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

Anna Mertaoja,Daphne Groothuis,Gerald Mascher,Miia Lindström,Nigel P Minton,Maria B Nowakowska,Viivi Heljanko

doi:10.3389/fmicb.2021.617269

Abstract

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.

Highlights

Clostridium botulinum is a Gram-positive, strictly anaerobic, spore-forming bacterium
The functional copy of spo0A was not the native gene as this would result in a complemented strain that would be indistinguishable from the progenitor wild type (WT) cell
When designing the watermark sequence, we considered the codon usage of C. botulinum species by replacing the WT codons with used synonymous ones

Summary

Introduction

Clostridium botulinum is a Gram-positive, strictly anaerobic, spore-forming bacterium. C. botulinum strains of Group I and Group II are usually associated with human botulism, which classically manifests as a food poisoning. This form of botulism results from consumption of foods where C. botulinum spores germinated and outgrew into a neurotoxigenic culture. Their toxinogenic potential at refrigeration temperatures makes C. botulinum Group II strains a particular food safety concern (Lindström et al, 2006). C. botulinum spores can colonize wounds or the digestive tract of infants and susceptible adults with compromised gut microbiota, causing the toxicoinfectious forms of wound and intestinal botulism, respectively (Harris, 2015)

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