A thiouracil desulfurase protects Clostridioides difficile RNA from 4-thiouracil incorporation, providing a competitive advantage in the gut.

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Published in final edited form as: Cell Host Microbe. 2025 Mar 25;33(4):573–588.e7. doi: 10.1016/j.chom.2025.03.001 Search in PMC Search in PubMed View in NLM Catalog Add to search A thiouracil desulfurase protects Clostridioides difficile RNA from 4-thiouracil incorporation providing a competitive advantage in the vertebrate gut Matthew J Munneke Matthew J Munneke 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Matthew J Munneke 1 , Yifeng Yuan Yifeng Yuan 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA Find articles by Yifeng Yuan 2 , Eva C Preisner Eva C Preisner 3 Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA Find articles by Eva C Preisner 3 , Catherine D Shelton Catherine D Shelton 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Catherine D Shelton 1 , Darian T Carroll Darian T Carroll 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Darian T Carroll 1 , Nicole S Kirchoff Nicole S Kirchoff 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Nicole S Kirchoff 1 , Ken P Dickson Ken P Dickson 3 Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA Find articles by Ken P Dickson 3 , Jose O Cantu Jose O Cantu 3 Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA Find articles by Jose O Cantu 3 , Martin V Douglass Martin V Douglass 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Martin V Douglass 1 , M Wade Calcutt M Wade Calcutt 4 Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA Find articles by M Wade Calcutt 4 , Katherine N Gibson-Corley Katherine N Gibson-Corley 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. Find articles by Katherine N Gibson-Corley 1 , Maribeth R Nicholson Maribeth R Nicholson 5 Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA Find articles by Maribeth R Nicholson 5 , Mariana X Byndloss Mariana X Byndloss 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. 6 Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA Find articles by Mariana X Byndloss 1, 6 , Robert A Britton Robert A Britton 3 Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA Find articles by Robert A Britton 3 , Valérie de Crécy-Lagard Valérie de Crécy-Lagard 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA Find articles by Valérie de Crécy-Lagard 2 , Eric P Skaar Eric P Skaar 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. 7 Lead contact Find articles by Eric P Skaar 1, 7, # Author information Article notes Copyright and License information 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA. 2 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA 3 Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA 4 Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA 5 Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232-7917, USA 6 Howard Hughes Medical Institute, Vanderbilt University Medical Center, Nashville, TN 37232, USA 7 Lead contact Author Contributions Conceptualization, M.J.M., V.C.L., E.P.S.; Investigation, M.J.M., Y.Y., E.C.P., C.D.S., D.T.C., K.P.D., J.O.C., M.V.D., N.S.K., M.W.C., M.R.N.; Formal Analysis, M.J.M., Y.Y., M.W.C. K.N.G.-C.; Visualization, M.J.M., Y.Y., K.N.G.-C.; Supervision, M.X.B., R.A.B., V.C.L., E.P.S.; Writing – original draft, M.J.M., E.P.S.; Writing – review and editing, all authors. # Corresponding author – [email protected] Issue date 2025 Apr 9. PMC Copyright notice PMCID: PMC11985272  NIHMSID: NIHMS2063643  PMID: 40139192 The publisher's version of this article is available at Cell Host Microbe Summary Nucleotides are essential building blocks for major cellular macromolecules and critical for life. Consequently, bacterial pathogens must acquire or synthesize nucleotides during infection. Clostridioides difficile is the most common hospital-acquired gastrointestinal infection, and nutrient acquisition is critical for pathogenesis. However, the impact of nucleotide metabolism on C. difficile infection remains unclear. Here we discover that 4-thiouracil (4-TU), a pyrimidine analog present in the human gut, is toxic to commensal bacteria. 4-TU hijacks the uracil salvage pathway for incorporation into RNA through the uracil phosphoribosyltransferase activity encoded by PyrR and Upp. C. difficile can salvage 4-TU as a pyrimidine source through the enzymatic action of a thiouracil desulfurase (TudS), thereby contributing to C. difficile fitness in mice fed 4-TU or MiniBioreactor models of infection containing exogenous 4-TU. Collectively, these results reveal a molecular mechanism for C. difficile to utilize a poisonous pyrimidine analog in the vertebrate gut to outcompete commensal microbes. Keywords: Clostridioides difficile , nucleotide, TudS, 4-thiouracil, PyrR, Upp, RNA, pyrimidine analog Graphical Abstract In Brief Clostridioides difficile is a master scavenger. Munneke et al. discover a pyrimidine analog in the vertebrate gut, 4-thiouracil can be used by C. difficile as a nutrient to outgrow neighboring bacteria. Their study identifies TudS, a thiouracil desulfurase, enables utilization of 4-thiouracil as a pyrimidine source, preventing incorporation into RNA. Introduction Cells require nucleic acids to store genetic information encoding for protein synthesis, an essential process for life. Nucleic acids are composed of nucleotides containing a nitrogenous base that dictates base-pairing in the macromolecule and defines the genetic code. In most bacteria, nucleotides can either be synthesized de novo or nucleotide precursors can be salvaged from the environment. Frequently, cellular energy demands dictate which of these processes is used, as de novo biosynthesis of purine and pyrimidine nucleotides requires up to 20 units of ATP per RNA nucleotide 1 . Nucleotides can also function in energy storage, serve as precursors to peptidoglycan synthesis, and act as signaling molecules, underscoring the importance of nucleotide acquisition for bacterial proliferation 2 , 3 . Nucleotide acquisition contributes to the virulence of several human bacterial and parasitic pathogens in the host environment 4 – 7 . Numerous bacteria, including human-associated lactic acid bacteria and Helicobacter pylori , can be auxotrophic for nucleotides suggesting that exogenous sources of nucleotides exist in the vertebrate host 8 – 10 . One possible reservoir is the human gastrointestinal tract, as humans are estimated to consume 1–2 g of dietary-derived nucleotides per day 11 . In the Drosophila gut, luminal uridine is salvaged by pathogens to generate and release uracil, activating host dual oxidase and inducing gut inflammation 12 , 13 . In vertebrates, the contribution of pyrimidine nucleotide de novo biosynthesis to gut colonization by Escherichia coli , Salmonella Typhimurium, and Klebsiella pneumoniae has been described 14 – 16 . However, molecular mechanisms of salvage by human gut pathogens are not well understood. Broad-spectrum antibiotics dramatically shift the microbiota and nutrient milieu in the vertebrate gastrointestinal tract, and treatment of mice with antibiotics alters the abundance of nucleotides and nucleotide precursors 17 . The Gram-positive human gastrointestinal pathogen Clostridioides difficile thrives in the antibiotic-perturbed gut and causes nearly half a million infections and 29,000 deaths annually 18 . C. difficile is a master scavenger of host-, microbiota-, and dietary-derived molecules that support macromolecular synthesis and virulence 19 – 23 . However, the mechanisms by which C. difficile acquires nucleotides in the antibiotic-perturbed gut and the impact of nucleotide metabolism on fitness have yet to be determined. Here we reveal the importance of pyrimidine nucleotide acquisition for C. difficile infection (CDI) and describe a molecular mechanism by which C. difficile salvages a modified pyrimidine nucleobase, 4-thiouracil (4-TU) present in human stool. We discovered that t hio u racil d e s ulfurase (TudS) is a C. difficile enzyme that is necessary and sufficient for 4-TU salvage and detoxification. Quantification of 4-thiouridine (s 4 U) in RNA revealed that TudS prevents incorporation of 4-TU into C. difficile RNA. Furthermore, TudS-mediated 4-TU detoxification is critical for pathogenesis in the vertebrate gut containing 4-TU, and TudS provides C. difficile a fitness advantage against competing microbes when 4-TU is present. Taken together, these findings suggest that 4-TU benefits C. difficile in the gut by inhibiting competing microbes and serving as a pyrimidine source for growth. Results Pyrimidine metabolism contributes to C. difficile infection. Based on the importance of nucleotide acquisition for virulence in several human pathogens 24 , 25 , we hypothesized that nucleotide acquisition is critical for CDI. To begin investigating how nucleotides impact CDI, pediatric stool samples from healthy or CDI donors lacking comorbidities were used to quantify the abundance of a pyrimidine nucleotide precursor, uracil, by liquid chromatography-tandem mass spectrometry (LC-MS/MS) ( Table S1 ). These results revealed a significant decrease in the abundance of uracil during CDI relative to healthy control stool ( Figure 1A ). We next sought to develop a murine model to determine the contribution of pyrimidine nucleotides to CDI. A cefoperazone mouse model of infection was employed and the abundance of uracil was quantified prior to infection ( Figure 1B ). Consistent with previous reports, the abundance of uracil decreases in the antibiotic-treated murine gut 17 , 26 ( Figure 1C ). Since uracil is depleted in the antibiotic-treated gut environment, we hypothesized that C. difficile must utilize the de novo synthesis pathway or salvage an alternative pyrimidine source during infection to meet the cellular requirement for pyrimidines. To test the former hypothesis, a mutant lacking the gene encoding orotate phosphoribosyl transferase (Δ pyrE ), an essential enzyme for pyrimidine biosynthesis, was generated in C. difficile strain CD196 ( Figure 1D ). Mice were infected with wildtype (WT) or Δ pyrE C. difficile and disease progression was monitored for 4 days post-infection. Gut colonization was similar between WT and Δ pyrE strains (data not shown). Mice infected with WT had decreased survival and lost more weight than mice infected with Δ pyrE , suggesting that pyrimidine biosynthesis is required for virulence in the murine gastrointestinal tract ( Figures 1E and 1F ). Collectively, these data establish the importance of pyrimidine nucleotide metabolism to C. difficile during infection. Figure 1. Pyrimidine biosynthesis contributes to C. difficile pathogenesis. Open in a new tab ( A ) LC-MS/MS quantification of uracil in human feces from healthy control (HC) and C. difficile infected (CDI) donors. N = 50 (HC) N = 10 (CDI). ( B ) Cefoperazone (cfp) mouse model of CDI. ( C ) LC-MS/MS quantification of uracil in stool from mice treated with cfp and untreated controls. N = 5. The