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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 inClostridioides difficile, 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, especiallyBacillus subtilis, 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 ofC. difficile.IMPORTANCEClostridioides difficileis 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 organismBacillus subtilisrevealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that killC. difficilewithout decimating the healthy microbiota needed to keepC. difficilein check. 

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created byL,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogenClostridioides difficile,about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability inC. difficile. We also show thatC. difficilehas five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non–C. difficileVanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability inC. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs inC. difficilemakes them potential targets for antibiotics that killC. difficileselectively.