Cargando…

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious age...

Descripción completa

Detalles Bibliográficos
Autores principales: Mertaoja, Anna, Nowakowska, Maria B., Mascher, Gerald, Heljanko, Viivi, Groothuis, Daphne, Minton, Nigel P., Lindström, Miia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873358/
https://www.ncbi.nlm.nih.gov/pubmed/33584620
http://dx.doi.org/10.3389/fmicb.2021.617269
_version_ 1783649366791159808
author Mertaoja, Anna
Nowakowska, Maria B.
Mascher, Gerald
Heljanko, Viivi
Groothuis, Daphne
Minton, Nigel P.
Lindström, Miia
author_facet Mertaoja, Anna
Nowakowska, Maria B.
Mascher, Gerald
Heljanko, Viivi
Groothuis, Daphne
Minton, Nigel P.
Lindström, Miia
author_sort Mertaoja, Anna
collection PubMed
description The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.
format Online
Article
Text
id pubmed-7873358
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-78733582021-02-11 CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research Mertaoja, Anna Nowakowska, Maria B. Mascher, Gerald Heljanko, Viivi Groothuis, Daphne Minton, Nigel P. Lindström, Miia Front Microbiol Microbiology The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management. Frontiers Media S.A. 2021-01-27 /pmc/articles/PMC7873358/ /pubmed/33584620 http://dx.doi.org/10.3389/fmicb.2021.617269 Text en Copyright © 2021 Mertaoja, Nowakowska, Mascher, Heljanko, Groothuis, Minton and Lindström. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Mertaoja, Anna
Nowakowska, Maria B.
Mascher, Gerald
Heljanko, Viivi
Groothuis, Daphne
Minton, Nigel P.
Lindström, Miia
CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title_full CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title_fullStr CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title_full_unstemmed CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title_short CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research
title_sort crispr-cas9-based toolkit for clostridium botulinum group ii spore and sporulation research
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873358/
https://www.ncbi.nlm.nih.gov/pubmed/33584620
http://dx.doi.org/10.3389/fmicb.2021.617269
work_keys_str_mv AT mertaojaanna crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT nowakowskamariab crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT maschergerald crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT heljankoviivi crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT groothuisdaphne crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT mintonnigelp crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch
AT lindstrommiia crisprcas9basedtoolkitforclostridiumbotulinumgroupiisporeandsporulationresearch