dotted line represents the limit of detection. ( D ) Bacterial de novo pyrimidine synthesis and salvage pathways. ( E ) Survival analysis and ( F ) weight loss of mice infected with WT or Δ pyrE C. difficile . N = 10. Each dot represents an individual mouse or human donor. All data are represented as mean ±SD. Statistical significance was determined using a two-tailed Mann-Whitney U test ( A ) and ( C ), Log-rank (Mantel-Cox) test ( E ), and two-way ANOVA with Dunnett’s multiple comparison post-test ( F ). P values are denoted, ns = not significant. Due to the energy expenditure required to biosynthesize pyrimidines, we hypothesized that salvaging from the gut environment rather than synthesizing de novo may be a favorable alternative to obtain pyrimidine nucleotides. To assess the capacity of C. difficile to salvage pyrimidine nucleotide precursors other than uracil, growth of a Δ pyrE strain was measured when exposed to a variety of pyrimidines as the sole pyrimidine source in vitro . Of the molecules tested, only uracil and 4-thiouracil (4-TU) restored growth of Δ pyrE ( Table S2 ; Figure 2A ). Since 20 μg/mL uracil and 20 μg/mL 4-TU restored growth of the Δ pyrE strain to WT levels, 20 μg/mL was selected for future growth assays. We next hypothesized that C. difficile encounters 4-TU in the gastrointestinal tract. To test this, 4-TU was quantified in human pediatric stool of healthy and CDI donors by LC-MS/MS. Indeed, 4-TU is present within human stool, and decreases during CDI ( Figure 2B ). These data suggest that C. difficile possesses a molecular mechanism to salvage 4-TU, a modified pyrimidine present in the human gastrointestinal tract, and incorporate it as a uracil source. Figure 2. TudS enables utilization and detoxification of 4-TU. Open in a new tab ( A ) Growth of WT and Δ pyrE C. difficile with 4-TU as the sole pyrimidine source. ( B ) LC-MS/MS quantification of 4-TU in human feces from healthy control (HC) and C. difficile infected (CDI) donors. N = 50 (HC) N = 10 (CDI). The dotted line represents the limit of detection. ( C ) Weight loss of CDI mice given WT, Δ pyrE , or Δ pyrE with 4-TU. N = 10. ( D ) Edema scores three days post-infection. N = 6 (WT), 6 (Δ pyrE ), 10 (Δ pyrE + 4-TU). ( E-H ) Representative photomicrographs of H&E-stained cecal sections three days post-infection. Stars (*) indicate edema with brackets identifying the relative amount of edema, arrows identifying inflammatory infiltrates, and arrowheads indicating sloughing of enterocytes. Scale bar = 100 μm ( I ) Growth of Δ pyrE and Δ pyrE Δ tudS C. difficile harboring empty vector (EV) or vector containing tudS (p tudS ) with 4-TU as the sole pyrimidine source. ( J ) Growth of Δ pyrE E. coli harboring EV or p tudS with either uracil or 4-TU as the sole pyrimidine source. ( K ) Growth of WT and Δ tudS C. difficile harboring EV or p tudS in media containing 4-TU. ( L ) Growth of WT E. coli harboring EV or p tudS in vehicle or 4-TU treated media. ( M ) Working model for the role of TudS in C. difficile . Data are represented as mean±SD of biological and technical triplicate. Individual data points represent individual mice or human donors. The concentration of 4-TU and uracil for all growth experiments was 20 μg/mL. Statistical significance was determined using a two-tailed Mann-Whitney U test ( B ), two-way ANOVA with Tukey’s multiple comparisons test ( C ), and one-way ANOVA with Dunnett’s multiple comparisons test ( D ). P values are denoted. Considering the observation that 4-TU can be utilized by C. difficile as a pyrimidine source for growth in vitro , we reasoned that salvage of 4-TU from the vertebrate gastrointestinal tract would restore the virulence defect exhibited by Δ pyrE . To test this, mice were infected with WT, Δ pyrE , or Δ pyrE with 4-TU introduced by oral gavage. Consistently, mice infected with WT lost more weight than mice infected with Δ pyrE , and 4-TU supplementation led to Δ pyrE causing similar weight loss to WT ( Figure 2C ). To further characterize the inflammatory environment, histopathological analysis was performed. Mice infected with WT displayed marked submucosal edema, neutrophilic infiltration, and enterocyte shedding while submucosal edema was significantly reduced in Δ pyrE -infected mice ( Figures 2D – H ). Notably, Δ pyrE -infected mice given 4-TU did not display substantial differences in submucosal edema relative to WT-infected mice ( Figures 2D , 2F , and 2H ). Collectively, these data suggest that C. difficile can salvage 4-TU as a pyrimidine source in the vertebrate gut. TudS enables C. difficile to utilize 4-TU as a pyrimidine source. Given that C. difficile can use 4-TU as a uracil source, we hypothesized that C. difficile encodes an enzyme capable of converting 4-TU to uracil. Proteins containing domain of unknown function 523 (DUF523) have been described in Gram-negative environmental bacteria as having the ability to convert thiouracil to uracil 27 , 28 . However, a molecular mechanism for thiouracil desulfuration has not been described in a pathogen. Members of the DUF523 family are present in organisms spanning all domains of life and are widespread across the bacterial tree ( Figure S1A ). Many organisms encode several members of the family (1 to 4) and DUF523 is conserved in numerous human pathogens including several Acinetobacter , Clostridium , and Clostridioides species ( Figure S1B ; Table S3 ). C. difficile encodes two DUF523 family members, CD196_RS15345 (CD630_30720; Uniprot id Q184Q8 ) and CD196_RS03875 (CD630_07100; Uniprot id Q189Q8 ) ( Figure S1B ). We have named CD196_RS03875 tudS ( t hio u racil d e s ulfidase) based on the data described below. We hypothesized that the C. difficile TudS is responsible for utilization of 4-TU as a pyrimidine source as it is in the same orthologous group as one of the only two biochemically characterized TudS enzymes ( Figure S1 ). To test this, a mutant strain lacking tudS was generated in the uracil auxotroph C. difficile background (Δ pyrE Δ tudS ) and this strain was provided 20 μg/mL 4-TU as the sole pyrimidine source. Indeed, Δ pyrE Δ tudS was unable to grow with 4-TU as the sole pyrimidine source, and this phenotype was complemented by expression of tudS in trans (Δ pyrE Δ tudS -p tudS ) ( Figure 2I ). To investigate if TudS is sufficient for utilization of 4-TU as a pyrimidine source, tudS was heterologously expressed in E. coli K12. E. coli lacks a DUF523 homolog, and Δ pyrE E. coli is unable to grow with 4-TU provided as the sole pyrimidine source ( Figure 2J ). However, heterologous expression of tudS in Δ pyrE E. coli enabled growth with 4-TU as the sole pyrimidine source ( Figure 2J ). These data demonstrate that tudS is both required and sufficient to use 4-TU as a pyrimidine source. To define the molecular mechanism by which TudS enables utilization of 4-TU as a pyrimidine source, the contribution of predicted active site residues to the ability of TudS to enable growth using 4-TU was examined. A multiple sequence alignment of TudS from C. difficile with homologs from diverse bacterial species revealed regions of high amino acid conservation that are consistent with catalytic motifs ( Figure S2A ) 27 . Structural determination of TudS from an uncultured Aeromonas species (Vcz) identified that conserved active site cysteines support coordination of an iron-sulfur cluster, and glutamate participates in hydrogen bonding with a water molecule that facilitates nucleophilic attack of the C-S bond of thiouracil 29 . Structural modeling of TudS from C. difficile overlaid with the crystal structure of TudS from Aeromonas revealed multiple cysteine residues (C38 and C99) and a glutamate residue (E40) in the predicted active site of TudS from C. difficile ( Figure S2B ). Residues C38, C99, and E40 were mutated to alanine and individual point mutant FLAG-TudS fusion proteins were expressed in Δ pyrE E. coli . Mutation of predicted active site residues did not affect TudS protein abundance relative to WT FLAG-TudS ( Figure S2C ), and mutation of each residue abolished the ability of TudS to enable growth using 4-TU as a pyrimidine source ( Figure S2D ). These results demonstrate that residues C38, C99, and E40 in C. difficile TudS are critical for utilization of 4-TU as a nutrient. TudS detoxifies 4-TU. Pyrimidine analogs such as 5-fluorouracil (5-FU), gemcitabine, and zidovudine are toxic to bacteria 30 – 32 , and a bacterial enzyme capable of inactivating 5-FU has recently been discovered 33 . Similarly, 4-TU has antibacterial activity 34 , 35 , however, a bacterial mechanism to detoxify 4-TU has not been described. To investigate if TudS detoxifies 4-TU, a mutant in the gene encoding TudS (Δ tudS ) was generated and challenged with 20 μg/mL 4-TU. Δ tudS was unable to grow in the presence of 4-TU, and this phenotype could be complemented by providing tudS in trans (Δ tudS -p tudS ) ( Figure 2K ). To investigate the ubiquity of TudS, we searched the genomes of pathogenic and commensal Clostridial strains for TudS homologs. Notably, the prevalence of TudS is higher in pathogenic strains (18 out of 18) than commensal strains (10 out of 24) ( Table S4 ). Further examination of the conservation of TudS across C. difficile strains using representative strains from all five C. difficile clades revealed TudS is conserved across all clades with high sequence identity, including several recent clinical isolates ( Figure S3A ). These observations motivated the hypothesis that TudS detoxifies 4-TU in C. difficile clinical isolates. To address this, strains isolated from patients with asymptomatic C. difficile colonization (AC) and symptomatic (S) CDI were treated with 4-TU. Regardless of symptom status, C. difficile clinical isolates demonstrated similar levels of resistance to 4-TU as the WT CD196 laboratory strain ( Figures S3B and S3C ). The finding that TudS and 4-TU resistance are maintained in C. difficile clinical isolates implies that 4-TU metabolism plays a role during CDI in humans. Consistent with previous reports 34 , 35 , growth of WT E. coli is inhibited by 20 μg/mL 4-TU ( Figure 2L ). We therefore sought to interrogate if TudS is sufficient to protect E. coli from 4-TU toxicity. Heterologous expression of tudS in WT E. coli (WT-p tudS ) enabled growth at a concentration of 4-TU that inhibited the growth of the empty vector control strain (WT-EV) ( Figure 2L ). To determine if TudS active site residues required to use 4-TU as a pyrimidine source are required to detoxify 4-TU, C38A, E40A, and C99A FLAG-TudS mutant fusions were expressed in WT E. coli ( Figure S2E ), and these strains were treated with 4-TU. WT E. coli strains harboring C38A, E40A, and C99A TudS mutants were unable to grow in the presence of 4-TU ( Figure S2F ). Both tagged and untagged TudS enabled WT E. coli to grow in the presence of 4-TU with similar dynamics to the untreated control strains ( Figure S2G ). Taken together, these data suggest that TudS detoxifies 4-TU by converting 4-TU to uracil ( Figure 2M ). TudS prevents