Journal article
Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq
Journal of bacteriology, Vol.207(10), e00220-25
10/23/2025
DOI: 10.1128/jb.00220-25
PMCID: PMC12548448
PMID: 40919935
Abstract
Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in
, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon insertion site sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially
, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, 3 of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of
.IMPORTANCE
is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here, we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism
revealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill
without decimating the healthy microbiota needed to keep
in check.
Details
- Title: Subtitle
- Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq
- Creators
- Maia E Alberts - University of IowaMicaila P Kurtz - University of IowaUte Müh - University of IowaJonathon P Bernardi - University of IowaKevin W Bollinger - University of IowaHoria A Dobrila - University of IowaLeonard Duncan - University of IowaHannah M Laster - University of IowaAndres J Orea - University of IowaAnthony G Pannullo - University of IowaJuan G Rivera-Rosado - University of IowaFacundo V Torres - University of IowaCraig D Ellermeier - University of IowaDavid S Weiss - University of Iowa
- Resource Type
- Journal article
- Publication Details
- Journal of bacteriology, Vol.207(10), e00220-25
- DOI
- 10.1128/jb.00220-25
- PMID
- 40919935
- PMCID
- PMC12548448
- NLM abbreviation
- J Bacteriol
- ISSN
- 0021-9193
- eISSN
- 1098-5530
- Publisher
- AMER SOC MICROBIOLOGY
- Grant note
- National Science Foundation: R21 AI159071, R01 AI155492 Public Health Service: DBI-2244169 NSF REU
This work was supported by Public Health Service Grants R21 AI159071 (D.S.W) and R01 AI155492 (C.D.E. and D.S.W). A.J.O. and F.V.T. were supported by NSF REU DBI-1852070. H.M.L. and J.G.R.-R. were supported by NSF REU DBI-2244169. We thank members of the Ellermeier and Weiss laboratories for helpful discussions, John Cronan for information on gpsA, Erin Purcell for information on C. difficile rhs/relA, and two anonymous reviewers for helpful suggestions.
- Language
- English
- Electronic publication date
- 09/08/2025
- Date published
- 10/23/2025
- Academic Unit
- Microbiology and Immunology; Anesthesia
- Record Identifier
- 9984962538702771
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