN-L)
DOI: 10.1016/j.mimet.2007.05.021
OpenAlex: W2099047627
Cited by: 663
References: 37
Citations by year: 2026: 14 · 2025: 15 · 2024: 16 · 2023: 22 · 2022: 31 · 2021: 38 · 2020: 29 · 2019: 34 · 2018: 41 · 2017: 26 · 2016: 51 · 2015: 51 · 2014: 56 · 2013: 55 · 2012: 47
FWCI: 15.35
Type: journal article
Language: en
Open access: closed
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/legal/tdmrep-license (tdm)
Locations: Journal of Microbiological Methods (publishedVersion)
PubMed (publishedVersion)
Funders: Biotechnology and Biological Sciences Research Council (BB/D522289/1, BB/D522797/1)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Health Sciences › Medicine › Infectious Diseases › Clostridium difficile and Clostridium perfringens research
Keywords: Clostridium, gene inactivation, Clostridium acetobutylicum, Clostridium difficile, Clostridium botulinum, Clostridium sporogenes, mutant stability, functional genomics
MeSH terms: Bacterial Proteins, Base Sequence, Clostridium, Conjugation, Genetic, Introns, Isopropyl Thiogalactoside, Plasmids, Promoter Regions, Genetic, Recombination, Genetic, RNA-Directed DNA Polymerase, Lactococcus lactis, Mutagenesis, Insertional, Gene Deletion, Retroelements

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The ClosTron: a universal gene knock-out system for the genus Clostridium

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