incorporation of 4-TU into RNA. Although 4-TU is antibacterial 34 , 35 , the mechanism of 4-TU toxicity has remained elusive. Due to the structural similarity between uracil and 4-TU, we hypothesized that 4-TU may hijack the uracil salvage pathway leading to incorporation into cellular RNA pools. WT and Δ tudS C. difficile were treated with 4-TU, and 4-thiouridine (s 4 U) was quantified in RNA by high performance liquid chromatography (HPLC) ( Figure 3A ). WT treated with 4-TU lacked detectable s 4 U in RNA, however, RNA isolated from Δ tudS contained a peak corresponding to s 4 U ( Figure 3B ). Quantification of the amount of uridine (U) substituted by s 4 U in RNA revealed that ~4% of U was replaced by s 4 U in Δ tudS ( Figure 3C ). These data suggest that 4-TU is incorporated into cellular RNA pools, and TudS prevents incorporation of 4-TU into RNA. Figure 3. TudS and uracil prevent incorporation of 4-TU into RNA. Open in a new tab ( A ) Workflow for assessing 4-thiouridine (s 4 U) incorporation in RNA. ( B ) WT and Δ tudS C. difficile were treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil and s 4 U incorporation was determined by HPLC. ( C ) Percent of uridine (U) substituted for s 4 U in C. difficile RNA from WT and Δ tudS strains treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil. ( D ) Percentage of mapped reads harboring a T->C conversion across the sequence of the housekeeping gene, rpoB from WT or Δ tudS C. difficile treated with 4-TU. ( E ) Growth of WT and Δ tudS C. difficile treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 40 μg/mL uracil. ( F ) WT E. coli was treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil and s 4 U incorporation was determined by HPLC. ( G ) Percent of uridine substituted for s 4 U in WT E. coli RNA treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil. ( H ) Growth of WT E. coli treated with vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil. Data are represented as mean±SD of biological and technical triplicate. Chromatograms are representative of three independent experiments. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test ( C ) and ( G ). P values are denoted. As an orthogonal approach to detect s 4 U in RNA, and specifically assess if s 4 U is present in mRNA, thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) was employed 36 . During cDNA synthesis, alkylated s 4 U in RNA results in T->C conversions. Following sequencing of cDNA libraries, reads were mapped to the gene encoding the beta subunit of RNA polymerase ( rpoB ). To avoid transcriptional differences during 4-TU treatment biasing results, rpoB was chosen 37 . This analysis revealed an abundance of T->C conversions (211 bases out of 1,171 possible bases) in Δ tudS treated with 4-TU, with no mutations detected in WT treated with 4-TU ( Figure 3D ). Collectively, these data demonstrate that 4-TU is incorporated into mRNA, and suggest that RNA polymerase cannot discriminate between uracil and 4-TU during transcription in C. difficile . Uracil prevents incorporation of 4-TU into RNA and alleviates toxicity. Since 4-TU is an antimicrobial derivative of a commonly encountered nutrient, uracil, we hypothesized that uracil may outcompete 4-TU for incorporation into RNA and alleviate toxicity. To test this, WT and Δ tudS C. difficile were treated with both 100 μg/mL 4-TU and 20 μg/mL uracil, and s 4 U in RNA was quantified by HPLC. Treatment of Δ tudS with uracil in combination with 4-TU diminished s 4 U levels in RNA ( Figure 3B ), and significantly reduced the amount of uridine substituted by s 4 U ( Figure 3C ). These data suggest that uracil competes with 4-TU for incorporation into RNA. To investigate if uracil is sufficient to restore C. difficile growth in the presence of 4-TU, WT and Δ tudS cultures were treated with 4-TU and uracil and growth was monitored. Treatment of Δ tudS with uracil decreased sensitivity to 4-TU relative to Δ tudS treated with only 4-TU ( Figure 3E ). Furthermore, the sensitivity of Δ tudS to 4-TU is reduced when cultured in rich media ( Figures S3D and S3E ), likely due to the presence of uracil. To determine if incorporation of 4-TU into RNA is conserved across bacterial species, WT E. coli was treated with 4-TU and s 4 U in RNA was quantified by HPLC. A peak corresponding to s 4 U was observed in RNA isolated from WT E. coli treated with 4-TU ( Figure 3F ), and uracil decreased s 4 U levels in RNA ( Figures 3F and 3G ). To assess if uracil alleviates 4-TU toxicity in E. coli , WT E. coli was treated with both 4-TU and uracil and growth was monitored. Consistent with Δ tudS C. difficile , uracil decreased the sensitivity of E. coli to 4-TU ( Figure 3H ). Cumulatively, these data suggest that the mechanism of action of 4-TU is nutrient dependent, uracil competes with 4-TU for incorporation into RNA, and 4-TU hijacks the uracil salvage pathway. A genetic selection reveals mutations that suppress 4-TU toxicity. To uncover the molecular mechanism by which 4-TU is incorporated into RNA, a genetic selection using strains that are sensitive to 4-TU (WT E. coli and Δ tudS C. difficile ) was conducted to isolate suppressor mutants that are resistant to 4-TU. Sequencing of spontaneous suppressor mutants revealed 11 mutations in WT E. coli and 15 mutations in Δ tudS C. difficile ( Table S5 ). Three types of mutations were isolated from the selection: nonsense, nonsynonymous mutations, and frame shifts, and all mutations relate to gene products that are predicted to be involved in nucleotide metabolism. Of the 26 mutations identified between E. coli and Δ tudS C. difficile , 21 mapped to either the open reading frame or promoter for the gene encoding uracil phosphoribosyltransferase (Upp). In Δ tudS C. difficile , two nonsynonymous mutations and one nonsense mutation were mapped to the regulator of pyrimidine biosynthesis, PyrR. Based on the finding that 81% of all mutations emerging from the selection were in Upp, we sought to determine the contribution of Upp to 4-TU metabolism in bacteria. Uracil phosphoribosyltransferase facilitates incorporation of 4-TU into RNA. Upp is a component of the uracil salvage pathway that catalyzes the conversion of uracil to uridine monophosphate (UMP), a common precursor for pyrimidine nucleotides that are incorporated into RNA ( Figure 1D ). To begin investigating how mutations in upp suppress 4-TU toxicity, the growth phenotypes of upp suppressor mutants in E. coli and Δ tudS C. difficile were compared to the parental strains in the presence of an inhibitory concentration of 4-TU. For E. coli , robust resistance was observed for nearly all strains harboring mutations in upp ( upp *) including the mutant in the promoter ( upp ← / → purM intergenic), except for one mutant, V118L that experienced an extended lag phase in 4-TU ( Figure S4A – D ). For C. difficile , representative point mutants were chosen for growth analysis from each category of mutations. Similar to E. coli , nonsense and frameshift mutations in upp in Δ tudS C. difficile (Δ tudS upp *) conferred resistance to 4-TU, and a nonsynonymous mutation, S139F resulted in an extended lag phase in 4-TU relative to WT ( Figure S4E and F ). To validate the 4-TU suppressor screen, E. coli lacking the gene encoding Upp (Δ upp ) was challenged with an inhibitory dose of 4-TU. Consistent with reports that inactivation of Upp confers resistance to 5-FU 33 , 38 , Δ upp growth was unaffected by the presence of 4-TU ( Figure 4A ). Since mutations in the promoter of upp were observed, we hypothesized that expression levels of upp may dictate 4-TU sensitivity. To test this, upp was overexpressed in WT E. coli ( E. coli -p upp ) and E. coli -p upp was treated with a sub-inhibitory concentration of 4-TU. Indeed, E. coli -p upp was more sensitive to 4-TU than the empty vector control strain ( E. coli -EV) ( Figure 4B ). To evaluate if Upp is involved in 4-TU incorporation into RNA, WT and a representative upp E. coli suppressor mutant ( upp* ) were treated with 4-TU and s 4 U incorporation into RNA was assessed by HPLC. Expectedly, WT harbored a peak corresponding to s 4 U, however, upp* lacked detectable s 4 U in RNA ( Figure 4C ). These data demonstrate that mutations in upp confer resistance to 4-TU by preventing incorporation into RNA. Figure 4. Upp facilitates incorporation of 4-TU into RNA. Open in a new tab ( A ) Growth of WT and Δ upp E. coli treated with vehicle or 20 μg/mL 4-TU. ( B ) Growth of WT E. coli harboring empty vector (EV) or vector containing E. coli upp (p upp ) treated with vehicle or 1 μg/mL 4-TU. ( C ) WT and a representative upp suppressor mutant ( upp *) E. coli were treated with vehicle or 100 μg/mL 4-TU and ribonucleosides were separated by HPLC. ( D ) Predicted structure of 4-TU bound C. difficile Upp with mutations mapped in red and magenta. ( E ) Recombinant C. difficile WT Upp and a representative suppressor mutant (S139F) were incubated with 100 μM 4-TU and phosphoribosyl transfer was monitored by HPLC. ( F ) Model for the role of Upp in 4-TU metabolism. Data are represented as mean±SD of biological and technical triplicate. Chromatograms are representative of three independent experiments. To interrogate the mechanism by which mutations in upp confer resistance to 4-TU, nonsynonymous mutations were mapped to a model of the three-dimensional structure of Upp from C. difficile ( Figure 4D ). This analysis revealed that S139 and A80 lie in the substrate binding pocket of Upp, where 4-TU is predicted to fit into a positively charged groove ( Figure 4D ). To investigate if Upp can use 4-TU as a substrate, recombinant Upp from C. difficile was expressed and purified and a biochemical assay was developed to monitor phosphoribosyl transfer to 4-TU by HPLC. Upp completely depleted 4-TU levels, generating 4-thiouridine monophosphate (s 4 UMP) ( Figure 4E ). Based on these findings, we reasoned that uracil competes with 4-TU for Upp-mediated phosphoribosyl transfer. To test this, recombinant Upp was treated with equimolar concentrations of uracil and 4-TU and s 4 UMP was quantified. Uracil diminished the ability of Upp to generate s 4 UMP ( Figures S5A and S5B ). These data suggest that uracil competes with 4-TU for the activity of Upp. We hypothesized that S139F and A80V mutations impede 4-TU binding to Upp due to a transition to bulky, hydrophobic amino acid residues in the substrate binding site, reducing the ability of Upp to convert 4-TU to s 4 UMP. To test this, a representative Upp point mutant, S139F was purified and the consequence of S139F on Upp phosphoribosyl transfer was monitored. Indeed, S139F hindered the ability of Upp to generate s 4 UMP from 4-TU, indicating that S139 is a critical residue for Upp phosphoribosyl transfer activity ( Figure 4E ). Taken together, these data suggest that Upp facilitates incorporation of 4-TU into RNA ( Figure 4F ). Mutations in the regulator of pyrimidine biosynthesis confer resistance to 4-TU. Mutations in the gene encoding the regulator of pyrimidine biosynthesis (PyrR) conferred