Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em>
Targeted mutations in the anaerobic methylotroph <i>Eubacterium limosum</i> have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from <i>Eubacterium callanderi</i> was placed under the control of an anhydrotetracycline-sensi...
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MDPI AG
2023-05-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/5/1256 |
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| author | James Millard Alexander Agius Ying Zhang Philippe Soucaille Nigel Peter Minton |
| author_facet | James Millard Alexander Agius Ying Zhang Philippe Soucaille Nigel Peter Minton |
| author_sort | James Millard |
| collection | DOAJ |
| description | Targeted mutations in the anaerobic methylotroph <i>Eubacterium limosum</i> have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from <i>Eubacterium callanderi</i> was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in <i>Eubacterium limosum</i> B2. The genes targeted in this study were those encoding the histidine biosynthesis gene <i>hisI</i>, the methanol methyltransferase and corrinoid protein <i>mtaA</i> and <i>mtaC</i>, and <i>mtcB</i>, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within <i>hisI</i> brought about the expected histidine auxotrophy, and deletions of <i>mtaA</i> and <i>mtaC</i> both abolished autotrophic growth on methanol. Deletion of <i>mtcB</i> was shown to abolish the growth of <i>E. limosum</i> on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of <i>E. limosum</i>. |
| first_indexed | 2024-03-11T03:28:07Z |
| format | Article |
| id | doaj.art-24f630959dea47aa870d910bc4bb5d2b |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-11T03:28:07Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-24f630959dea47aa870d910bc4bb5d2b2023-11-18T02:34:06ZengMDPI AGMicroorganisms2076-26072023-05-01115125610.3390/microorganisms11051256Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em>James Millard0Alexander Agius1Ying Zhang2Philippe Soucaille3Nigel Peter Minton4BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UKBBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UKBBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UKBBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UKBBSRC/EPSRC Synthetic Biology Research Centre (SBRC), Biodiscovery Institute, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UKTargeted mutations in the anaerobic methylotroph <i>Eubacterium limosum</i> have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from <i>Eubacterium callanderi</i> was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in <i>Eubacterium limosum</i> B2. The genes targeted in this study were those encoding the histidine biosynthesis gene <i>hisI</i>, the methanol methyltransferase and corrinoid protein <i>mtaA</i> and <i>mtaC</i>, and <i>mtcB</i>, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within <i>hisI</i> brought about the expected histidine auxotrophy, and deletions of <i>mtaA</i> and <i>mtaC</i> both abolished autotrophic growth on methanol. Deletion of <i>mtcB</i> was shown to abolish the growth of <i>E. limosum</i> on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of <i>E. limosum</i>.https://www.mdpi.com/2076-2607/11/5/1256<i>Eubacterium limosum</i><i>Eubacterium callanderi</i>toxin–antitoxin systemsRelBECRISPRgene editing |
| spellingShingle | James Millard Alexander Agius Ying Zhang Philippe Soucaille Nigel Peter Minton Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> Microorganisms <i>Eubacterium limosum</i> <i>Eubacterium callanderi</i> toxin–antitoxin systems RelBE CRISPR gene editing |
| title | Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> |
| title_full | Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> |
| title_fullStr | Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> |
| title_full_unstemmed | Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> |
| title_short | Exploitation of a Type 1 Toxin–Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen <em>Eubacterium limosum</em> |
| title_sort | exploitation of a type 1 toxin antitoxin system as an inducible counter selective marker for genome editing in the acetogen em eubacterium limosum em |
| topic | <i>Eubacterium limosum</i> <i>Eubacterium callanderi</i> toxin–antitoxin systems RelBE CRISPR gene editing |
| url | https://www.mdpi.com/2076-2607/11/5/1256 |
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