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1. Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile

   https://doi.org/10.1128/spectrum.02120-23  

   Sidner, Ben; Lerma, Armando; Biswas, Baishakhi; Do, Thi Van; Yu, Yafan; Ronish, Leslie A.; McCullough, Hugh; Auchtung, Jennifer M.; Piepenbrink, Kurt H. (December 2023, Microbiology Spectrum)

   Atack, John M. (Ed.)

   ABSTRACT Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others.Clostridioides difficileis an anaerobic bacterium, which colonizes the mammalian gastrointestinal (GI) tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. AlthoughC. difficiletoxicity stems from secreted toxins, colonization is a prerequisite forC. difficiledisease. WhileC. difficileis known to associate with the mucous layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well understood. To understand the molecular mechanisms by whichC. difficileinteracts with mucins, we usedex vivomucosal surfaces to test the ability ofC. difficileto bind to mucins from different mammalian tissues. We found significant differences inC. difficileadhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed defects in adhesion by mutants deficient in flagella but not type IV pili. These results imply that interactions between host mucins andC. difficileflagella facilitate the initial host attachment ofC. difficileto host cells and secreted mucus. IMPORTANCEClostridioides difficileis one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by whichC. difficilecolonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants. 

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2. Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq

   https://doi.org/10.1128/jb.00220-25  

   Alberts, Maia E; Kurtz, Micaila P; Müh, Ute; Bernardi, Jonathon P; Bollinger, Kevin W; Dobrila, Horia A; Duncan, Leonard; Laster, Hannah M; Orea, Andres J; Pannullo, Anthony G; et al (October 2025, Journal of Bacteriology)

   Henkin, Tina M (Ed.)

   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. 

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3. Specialization of independently acquired flagellar FliC proteins in plant-associated Sphingomonas balances swimming and immunogenicity

   https://doi.org/10.1073/pnas.2535583123  

   Russ, Dor; Saha, Chinmay; Paul, Karnelia; Zheng, Zhiyu; Law, Theresa F; Anguita-Maeso, Manuel; Lundberg, Derek S; Fitzpatrick, Connor R; Dangl, Jeffery L (March 2026, Proceedings of the National Academy of Sciences)

   Plants monitor their environment for microbial invaders using pattern-recognition receptors that detect microbe-associated molecular patterns (MAMPs). Flagellin, the main component of bacterial flagellum, contains the flg22 epitope recognized by the plant immune receptor FLS2. Immune recognition can create an evolutionary conflict, requiring bacteria to balance flagellar function and immune evasion. Here, we show that plant-associated Sphingomonas resolve this constraint by partitioning two flagellar functions, motility and colonization, across two divergent and independently acquired flagellin genes. Comparative genomics revealed widespread coexistence of FliC proteins expressing either an immunogenic variant (FliC-H) or a nonimmunogenic variant (FliC-L). The nonimmunogenic FliC-L is necessary and sufficient for full directional swimming, whereas FliC-H is dispensable for swimming, but sufficient for full attachment and colonization. Flagellin expression patterns mirror these functions. Thus, FLS2 recognizes the flagellar variant required for colonization rather than motility, potentially restricting colonizing bacteria from entering internal leaf and root tissues. 

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4. Nascent flagellar basal bodies are immobilized by rod assembly in Bacillus subtilis

   https://doi.org/10.1128/mbio.00530-25  

   Dunn, Caroline M; Foust, Daniel J; Gao, Yongqiang; Biteen, Julie S; Shaw, Sidney L; Kearns, Daniel B (June 2025, mBio)

   Salama, Nina R (Ed.)

   ABSTRACT Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation inBacillus subtilisusing a fluorescent fusion to the C-ring protein FliM. We find thatB. subtilisbasal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site.Bacillus subtilisassembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show thatB. subtilisbasal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella ofB. subtilisis different from the polar patterning of other bacteria, and we infer that theB. subtilisrod probes the PG for holes that can accommodate the machine. 

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5. Flagellum-driven motility enhances Pseudomonas aeruginosa biofilm formation by altering cell orientation

   https://doi.org/10.1128/aem.00821-25  

   Wei, Guanju; Palalay, Jessica-Jae S; Sanfilippo, Joseph E; Yang, Judy Q (July 2025, Applied and Environmental Microbiology)

   van_Kessel, Julia C (Ed.)

   ABSTRACT Bacterial motility plays a crucial role in biofilm development, yet the underlying mechanism remains not fully understood. Here, we demonstrate that the flagellum-driven motility ofPseudomonas aeruginosaenhances biofilm formation by altering the orientation of bacterial cells, an effect controlled by shear stress rather than shear rate. By tracking wild-typeP. aeruginosaand its non-motile mutants in a microfluidic channel, we demonstrate that while non-motile cells align with the flow, many motile cells can orient toward the channel sidewalls, enhancing cell surface attachment and increasing biofilm cell density by up to 10-fold. Experiments with varying fluid viscosities further demonstrate that bacterial swimming speed decreases with increasing fluid viscosity, and the cell orientation scales with the shear stress rather than shear rate. Our results provide a quantitative framework to predict the role of motility in the orientation and biofilm development under different flow conditions and viscosities.IMPORTANCEBiofilms are ubiquitous in rivers, water pipes, and medical devices, impacting the environment and human health. While bacterial motility plays a crucial role in biofilm development, a mechanistic understanding remains limited, hindering our ability to predict and control biofilms. Here, we reveal how the motility ofPseudomonas aeruginosa, a common pathogen, influences biofilm formation through systematically controlled microfluidic experiments with confocal and high-speed microscopy. We demonstrate that the orientation of bacterial cells is controlled by shear stress. While non-motile cells primarily align with the flow, many motile cells overcome the fluid shear forces and reorient toward the channel sidewalls, increasing biofilm cell density by up to 10-fold. Our findings provide insights into how bacterial transition from free-swimming to surface-attached states under varying flow conditions, emphasizing the role of cell orientation in biofilm establishment. These results enhance our understanding of bacterial behavior in flow environments, informing strategies for biofilm management and control. 

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