resistance to 4-TU in Δ tudS C. difficile ( Table S5 ). In the presence of uridine nucleotides, PyrR binds to hairpin structures in RNA causing transcriptional termination of target genes, thus acting as a negative regulator of de novo pyrimidine biosynthesis 39 . In addition to regulating the expression of pyrimidine biosynthesis genes, PyrR from Mycobacterium tuberculosis (Mtb) and Bacillus subtilis possesses uracil phosphoribosyltransferase activity 38 , 40 . Nonsynonymous and frameshift mutations in pyrR were identified ( Table S5 ), and nonsynonymous mutations were mapped to the predicted three-dimensional structure of PyrR from C. difficile ( Figure 5A ). Interestingly, both nonsynonymous mutations observed in the genetic selection lie outside of the predicted 4-TU binding pocket ( Figure 5A ). Figure 5. Mutation of PyrR confers resistance to 4-TU. Open in a new tab ( A ) Predicted structure of 4-TU bound C. difficile PyrR with mutations mapped in red. ( B ) Schematic of the pyr operon in C. difficile . ( C ) pyrF transcription in WT, Δ tudS , and a representative pyrR suppressor mutant (Δ tudS pyrR *) treated with vehicle or 100 μg/mL 4-TU. ( D ) WT, Δ tudS, and Δ tudS pyrR * C. difficile were treated with vehicle or 100 μg/mL 4-TU and ribonucleosides were separated by HPLC. ( E ) Growth of WT, Δ tudS, and pyrR point mutants treated with vehicle or 100 μg/mL 4-TU. ( F ) Recombinant C. difficile WT PyrR and PyrR suppressor mutants were incubated with 100 μM 4-TU and phosphoribosyl transfer was monitored by HPLC. ( G ) Model for C. difficile 4-TU metabolism. Data are represented as mean±SD of biological and technical triplicate. Chromatograms are representative of three independent experiments. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test ( C ). P values are denoted. Based on the bifunctionality of PyrR observed in other bacterial species, we hypothesized that C. difficile PyrR is also bifunctional, and mutations in PyrR may confer resistance to 4-TU by abrogating conversion of 4-TU to s 4 UMP and increasing flux through the de novo pyrimidine biosynthesis pathway. Notably, E. coli strains exposed to sub-inhibitory concentrations of 5-FU during serial passage overexpress the de novo pyrimidine biosynthesis pathway to overcome toxicity 41 . To test this hypothesis, the genome of C. difficile CD196 was mined for pyrimidine biosynthesis genes. C. difficile encodes for two paralogs of the two subunits of carbamoyl phosphate synthetase (CarAB), which are localized in the chromosome next to the gene encoding orotate 5’-phosphate decarboxylase (PyrF) in a predicted operon ( Figure 5B ). To determine if PyrR regulates the expression of this operon, the 5’ untranslated region (UTR) was analyzed and a predicted PyrR binding site was identified in this region with high confidence (E value = 4.1e-7). Additionally, a 116 base pair deletion was observed in the 5’ UTR of pyrF that overlaps with the predicted PyrR binding site in a Δ tudS C. difficile 4-TU suppressor strain ( Table S5 ). To investigate if mutation of pyrR impacts the regulation of pyrimidine biosynthesis genes, the transcript abundance for pyrF , the first gene in the predicted de novo pyrimidine biosynthesis operon was quantified in WT, Δ tudS , and a nonsynonymous pyrR mutant (D135Y) in the Δ tudS background (Δ tudS pyrR* ) ( Figure 5B ). An increase in pyrF transcript abundance in the Δ tudS pyrR* strain was observed ( Figure 5C ), indicating that PyrR regulates pyrF expression. These data suggest that mutation of pyrR increases de novo pyrimidine biosynthesis, potentially enabling endogenous UMP to compete with s 4 UMP for incorporation into RNA. To test if mutations in pyrR suppress 4-TU toxicity by decreasing incorporation of 4-TU into RNA, RNA from WT, Δ tudS , and Δ tudS pyrR* treated with vehicle or 4-TU was isolated and s 4 U was quantified by HPLC. This analysis revealed less s 4 U in RNA isolated from Δ tudS pyrR* relative to the Δ tudS parental strain ( Figure 5D ). We next hypothesized that mutations in pyrR improve growth in the presence of 4-TU. Nonsynonymous mutations in pyrR (G149E and D135Y) confer intermediate resistance to 4-TU relative to WT ( Figure 5E ). Since PyrR homologs have been reported to possess uracil phosphoribosyltransferase activity, we hypothesized that C. difficile PyrR can use 4-TU as a substrate to generate s 4 UMP. To test this, recombinant C. difficile PyrR was incubated with 4-TU and phosphoribosyl transfer was monitored by HPLC. This analysis identified the generation of s 4 UMP by PyrR ( Figure 5F ), indicating that both PyrR and Upp contribute to s 4 UMP pools in strains lacking tudS . To determine the impact of nonsynonymous mutations in pyrR to phosphoribosyl transfer activity, 4-TU phosphoribosyl transfer activity was determined for recombinant D135Y and G149E PyrR. This experiment revealed a decrease in s 4 UMP production relative to the WT enzyme ( Figure 5F ), suggesting that mutations in pyrR may suppress 4-TU toxicity by decreasing s 4 UMP pools available for RNA incorporation. To investigate if uracil competes for the active site of PyrR similar to Upp, PyrR was incubated with equimolar concentrations of uracil and 4-TU. A decrease in the production of s 4 UMP was observed when uracil and 4-TU were simultaneously provided to PyrR as substrates ( Figure S5C and S5D ). These data suggest that mutations in pyrR confer resistance to 4-TU by increasing pyrimidine biosynthesis and decreasing s 4 UMP production. We hypothesize that both Upp and PyrR contribute to the incorporation of 4-TU into RNA by utilizing 4-TU as a substrate for phosphoribosyl transfer, which is prevented by the TudS-dependent conversion of 4-TU to uracil ( Figure 5G ). TudS-mediated 4-TU detoxification is required for C. difficile gut pathogenesis. C. difficile competes with the resident microbiota and immune system for nutrients encountered in the gastrointestinal tract of susceptible hosts 26 , 42 . We hypothesized that TudS-mediated 4-TU metabolism gives C. difficile an advantage in the gut by inhibiting competing microbes while serving as a nutrient source. Based on the observation that 4-TU is present in human stool ( Figure 2B ), we sought to develop a model to determine the contribution of 4-TU metabolism to CDI. C57BL/6J mice consuming chow possess low levels of 4-TU in the gut, therefore, 4-TU was given by oral gavage for three consecutive days following antibiotic treatment to establish 4-TU in the murine gut ( Figure 6A ). Indeed, 4-TU treatment elevated 4-TU levels in the feces to levels that mimic human stool ( Figure 2B ; Figure 6B ). In the absence of 4-TU, mice infected with WT and Δ tudS C. difficile harbored similar bacterial burdens and had comparable weight loss 24 hours post-infection ( Figure 6C and 6D ). To assess the contribution of TudS-mediated 4-TU detoxification to CDI in the vertebrate gut containing 4-TU, mice were administered 4-TU and infected with WT or Δ tudS C. difficile . Mice infected with Δ tudS showed reduced bacterial burdens and lost less weight than mice infected with WT 24 hours post-infection ( Figure 6E and 6F ). Mice treated with 4-TU and infected with WT or Δ tudS strains showed similar disease at later timepoints, likely due to a reduction in 4-TU levels (data not shown). These data suggest that TudS promotes C. difficile survival in the host when 4-TU is present. Figure 6. TudS provides C. difficile a competitive advantage against commensal microbes. Open in a new tab ( A ) Cefoperazone and 4-TU mouse model of CDI. ( B ) LC-MS/MS quantification of 4-TU in the feces of uninfected mice. N = 5. The dotted line represents the limit of detection. ( C ) Colony forming unit (CFU) analysis and ( D ) weight loss one day post-infection of mice treated with vehicle and infected with WT or Δ tudS C. difficile . N = 10. ( E ) CFU analysis and ( F ) weight loss one day post-infection of mice treated with 15 mg 4-TU and infected with WT or Δ tudS . N = 17–20. ( G-J ) Representative photomicrographs of H&E stained cecal sections 24 hours post-infection. Stars (*) indicate submucosal edema and arrows indicate inflammatory cell infiltration. Bars = 100 μm (inset bar = 50 μm) ( K ) Neutrophilic infiltration 24 hours post-infection. N = 8–10. ( L ) LC-MS/MS quantification of uracil in the feces of 4-TU-treated mice infected with WT or Δ tudS C. difficile N=8–9. ( M ) Competitive index of WT and Δ tudS in the presence and absence of human fecal microbiotas treated with vehicle or 250 μg/mL 4-TU. ( N ) Competitive index of WT B. thetaiotaomicron ( Bt ) and WT or Δ tudS C. difficile ( Cd ), ( O ) WT E. coli Nissle ( Ec N) and WT or Δ tudS C. difficile ( Cd ), and ( P ) WT E. coli Nissle ( Ec N) with empty vector (EV) or expressing C. difficile tudS (p tudS ) and WT C. difficile ( Cd ) treated with vehicle or 100 μg/mL 4-TU. Data are represented as mean±SD of biological and technical triplicate. Individual data points are representative of biological replicates or individual mice. Statistical significance was determined using a two-tailed Mann-Whitney U test ( B-F ) and ( L ) and one-way ANOVA with Tukey’s multiple comparison post-test ( K ) and ( M - P ). P values are denoted, ns = not significant. The uneven number of mice between groups is due to mortality during oral gavage. To further investigate the inflammatory environment during infection of the murine gut containing 4-TU, histopathological analysis was performed 24 hours post-infection. These analyses revealed neutrophilic infiltration and mild edema in WT infected mice treated with vehicle or 4-TU, and vehicle treated mice infected with Δ tudS ( Figure 6G – 6I ). However, 4-TU treated mice infected with Δ tudS exhibited little to no inflammation or edema ( Figure 6J ). In addition, no significant differences in neutrophilic infiltration were observed between vehicle treated mice infected with WT or Δ tudS , and mice treated with 4-TU and infected with Δ tudS displayed reduced neutrophilic infiltration relative to mice infected with WT ( Figure 6K ). To investigate the potential of TudS-mediated 4-TU metabolism augmenting uracil availability in the gut, mice given 4-TU were infected with WT or Δ tudS and uracil was quantified by LC-MS/MS. The abundance of uracil was higher in mice infected with WT than mice infected with Δ tudS ( Figure 6L ). Taken together, these data suggest that the metabolism of 4-TU by TudS increases uracil availability and contributes to C. difficile fitness and pathogenesis in the murine gut containing 4-TU. To determine if 4-TU metabolism confers a fitness advantage to C. difficile in the presence of commensal microbes, WT was competed against Δ tudS in a human fecal minibioreactor model of CDI 43 ( Figure S6A ). Following perturbation of a complex human microbial community, WT and Δ tudS were added to reactors supplied with media containing vehicle or 4-TU and the abundance of each strain was assessed ( Figure S6B and S6C ). In 4-TU-treated reactors containing human microbial communities