The ClosTron: mutagenesis in Clostridium refined and streamlined
Abstract
The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully.
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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., 2010
The ClosTron technique was further optimized to generate multiple insertional mutations in the same strain by recycling of marker through the actions of FLP recombinase, and delivery of cargo sequence of up to 1 kb was successfully demonstrated in C. sporogenes.
“This technique was further optimized to generate multiple insertional mutations in the same strain by recycling of marker through the actions of FLP recombinase, and the delivery of cargo sequence of up to 1 kb was successfully demonstrated in C. sporogenes (Heap et al., 2010).”
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., 2010
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., 2010
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- Authors
- Heap, J. T., Kuehne, S. A., Ehsaan, M., Cartman, S. T., Cooksley, C. M., Scott, J. C., & Minton, N. P.
- Affiliations
- University of Nottingham (GB)
- Corresponding author
- Minton, Nigel P.
- Venue
- Journal of Microbiological Methods
- Publisher
- Elsevier BV
- Published
- 2010-01
- Volume
- 80
- Issue
- 1
- Pages
- 49-55
- ISSN
- 0167-7012 (ISSN-L)
- OpenAlex
- W2076243547
- Cited by
- 418
- References
- 33
- Citations by year
- 2026: 12 · 2025: 12 · 2024: 14 · 2023: 15 · 2022: 13 · 2021: 29 · 2020: 13 · 2019: 25 · 2018: 28 · 2017: 24 · 2016: 33 · 2015: 34 · 2014: 53 · 2013: 34 · 2012: 35
- FWCI
- 10.52
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Microbiological Methods (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Biotechnology and Biological Sciences Research Council (BB/D522289/1, BB/D522797/1, B/D522289/1, BB/G016224/1, BB/E021271/1, BB/F003390/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- Clostridium, FLP/FRT recombination, transgene delivery, Clostridium acetobutylicum, Clostridium sporogenes, Clostridium beijerinckii, Clostridium difficile, mutant isolation, blue-white screening, plasmid design
- MeSH terms
- Clostridium, Genetic Techniques, Introns, Plasmids, Mutagenesis
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