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Updated: Mar 20, 2026
MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
RNA-seq analysis of virR and revR mutants of Clostridium perfringens
Lee-Yean Low1, Paul F Harrison2, Ya-Hsun Lin1
1Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, 3800, Australia.
Background:
Clostridium perfringens causes toxin-mediated diseases, including gas gangrene (clostridial myonecrosis) and food poisoning in humans. The production of the toxins implicated in gas gangrene, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. In addition, RevR, an orphan response regulator, has been shown to affect virulence in the mouse myonecrosis model. RevR positively regulates the expression of genes that encode hydrolytic enzymes, including hyaluronidases and sialidases.
Results:
To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis was carried out with strand-specific RNA-seq on C. perfringens strain JIR325 and its isogenic virR and revR regulatory mutants. Using the edgeR analysis package, 206 genes in the virR mutant and 67 genes in the revR mutant were found to be differentially expressed. Comparative analysis revealed that VirR acts as a global negative regulator, whilst RevR acts as a global positive regulator. Therefore, about 95 % of the differentially expressed genes were up-regulated in the virR mutant, whereas 81 % of the differentially expressed genes were down-regulated in the revR mutant. Importantly, we identified 23 genes that were regulated by both VirR and RevR, 18 of these genes, which included the sporulation-specific spoIVA, sigG and sigF genes, were regulated positively and negatively by RevR and VirR, respectively. Furthermore, analysis of the mapped RNA-seq reads visualized as depth of coverage plots showed that there were 93 previously unannotated transcripts in intergenic regions. These transcripts potentially encode small RNA molecules.
Conclusion:
In conclusion, using strand-specific RNA-seq analysis, this study has identified differentially expressed chromosomal and pCP13 native plasmid-encoded genes, antisense transcripts, and transcripts within intergenic regions that are controlled by the VirSR or RevR regulatory systems.
Insights
Clostridium perfringens virulence is controlled by the VirSR and RevR regulatory systems. This study used RNA-seq to identify genes regulated by VirSR and RevR, revealing their roles in toxin production and disease.
Area of Science:
- Microbiology
- Molecular Biology
- Genomics
Background:
- Clostridium perfringens causes serious diseases like gas gangrene and food poisoning through toxin production.
- The VirSR two-component system and the RevR regulator are key in controlling toxins like alpha-toxin and perfringolysin O.
- RevR also influences virulence by regulating genes for hydrolytic enzymes such as hyaluronidases and sialidases.
Purpose of the Study:
- To comprehensively characterize the regulatory networks of VirSR and RevR in Clostridium perfringens.
- To identify genes and transcripts controlled by these two crucial regulatory systems.
Main Methods:
- Comparative transcriptomic analysis using strand-specific RNA-sequencing (RNA-seq).
- Analysis of gene expression in wild-type C. perfringens strain JIR325 and its isogenic virR and revR mutants.
- Utilized the edgeR package for differential gene expression analysis.
Main Results:
- Identified 206 differentially expressed genes in the virR mutant and 67 in the revR mutant.
- Revealed VirR as a global negative regulator and RevR as a global positive regulator.
- Discovered 93 unannotated transcripts in intergenic regions, potentially encoding small RNAs, and identified 23 genes regulated by both VirR and RevR.
Conclusions:
- Strand-specific RNA-seq successfully identified differentially expressed genes, antisense transcripts, and intergenic transcripts regulated by VirSR and RevR.
- This study provides a detailed understanding of the regulatory roles of VirSR and RevR in C. perfringens.
- The findings contribute to understanding the complex gene regulation underlying C. perfringens virulence.