or no community controls, WT outcompeted Δ tudS by ~50,000 fold ( Figure 6M ). Next, co-cultures of C. difficile and representative commensals from the Proteobacteria and Bacteroidetes phyla were treated with vehicle or 4-TU. WT C. difficile outcompeted Bacteroides thetaiotaomicron ( Bt ) and E. coli Nissle ( Ec N) in the presence of 4-TU ( Figure 6N and 6O ). To determine the contribution of TudS to the competitive advantage observed in 4-TU, Δ tudS C. difficile was co-cultured with Bt and Ec N. Both Ec N and Bt strains outcompeted Δ tudS in the presence and absence of 4-TU ( Figure 6N and 6O ). Moreover, expression of C. difficile tudS in Ec N abolished the competitive advantage conferred by tudS to C. difficile ( Figure 6P ). These data demonstrate that TudS is required for C. difficile to outcompete commensal microbes in the presence of 4-TU, and expression of tudS in a commensal strain is sufficient to diminish the competitive advantage conferred by 4-TU to C. difficile . Collectively, these findings reveal the importance of TudS-mediated 4-TU metabolism to C. difficile fitness in the presence of commensal microbes and in the vertebrate gastrointestinal tract. Discussion Microbes are constantly exposed to a wide array of molecules in the environment that they inhabit. Consequently, systems to sense and metabolize molecules encountered in the colonization niche are maintained, and have the potential to influence host health and physiology. One such example is bacteria adapting nucleotide salvage pathways to their environment. Uropathogenic E. coli salvages pseudouridine, an abundant modified pyrimidine in urine, by maintaining a pseudouridine kinase and pseudouridine-5’-phosphate glycosidase 44 . Chlamydia trachomatis , an obligate intracellular pathogen, salvages queuine (q) and queuosine (Q) that are present in host cells but is unable to salvage the Q precursors preQ 0 and preQ 1 that are absent in host cells 45 . We found that pyrimidine salvage in C. difficile is selective, and of the pyrimidine sources screened, only uracil and 4-TU could be salvaged ( Table S2 ). Intriguingly, 2-thiouracil (2-TU) could not be salvaged by C. difficile , which is consistent with the soil-dwelling bacterium Pseudomonas putida utilizing 4-TU but not 2-TU as a pyrimidine source 28 . Additionally, purified TudS from an uncultured Aeromonas species more efficiently converts 4-TU to uracil than 2-TU, suggesting that 4-TU is the preferred substrate for TudS 29 . This study reports a detoxification function for TudS, preventing incorporation of 4-TU into RNA. s 4 U is a prominent modification in tRNA that is conserved in bacteria and archaea 46 , and functions as a photosensor for near-UV stress 47 – 50 . In bacteria, two enzymes are required for the biosynthesis of s 4 U in tRNA, cysteine desulfurase IscS and tRNA uridine 4-sulfurtransferase ThiI 51 – 55 . Although the biosynthesis enzymes for s 4 U have been described, “erasers” or enzymes that recycle s 4 U have remained elusive. TudS has been suggested to play a role in tRNA recycling and detoxifying tRNA-derived s 4 UMP, since TudS can use s 4 UMP as a substrate 28 . In addition, RudS is a tRNA modifying enzyme containing a TudS fused to domain of unknown function 1722 (DUF1722) that converts s 4 UMP to UMP in tRNA 56 . However, expression of tudS in E. coli and deletion of tudS in P. putida does not alter the abundance of s 4 U in tRNA 28 , 56 . These findings suggest that s 4 UMP-containing tRNA is not the primary substrate of TudS. Although 4-TU is widely used in eukaryotic systems to identify protein-RNA interactions, the utility of 4-TU in bacteria has not been extensively investigated. In archaea, a recent study demonstrated the efficient labeling of RNA using 4-TU and reported toxicity to the model archaea Haloferax volcanii and Sulfolobus acidocaldarius 57 . In eukaryotes, s 4 U can inhibit ribosomal RNA synthesis and cause a nucleolar stress response 58 . The findings that 4-TU is incorporated into RNA in bacteria suggests that low doses of 4-TU can be used for experiments to identify RNA-protein interactions. In addition, the application of SLAM-seq to bacteria in this work highlights the potential of using 4-TU to study bacterial RNA kinetics. Although the mechanism by which s 4 U in RNA causes bacterial growth inhibition is unknown, it is tempting to speculate that s 4 U induces complications during translation. Future studies investigating the consequences of s 4 U in RNA, and the mechanisms by which s 4 U in RNA inhibits bacterial growth will fill these gaps in knowledge. Furthermore, the mechanism of 4-TU toxicity may be multifaceted as s 4 UMP may be integrated into other cellular pathways requiring uridine containing metabolites such as cell wall and glycogen synthesis. In archaea and eukarya, incorporation of 4-TU into RNA relies on uracil phosphoribosyltransferase (UPRTase) 57 . In systems lacking a UPRTase, heterologous expression of UPRTase from Toxoplasma gondii is a strategy utilized in 4-TU tagging to label newly synthesized RNA 59 . Recombinant UPRTase from Leishmania donovani can use 4-TU as a substrate to generate s 4 UMP 60 . The ability of bacterial proteins harboring UPRTase activity to use 4-TU as a substrate has not been tested. The UPRTases Upp and PyrR from Mtb can use 5-FU as a substrate 38 , and our results demonstrate that Upp ( Figure 4E ) and PyrR ( Figure 5F ) can use 4-TU as a substrate. We found that mutations in PyrR provided only intermediate resistance to 4-TU ( Figure 5E ), likely due to the presence of a functional Upp. We propose that similar to Mtb, Upp provides the major UPRTase activity in C. difficile . Thiouracil has been used to treat hyperthyroidism and Graves’ disease in humans and in agriculture as an illegal growth promoter 61 , 62 . Diets containing glucosinolate-rich Brassicaceae increase levels of thiouracil in livestock 63 , and in vitro digestion of Brassicaceae with porcine fecal bacteria elevate thiouracil concentrations 64 . Furthermore, thiouracil is present in cruciferous vegetables including broccoli sprouts, and detection of thiouracil was dependent on treatment with myrosinase, an enzyme conserved in gut bacteria 65 , 66 . These findings suggest that the source of thiouracil is dietary-derived and dependent on the gut microbiota. We propose that C. difficile encounters 4-TU in the human gut, and this molecule provides C. difficile an advantage by inhibiting competing microbes while also serving as a nutrient in this environment. This work suggests that TudS enables growth of C. difficile in the presence of 4-TU. Although 4-TU is incorporated into RNA in strains lacking tudS , these experiments do not distinguish between the roles of TudS in using 4-TU as a nutrient and in detoxifying 4-TU. It is possible that strains lacking tudS are unable to grow in the presence of 4-TU because of an inability to utilize 4-TU as a pyrimidine source even considering the de novo pyrimidine biosynthesis pathway ( Figure 2K and 2L ). Furthermore, we hypothesize that uracil alleviates 4-TU toxicity by outcompeting 4-TU for incorporation into RNA. However, it is possible that uracil diminishes 4-TU toxicity by restoring pyrimidine metabolic homeostasis. In L. donovani , 4-TU is a substrate-inhibitor of UPRTase 60 . It is interesting to speculate that a mechanism of 4-TU toxicity may be due to the inhibition of enzymes involved in bacterial pyrimidine metabolism. Here we discovered an enzyme that enables detoxification and utilization of the unconventional pyrimidine 4-TU in the important human pathogen C. difficile . TudS prevents incorporation of 4-TU into RNA by converting 4-TU to uracil that can then be used as a pyrimidine source for growth. The absolute conservation of TudS across C. difficile clades including clinical isolates, and the observation that 4-TU is present in human stool suggests that C. difficile encounters 4-TU in the human host. Since TudS is not conserved in multicellular eukaryotes, we propose that TudS-mediated 4-TU acquisition has the potential to serve as a therapeutic target to treat CDI. Furthermore, the metabolism of 4-TU provides C. difficile with a fitness advantage against human commensals commonly encountered during infection. These findings increase our understanding of nutrient acquisition during CDI and provide evidence that an antimicrobial uracil analog present in the human gastrointestinal tract is incorporated into bacterial mRNA pools. Limitations of the study This work suggests that 4-TU is encountered in the human gut, and both uracil and 4-TU decrease during CDI. These data indicate either increased utilization of uracil and 4-TU by C. difficile , or antibiotic depletion of the microbiota decreases uracil and 4-TU abundance. Our data indicate C. difficile salvages 4-TU from the vertebrate gut, and antibiotics decrease uracil abundance, suggesting that both possibilities may be true. The sources of 4-TU and uracil, and the gut microbes responsible for generating 4-TU and uracil have remained elusive. Future studies should investigate the sources of 4-TU and uracil, and identify if C. difficile or antibiotics are the main factor in reducing the levels of 4-TU and uracil. Resource Availability Lead Contact Further information and request for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Eric P. Skaar ( [email protected] ) Materials Availability All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement. Data and Code Availability Raw sequencing files obtained from SLAM-seq experiments are available in the National Center for Biotechnology Information (NCBI) sequence read archive (SRA) under BioProject: PRJNA1219594. Whole genome sequencing data of E. coli 4-TU suppressor strains are available under BioProject: PRJNA1219637. Whole genome sequencing data of C. difficile 4-TU suppressor strains are available under BioProject: PRJNA1219607. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request STAR Methods: Experimental Model and subject details Bacterial strains and growth conditions Bacterial strains used in this study are listed in Table S6 . C. difficile strains were grown anaerobically at 37°C in an anaerobic chamber (90% nitrogen, 5% hydrogen, 5% carbon dioxide, Coy Lab Products) in brain-heart-infusion (BD Life Sciences) supplemented with 0.5% yeast extract (Sigma-Aldrich) and 0.1% cysteine (Sigma-Aldrich) broth (BHIS) or agar (BHISA) or in C. difficile minimal media (CDMM) as described previously 67 and supplemented with 20 μg/mL uracil (Sigma-Aldrich) when necessary. Unless otherwise noted, data from growth experiments were obtained in CDMM. Escherichia coli strains were grown in lysogeny broth (LB) or agar (LBA) or in CDMM at 37°C, supplemented with 20 μg/mL chloramphenicol (Cm), 100 μg/mL carbenicillin (Carb), and 20 μg/mL uracil when appropriate. Bacillus subtilis strains were grown on LBA or BHI broth supplemented with 5 μg/mL tetracycline (Tet) and/or 2.5 μg/mL Cm. Bacteroides thetaiotaomicron was grown anaerobically at 37°C in BHIS or BHISA or CDMM supplemented with 0.005% haemin (Frontier Scientific) and 0.0001% vitamin K, pH 7.4 (Sigma-Aldrich). All antibiotics were purchased from Sigma-Aldrich. Animal models All mouse experiments under protocol M2300018 were reviewed and approved by the Institutional Animal Care and use Committee of Vanderbilt University Medical Center. Procedures were performed according to the institutional policies, National Institutes of Health guidelines, and American Veterinary Medical Association guidelines on euthanasia. Five- to eight- week-old male C57BL/6 wild-type (cat# 000664) mice, originally obtained from Jackson Laboratory (Bar Harbor), were housed under specific pathogen-free conditions in groups of five and given one week to equilibrate their microbiota prior to experimentation. All experimental procedures were conducted in a biosafety level 2 laminar flow hood. Mice were fed a standard chow diet (LabDiets; Rodent Chow Diet 5001) and maintained at Vanderbilt University Medical Center Animal Facilities. Human fecal samples Fecal samples were obtained from pediatric patients at Monroe Carell Jr. Children’s Hospital at Vanderbilt diagnosed with CDI from July 2017 through December 2019 after informed parental consent and patient assent. The study was approved by the Vanderbilt Institutional Review Board. Children were characterized as symptomatic from C. difficile if they had unformed stools, an acute change in stool character, ≥ 3 bowel movements in 24 hours, and tested positive for C. difficile by nucleic acid amplification-based testing (NAAT). Children with asymptomatic C. difficile colonization tested positive by NAAT in the absence of symptoms. Stool samples from pediatric healthy controls were obtained through prospective surveillance during well-child visits as part of the Centers for Disease Control and Prevention (CDC) New Vaccine Surveillance Network (2012–2019) 68 . Healthy controls were immunocompetent, <18 years old, and without respiratory symptoms for three days or gastroenterology symptoms for 14 days prior to their visit. Neither CDI nor healthy control patients had a diagnosis of malignancy, inflammatory bowel disease, or history of solid organ or stem cell transplant. Method Details C. difficile mutant strain generation The C. difficile tudS -targeted CRISPR plasmid was constructed by amplifying 500 bp upstream and downstream of the tudS gene and fragments were cloned into the NotI and XhoI sites of pKM197 using Gibson assembly. The tudS targeting guide RNA (gRNA) and scaffold sequence was synthesized and inserted into the KpnI and MluI sites by Gibson assembly. The C. difficile pyrE -targeting CRISPR plasmid was previously constructed and validated 69 . The resulting plasmids were transformed into E. coli DH5α and confirmed by Sanger sequencing. Constructs were then transformed into recA + E. coli MG1655 to generate multimers that improve transformation into B. subtilis JH BS2 harboring a Tn916 conjugation system. B. subtilis strains containing pKM197_ tudS and pKM197_ pyrE were mated with C. difficile CD196 by mixing 100 μL of each strain on a BHISA plate followed by incubation for 16 hours at 37°C in the anaerobic chamber. Plates were scraped and transferred into 2 mL BHIS prior to plating 200 μL on BHISA containing 20 μg/mL thiamphenicol and 50 μg/mL kanamycin (BHISA tm20kan50 ). Single colonies were picked and patched onto new BHISA tm20kan50 and BHISA containing 5 μg/mL tetracycline. Tetracycline sensitive colonies were passed up to four times on BHISA tm20 containing 1% xylose to induce CRISPR-Cas9. Colonies were screened by colony PCR using a primer set located outside of the upstream and downstream homology regions of tudS or pyrE , and a primer set inside of tudS or pyrE . After deletion confirmation by PCR, plasmids were cured by serial passage in BHIS containing 0.5% xylose. Deletions were further confirmed by Sanger and whole genome sequencing. A complete list of oligonucleotides used in this study is listed in Table S6 . Complementation plasmids The C. difficile Δ tudS complementation plasmid was constructed by inserting the tudS gene into the BamHI and SacI sites of pAP114 by Gibson assembly. The resulting plasmid (pAP114- tudS ) was transformed into E. coli DH5α, confirmed by Sanger sequencing, and transformed into E. coli CA434 for conjugation with C. difficile . The plasmid (pAP114- tudS ) was transferred to C. difficile using a heat-shock conjugation method previously described 70 . Transconjugants were selected on BHISA containing 20 μg/mL thiamphenicol and 50 μg/mL colistin. Strains harboring pAP114 were cultured in BHIS tm20 to ensure plasmid retention and 1% xylose to induce expression of tudS . Protein expression plasmids Protein expression plasmids for C. difficile Upp and PyrR were constructed by inserting the pyrR or upp gene into the NdeI and BamHI sites of pET15b using Gibson assembly. The resulting plasmids were transformed into E. coli DH5α, confirmed by Sanger sequencing, and transformed into E. coli BL21 (DE3) pREL for expression and purification. Point mutation generation in pET15b_ pyrR and pET15b_ upp was performed with the NEB Q5 Site Directed Mutagenesis kit according to the manufacturer’s instructions using the primers listed in Table S6 . Heterologous expression experiments The C. difficile tudS gene was inserted into the NdeI and HindIII sites of pBAD33.1 using Gibson assembly. The resulting plasmid was transformed into E. coli DH5α and confirmed by Sanger sequencing. A FLAG-tag was synthesized and inserted into the NdeI site of pBAD33.1_ tudS by Gibson assembly to generate an N-terminal fusion. Point mutation generation in pBAD33.1_ tudS was performed with the NEB Q5 Site Directed Mutagenesis kit according to the manufacturer’s instructions using the primers listed in Table S6 . The resulting plasmids were transformed into E. coli K-12 BW25113 WT or Δ pyrE . E. coli K-12 strains harboring pBAD33.1_ tudS , pBAD33.1_ tudS C38A, pBAD33.1_ tudS E40A, pBAD33.1_ tudS C99A, or empty vector were inoculated into C. difficile minimal media (CDMM) with Cm 20 and incubated overnight at 37°C anaerobically. After 16 h of growth, cultures were subcultured 1:50 into fresh CDMM containing 1% sorbitol and 40 mM NaNO 3 and expression was induced by addition of 0.2% arabinose for 2 h prior to 1:50 inoculation into CDMM containing vehicle, 4-TU, or uracil at the indicated concentrations. All growth assays were performed in a 96-well plate in 200 μL of media at 37°C anaerobically. Optical density at 600 nm (OD 600 ) served as a measurement of growth and was measured every 30 minutes for the indicated total time in an EpochII microplate reader (BioTek). upp overexpression experiments E. coli upp with its native promoter and ribosome binding site was inserted into the NruI and HindIII site of pACYC184 using Gibson assembly. The resulting plasmid was transformed into E. coli DH5α, confirmed by Sanger sequencing, and transformed into E. coli K-12. E. coli K-12 strains harboring pACYC184_ upp or empty vector were inoculated into CDMM with Cm 20 and incubated overnight aerobically with shaking at 180 rpm. After 16 h of growth, cultures were subcultured 1:100 into fresh CDMM for 2 h prior to 1:50 inoculation into CDMM containing vehicle or 4-TU at the indicated concentrations. All growth assays were performed in a 96-well plate in 200 μL of media at 37°C aerobically. OD 600 served as a measurement of growth and was measured every 30 minutes for the indicated total time in an EpochII microplate reader (BioTek). C. difficile growth assays Freshly streaked C. difficile colonies were used to inoculate 5 mL BHIS and strains were grown overnight at 37°C anaerobically. After 16 h of growth, cultures were subcultured 1:50 into fresh BHIS or CDMM for 6 h prior to 1:50 inoculation into BHIS or CDMM containing vehicle, 4-TU, or uracil at the indicated concentrations. All growth assays were performed in a 96-well plate in 200 μL of media at 37°C aerobically. OD 600 served as a measurement of growth and was measured every 30 minutes for the indicated total time in an EpochII microplate reader (BioTek). Immunoblotting for protein abundance WT or Δ pyrE E. coli K-12 harboring pBAD33.1_ tudS , pBAD33.1_ tudS C38A, pBAD33.1_ tudS E40A, pBAD33.1_ tudS C99A, or empty vector were grown in CDMM containing 1% sorbitol, 40 mM NaNO 3 , and 0.2% arabinose for 8 h. Cells were normalized by OD 600 , pelleted, and resuspended in 1X PBS containing SDS sample buffer. Samples were incubated at 95°C for 10 min and centrifuged at maximum speed for 10 min. The resulting supernatants were loaded into wells of 4 to 20% gradient Mini-Protean SDS-PAGE gels (Bio-Rad) and run in the Mini-Protean electrophoresis system (Bio-Rad). Proteins were transferred to a nitrocellulose membrane and immunoblotting was performed using anti-FLAG M2 (Sigma-Aldrich) and IRDye 680 conjugated anti-mouse secondary. Protein bands were visualized on a ChemiDoc MP imaging system. E. coli Nissle and B. thetaiotaomicron co-cultures with C. difficile Freshly streaked C. difficile , E. coli , or B. thetaiotaomicron were used to inoculate 5 mL BHIS and strains were grown overnight at 37°C anaerobically. After 16 h of growth, a 1:1 ratio of bacterial strains was diluted to an OD 600 of 0.01 in 10 mL CDMM treated with vehicle or 100 μg/mL 4-TU and grown at 37°C anaerobically. Bacterial titers were quantified by performing 10-fold serial dilutions in 1X PBS and plating on taurocholate cefoxitin cycloserine fructose agar (TCCFA) for C. difficile and BHISA + 4 μg/mL vancomycin for E. coli Nissle and B. thetaiotaomicron . Plates were incubated at 37°C anaerobically for 16 h. C. difficile animal model of infection Mice were treated with 0.5 mg/mL cefoperazone in their drinking water for 5 or 10 days followed by 2 days of recovery with normal drinking water. For the 4-TU mouse model, mice were administered 15 mg 4-TU (Sigma-Aldrich) in 100 μL dimethyl sulfoxide (Sigma-Aldrich) or 100 μL dimethyl sulfoxide as a vehicle control by oral gavage for three consecutive days prior to infection. Mice were then infected with 10 5 C. difficile CD196 spores in 100 μL 1X PBS. Before infection, mice were confirmed to be C. difficile negative via plating. Body weight was monitored daily and mice that exhibited weight loss in excess of 20% were humanely euthanized. Depending on the infection model, C. difficile infection was allowed to proceed for up to 4 days. Each mouse was allocated a 30-minute time window for fecal pellet collection. If mice were unable to produce a fecal pellet in the allocated timeframe, they were not included in colony forming unit analysis. C. difficile burdens were determined by homogenizing fecal pellets in PBS, performing 5-fold serial dilutions, and plating on TCCFA anaerobically. Histopathology and scoring Murine ceca were cleared of contents, fixed in 10% neutral buffered formalin, and embedded in paraffin. Cecal tissue was sectioned at 5 μm and stained with hematoxylin and eosin (H&E). H&E stained sections were evaluated by a veterinary pathologist (K.N.G.-C.) blinded to the composition of the groups. Slides were semi-quantitatively scored at the same magnification (10X) for submucosal edema on a scale from 0–4, in which: 0, not present/within normal limits; 1, mild, rare, scattered; 2, moderate, multifocal; 3, marked, locally extensive; and 4, marked, severe, diffuse. Neutrophilic infiltration and edema scores were based on areas of the cecum most severely affected by C. difficile infection. The entire slide was evaluated, and the score assigned marked the most severe lesion present as lesions tended to often be multifocal to segmental in nature. Quantification of uracil and 4-TU from human and murine stool. Human or murine stool were flash frozen on liquid nitrogen and lyophilized to complete dryness (~48 hours). Uracil and 4-thiouracil were quantified using isotope dilution LC-MS/MS in the Vanderbilt University Mass Spectrometry Core Laboratory. Sample analyses were carried out using a Waters Acquity UPLC system (Waters, Milford, MA). A Waters Acquity BEH-C18 UPLC column (2.1 × 100 mm; 1.8 μm) was used for all chromatographic separations. The column temperature was not thermostatted. Mobile phases were made up of 0.2% AcOH and 15 mM ammonium acetate in (A) H 2 O/CH 3 CN (9:1) and in (B) CH 3 CN/H 2 O/CH 3 OH (90:5:5). Gradient conditions were as follows: 0–2 min, B = 30 %; 2–10 min, B = 30–100%; 10–11.5 min, B = 100%; 11.5–12 min, B = 100–30%; 12–16 min, B = 100%. The LC flow rate was maintained at 300 μL/min. A software-controlled divert valve was used to transfer eluent from 0–6 min and from 10.5–16 min of each chromatographic run to waste; the total chromatographic run time was 16 minutes. The injection volume was 10 μL. Single reaction monitoring (SRM) detection was performed using a Thermo TSQ Vantage triple-stage quadrupole mass spectrometer equipped with an Ion Max APCI ion source (Thermo-Fisher, Waltham, MA). The mass spectrometer was operated in negative APCI mode. Quantitation was based on SRM detection of pentafluorobenzyl derivatives (Uracil: m/z 291 --> 271, CE 12; Uracil- 13 C 1 15 N 2 : m/z 294 --> 271, CE 12; 4-Thiouracil: m/z 307 --> 287, CE 13; 2-Thiouracil- 13 C 1 15 N 2 : m/z 310-->290, CE 10). The following optimized source parameters were used for the detection of analytes and internal standards: N 2 sheath gas 40; N 2 auxiliary gas 5; corona discharge current 15.0 μA; aux heater temp 350 °C; s -lens 70; capillary temperature 300 °C. Calibration standards were prepared by diluting working stocks of each metabolite in aqueous 0.4 M Na 3 PO 4 to a final volume of 50 μL. Lyophilized stool was weighed and homogenized to a final density of 75 mg/mL in 0.4 M Na 3 PO 4 with vigorous shaking overnight at 4 °C. The calibration standards (50 μL) and stool extracts (50 μL) were spiked with a mixture of 200 ng uracil- 13 C 1 15 N 2 and 200 ng 2-thiouracil- 13 C 1 15 N 2 dissolved in 0.5 N NaOH. To improve the chromatographic resolution and MS ionization efficiency, the various uracil analogues were derivatized by extractive alkylation with pentafluorobenzyl bromide (PFB-Br) prior to analysis 71 . Samples and standards were diluted with 450 μL deionized water, 100 μL of PFB-Br dissolved in chloroform (4 % v/v), and 25 μL of the phase transfer catalyst tetrabutylammonium dihydrogen phosphate (1.0 M in H 2 O). After sonication in a 50 °C water bath for 20 min, the crude reaction products were extracted twice with 700 μL hexane / ethyl acetate (4:1). The organic extracts were combined, evaporated under a gentle stream of dry nitrogen, reconstituted in 150 μL HPLC-grade methanol, and centrifuged at 4°C for 10 minutes (18,000 × g ) to remove particulates. The supernatants were transferred to 200 μL spring-loaded inserts in autosampler vials equipped with Teflon-lined bonded rubber septa. Data acquisition and quantitative spectral analysis were performed using Thermo-Finnigan Xcalibur version 2.0.7 SP1 and Thermo-Finnigan LCQuan version 2.7.0.20, respectively. Calibration curves were constructed by plotting the peak area ratios (analyte/internal standard) against the analyte concentrations for a series of nine standards (0.005–50 total nmol). A weighting factor of 1/C 2 was applied in the linear least-squares regression analysis to maintain homogeneity of variance across the calibrated concentration range. Phylogenetic analysis of TudS The maximum likelihood tree of representative bacterial genomes was built as previously described 72 . 4,237 representative bacterial genomes were collected from BV-BRC database ( https://www.bv-brc.org/ , version 3.31.12) 73 in Oct 2023 and the sequences of 10 universally distributed ribosomal proteins (L2, L3, L4, L14, L16, L18, L22, L24, S3, and S8) were retrieved. The sequences of each ribosomal protein family were independently aligned using MUSCLE v5.1 74 and trimmed using BMGE v1.12 75 . The maximum likelihood tree was generated with the concatenated alignment using FastTree v2.1.11 76 with-lg -cat 20 model and bootstrap (100 replicates). For better visualization, the branches are grouped and colored by phyla. The branches with bootstrap values greater than 0.7 were indicated. DUF523 proteins in the representative bacterial genomes were searched by BLASTp v2.15.0 77 with an E-value cutoff of 1. The sequences then searched against Pfam database v2024-05-28 78 using hmmsearch v3.4 ( hmmer.org ) with default setting. Entries with single domains and E-value over 0.05 were verified by HHpred (toolkit.tuebingen.mpg.de/tools/hhpred) 79 . The resulted DUF523 proteins were listed in Table S3 . The selected DUF523 proteins were aligned using MUSCLE and visualized using Jalview v2.11.3.3 80 . For the tree of DUF523 proteins, the alignment was trimmed using BMGE and subjected to build the maximum likelihood tree using FastTree with -lg -cat 20 model with bootstrap (1000 replicates). The trees were visualized using the iTOL platform https://itol.embl.de/ (version 6.9.1) 81 . The structure of TudS Cd was generated by AlphaFold v4 82 and aligned with that of DUF523 Vzc (PDB 6Z93) using PyMOL v3.0.0. The structure of mutant Upp Cd and mutant PyrR Cd were modeled by SWISS-MODEL ( swissmodel.expasy.org/ ) 83 with the template of Bacillus caldolyticus Upp (PDB 1I5E) and Bacillus subtilis PyrR (PDB 4P83), respectively. The docking of proteins and 4-TU was generated by Open Babel v3.1.1 84 and AutoDock Vina v4 85 with setting -xr -p 7.4 – partialcharge eem and exhaustiveness=128, respectively. The structures were visualized using PyMOL. RNA isolation Freshly streaked C. difficile or E. coli colonies were used to inoculate 5 mL BHIS and strains were grown overnight at 37°C anaerobically. After 16 h of growth, cultures were subcultured 1:25 into fresh CDMM and grown to an OD 600 of 0.3 and vehicle, 100 μg/mL 4-TU, or 100 μg/mL 4-TU and 20 μg/mL uracil were added for 1 h. Following 1 h of treatment, bacteria were centrifuged and resuspended in 1 mL of TRIzol Reagent (Invitrogen), then stored at −80°C until time of RNA isolation. For RNA isolation, resuspended bacteria were homogenized in a bead beater with Lysing Matrix B beads (MP Biomedical) at a speed of 6 m/s for 45 seconds. Homogenized suspensions were centrifuged and the upper phase was collected and mixed with 200 μL chloroform (Acros Organics). Samples were incubated at room temperature for 2 min, centrifuged at 4°C for 15 min, and 400 μL of the upper aqueous phase was collected. Samples were mixed with 500 μL of ice-cold isopropyl alcohol (Sigma-Aldrich), incubated on ice for 10 minutes, and centrifuged at 4°C for 15 min. The supernatant was removed and 500 μL of ice-cold 75% ethanol (Sigma-Aldrich) with 1 mM dithiothreitol (DTT) (ThermoFisher) was added to the pellet. Samples were centrifuged at 4°C for 10 min, supernatants were discarded, and pellets were allowed to air dry for at least 10 min. Pellets were resuspended in 50 μL of DNase-Free, RNase-Free water (ThermoFisher) with 1 mM DTT. DNA contamination was removed using the Turbo DNA-free kit (Invitrogen) according to manufacturer’s instructions. RNA was stored at −80°C. s 4 U incorporation assay 10 μg of RNA isolated from C. difficile or E. coli was diluted into 30 μL of DNase-Free, RNase-Free water with 0.1 mM DTT. RNA was incubated at 95°C for 3 min followed by an incubation on ice for 5 min. Samples were treated with 1 μL (100U/μL) of nuclease P1 (NEB) and ammonium acetate pH 5.3 was added to 10 mM. Samples were incubated at 45°C for 2 h to digest RNA into individual nucleotides. Following incubation, 2 μL (0.001U/μL) of snake venom phosphodiesterase I (Worthington Biochemical) was added and ammonium bicarbonate was added to 100 mM and reactions were incubated at 37°C for 16 h. The resulting nucleotide mixture was treated with 1 μL of Antarctic phosphatase (NEB) and incubated at 37°C for 1 h to generate nucleosides. Nucleosides were combined with 90 μL DNase-Free, RNase-Free water and DTT was added to 0.1 mM and sodium acetate pH 5.2 was added to 100 mM. 100 μL of ice-cold absolute ethanol was added and samples were incubated at −80°C for 10 min. Samples were centrifuged for 5 min at room temperature and the supernatant was transferred to a new tube and DTT was added to 0.1 mM. 300 μL of ice-cold absolute ethanol was added and samples were incubated at −80°C for 10 min. Samples were centrifuged for 5 min at room temperature and the supernatant was transferred to a new tube. Samples were evaporated to complete dryness using a vacuum concentrator and pellets were reconstituted in 55 μL DNase-Free, RNase-Free water. Nucleosides derived from RNA and standards were analyzed on an Agilent 1260 Infinity II system. Analytes were separated by gradient high-performance liquid chromatography (HPLC) on a Supelco Ascentis Express C 18 column (25 cm × 2.1 mm, 5 μm) with a Phenomenex SecurityGuard C 18 cartridge (3.2 × 8 mm) at a flow rate of 0.5 mL/min using 0.1M triethyl ammonium acetate in 3% acetonitrile and 90% acetonitrile as the A and B mobile phases, respectively. The gradient was held at 100% A for 15 minutes followed by a 5-minute ramp to 100% B. The column was washed at 100% B for 5 minutes followed by a 20-minute equilibration at 100% A. Uridine, cytosine, guanosine, and adenine were detected at 260 nm and 4-thiouridine was detected at 330 nm, and retention times were confirmed using each standard. The percentage of uridine substituted by 4-thiouridine was determined using the following formula: % U substituted by S4U = ( Area4SU , 330 nm / ε 4SU , 330 nm ) × ( ε U , 260 nm / AreaU , 260 nm ) × 100 s 4 U RNA-Sequencing RNA was alkylated as previously described with a few minor modifications 86 . Reactions containing 500 ng of RNA, 10 mM iodoacetamide (IAA), 50 mM NaPO 4 , and 50% DMSO were incubated at 50°C for 15 min. Reactions were quenched via addition of 1 μL of 1 M DTT to a total reaction volume of 50 μL (20 mM final concentration of DTT). Following addition of 4 μL linear acrylamide (5 mg/mL), 5 μL 3M NaOAc pH 5.2, and 125 μL 100% EtOH, RNA was precipitated for 30 min at −80°C. Precipitated RNA was pelleted by centrifugation at 16,000 × g for 30 min and the RNA pellet was washed and with 1 mL 75% EtOH. Following centrifugation at 16,000 × g for 10 min, pellets were allowed to dry for 10 min and resuspended in 10 μL DNase-Free, RNase-Free water. RNA-seq libraries were prepared using the Lexogen RiboCop rRNA Depletion Kit for Gram-positive Bacteria and CORALL RNA-seq V2 Library Prep Kit with UDIs according to the manufacturer’s instructions. cDNA libraries were multiplexed and sequenced via NovaSeq X Series PE150. Reads were mapped to a reference file containing only the sequence for rpoB from C. difficile CD196 and T->C conversions were identified using the Breseq pipeline 87 . The percentage of mapped reads harboring a T->C conversion at a particular location in the rpoB sequence was calculated by dividing the mapped reads harboring a T->C conversion by the total number of reads mapped at that location. Protein expression and purification E. coli BL21 (DE3) pREL containing the pET15b_ pyrR or pET15b_ upp plasmids were grown overnight in 5 mL LB containing Cm 20 and Carb 100. After 16 h of growth, cells were subcultured 1:100 into Terrific broth (ThermoFisher) containing Carb 100 and grown to an OD 600 of 0.5 at 37°C prior to the addition of 0.5 mM isopropyl-1-thiol-D-galactopyranoside (IPTG). Growth was continued at 30°C for 3 h and cells were harvested by centrifugation (8,000 × g for 20 minutes) and pellets were washed in 40 mL 20 mM Tris-HCl pH 8 and saved at −80°C. Cell pellets were resuspended in 25 mL 50 mM Tris-HCl pH 8, 300 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mg/mL lysozyme, 40 μg/mL DNase I, and the addition of 1 Pierce protease inhibitor (EDTA free) (ThermoFisher) and incubated at 4°C for 30 minutes rotating end over end. Cells were lysed by sonication for a total of 18 cycles (30s on, 2 min off) at 90% amplitude. Following sonication, lysates were centrifuged at 4,000 × g for 15 min at 4°C to pellet unlysed cells. The resulting supernatants were centrifuged at 40,000 × g for 30 min to pellet insoluble debris and clarify the lysates. Lysates were combined with equilibrated Ni-charged Profinity IMAC resin (Biorad) and allowed to bind for 1 h prior to addition to a gravity column. The column was washed and bound proteins were eluted using a stepwise gradient of 40–250 mM imidazole in 50 mM Tris-HCl pH 8, 300 mM NaCl, and 10% glycerol. Protein fractions were run on 4 to 20% gradient Mini-Protean SDS-PAGE gels (Bio-Rad) in the Mini-Protean electrophoresis system (Bio-Rad) and analyzed with SimplyBlue SafeStain (ThermoFisher). Phosphoribosyl Transfer Assay Phosphoribosyl transfer activity was quantified as previously described with some minor modifications 88 . Enzymatic reactions were initiated by adding 3 μg of recombinant enzyme to reaction mixtures containing 100 μM uracil or 100 μM 4-TU (or both uracil and 4-TU), 1 mM PRPP, 10 mM MgCl 2 , and 50 mM Tris-HCl pH 7.8 in a final volume of 0.2 mL. Reactions were incubated at 37°C for the indicated time courses and quenched by boiling at 100°C for 3 min. Reactions were passed through Amicon Ultra 0.5 mL centrifugal filters with a 3 kDa molecular weight cut off. The resulting flow through was analyzed on an Agilent 1260 Infinity II system. Analytes were separated by isocratic HPLC on a Supelco Ascentis Express C 18 column (25 cm × 2.1 mm, 5 μm) with a Phenomenex SecurityGuard C 18 cartridge (3.2 × 8 mm) at a flow rate of 0.5 mL/min using 5 mM potassium phosphate pH 4 with 5% acetonitrile as the mobile phase for 15 minutes. Uracil and uridine monophosphate were detected at 260 nm, and 4-thiouracil and 4-thiouridine monophosphate were detected at 330 nm. Retention times were determined using each standard. qRT-PCR cDNA was generated from 200 ng of RNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to manufacturer’s instructions and subjected to qRT-PCR using iQ SYBR green supermix (Bio-Rad) with the primer pairs listed in Table S6 . Amplification of cDNA template was performed on a CFX96 qPCR cycler (Bio-Rad) using a 3-step melt curve program. Threshold cycle (CT) values for each transcript were normalized by the rpoB gene. Genetic Selections For C. difficile , overnight cultures of WT and Δ tudS were diluted 1:5 in BHIS and 200 μL was plated on TCCFA containing 500 μg/mL 4-TU and incubated at 37°C anaerobically for 48 h. Individual Δ tudS suppressor colonies were isolated on BHISA and replated on TCCFA with 500 μg/mL 4-TU to confirm stable resistance. For E. coli , overnight cultures started from independent colonies were subcultured 1:100 for 2 hours and diluted 1:50 into a 96 well plate containing vehicle or 20 μg/mL 4-TU in CDMM. Growth was monitored in an EpochII microplate reader (BioTek) and suppressor mutants from individual wells demonstrating growth in 4-TU were isolated on LBA. Genomic DNA (gDNA) was extracted using the Qiagen DNeasy Blood and Tissue kit according to manufacturer’s instructions. Purified gDNA was sequenced by SeqCenter (Pittsburgh, PA) using 150-bp paired end reads on Illumina NextSeq 2000. Single nucleotide polymorphisms (SNPs) were identified in C. difficile CD196 accession NZCP059592 or E. coli K12 BW25113 accession NZ_CP009273 using the bacterial and viral bioinformatic resource center (BV-BRC) variation analysis service 73 . Nonsynonymous mutations identified by whole genome sequencing were mapped to the AlphaFold model of C. difficile Upp or PyrR using PyMOL. Minibioreactor arrays Human fecal communities were established in continuous-flow minibioreactor arrays containing bioreactor medium 3 (BRM3) as described previously 89 , 90 . Briefly, aliquots of frozen fecal PBS slurries (25% wt/vol) from individual donors (n=4) were thawed, vortexed, and 4 ml were used to inoculate each BRM3 filled reactor. Fecal communities were allowed to grow in batch culture for 16 h prior to initiating continuous flow. Communities were equilibrated in continuous flow for 7 days and then treated with clindamycin (500 μg/mL) twice daily (morning and evening) for 4 days. 16 hours post clindamycin treatment, communities were supplied with BRM3 containing vehicle (dimethyl sulfoxide) or 4-TU (250 μg/mL). After 24 h of growth to allow washout of clindamycin, communities were challenged with a 1:1 mixture of exponentially growing C. difficile CD196 (WT and Δ tudS ) strains (2 × 10 4 CFU/mL). Samples were collected 2 days post-inoculation and the competitive index of WT:Δ tudS was determined by qPCR using strain specific primers listed in Table S6 ( Figure S6B and S6C ). Amplification of template DNA was performed on a CFX96 qPCR cycler (Bio-Rad) using iQ SYBR green supermix (Bio-Rad) with 40 cycles of amplification (95°C for 10 s and 57°C for 30 s). QUANTIFICATION AND STATISTICAL ANALYSIS Raw data were collected in Microsoft Excel and imported into GraphPad Prism for visualization and statistical analysis. Statistical significance was assessed using Student’s t -test, Mann-Whitney U test, One-way ANOVA with the Tukey correction for multiple comparisons, Two-way ANOVA with the Dunnett’s multiple comparison post-test, or Log-rank test. Exact statistical tests used, group sizes, and dispersion and precision of measurements are defined in the figure legends. Supplementary Material 1 Document S1. Figures S1 – S6 and Tables S1 – S2 , S4 – S5 NIHMS2063643-supplement-1.pdf (11.3MB, pdf) 2 Table S3. Genomic information of select DUF523 features, related to Figure S1 . NIHMS2063643-supplement-2.xlsx (66.6KB, xlsx) 3 Table S6. Strains and primers used in this study, related to STAR Methods. NIHMS2063643-supplement-3.xlsx (16.5KB, xlsx) Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-FLAG M2 Sigma Cat#F3165 Donkey anti-mouse IRDye 680 secondary antibody LI-COR Cat#926-68070 Bacterial and virus strains For bacterial strains used in this study see Supplementary Table 6 NA NA Biological samples Human fecal samples This study NA Chemicals, peptides, and recombinant proteins 4-thiouracil (4-TU) Sigma Cat#440736 4-thiouridine (s 4 U) Sigma Cat#T4509 Uracil- 13 C, 15 N2 Cayman Chemical Cat#33997 2-thiouracil- 13 C, 15 N2 LGC Standards Cat#TRC-T375408 4-thiouridine-5’-monophosphate (s 4 UMP) Jena Bioscience Cat#NU-1154S Uracil Sigma Cat#U0750 Uridine Sigma Cat#U3750 Uridine monophosphate (UMP) Sigma Cat#U6375 5-phospho-D-ribose 1-diphosphate (PRPP) Cayman Chemical Cat#18897 Dimethyl sulfoxide (DMSO) Sigma Cat#D8418 pentafluorobenzyl bromide (PFB-Br) ThermoFisher Cat#TS-58220 Iodoacetamide Sigma Cat#D8418 Linear Acrylamide Invitrogen Cat#AM9520 UltraPure Water Invitrogen Cat#10977-023 Acetonitrile (ACN), HPLC grade Fisher Cat#A998 Triethyl ammonium acetate Sigma Cat#625718 TRIzol reagent Ambion Cat#15596018 Kanamycin sulfate Sigma Cat#60615-5G Carbenicillin Fisher Cat#BP2648-5 Chloramphenicol Fisher Cat#BP904-100 Tetracycline hydrochloride Alfa Aesar Cat# B21408 Thiamphenicol Sigma Cat#0261 Colistin Sigma Cat#C4461 Cefoperazone Sigma Cat#C4292 Cefoxitin Sigma Cat#C4786 D-cycloserine Sigma Cat#C6880 Vancomycin Fisher Cat#BP29581 Profinity IMAC Resin, Ni-charged Biorad Cat#1560133 Hemin from porcine Sigma Cat#51280 Nuclease P1 NEB Cat#M0660S Antarctic phosphatase NEB Cat#M0289 Snake venom phosphodiesterase I (PDE I) Worthington Biochemical Cat# LS003926 Upp This study NA Upp S139F This study NA PyrR This study NA PyrR D135Y This study NA PyrR G149E This study NA Critical commercial assays Turbo DNA-free kit Invitrogen Cat#AM1907 DNeasy Blood and Tissue Kit Qiagen Cat#69506 RiboCop rRNA Depletion Kit Lexogen Cat#127.24 CORALL RNA-seq Library Prep Kit Lexogen Cat#171.24 GeneJET PCR Purification Kit Thermo Scientific Cat#K0702 GeneJET PCR Plasmid Miniprep Kit Thermo Scientific Cat#K0503 Q5 Site-Directed Mutagenesis Kit NEB Cat#E0554S NEBuilder HiFi DNA Assembly Master Mix New England Biolabs Cat#M5520A High-capacity cDNA Reverse Transcription Kit Bio-Rad Cat#1708891 iQ SYBR Green Supermix Bio-Rad Cat#1708882 Deposited data Raw sequencing data generated from WGS of E. coli suppressor strains This study NCBI SRA BioProject: PRJNA1219637 Raw sequencing data generated from WGS of Δ tudS C. difficile suppressor strains This study NCBI SRA BioProject: PRJNA1219607 Raw sequencing data generated from SLAM-seq of WT and Δ tudS C. difficile This study NCBI SRA BioProject: PRJNA1219594 Experimental models: Cell lines NA NA NA Experimental models: Organisms/strains Mouse: C57BL/6J The Jackson Laboratory RRID:IMSR_JAX:000664 Oligonucleotides For oligonucleotides used in this study see Supplemental Table 6 NA NA Recombinant DNA For plasmids used in this study see Supplemental Table 6 NA NA Software and algorithms Prism 10 GraphPad https://www.graphpad.com Canvas X Draw 7 Canvas GFX https://www.canvasgfx.com CLC Genomics Workbench Qiagen https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-genomics-workbench/ BV-BRC Bacterial and Viral Bioinformatics Resource Center https://www.bv-brc.org/ PyMol Schrodinger https://pymol.org/ Interactive Tree of Life (version 6.9.1) iTOL https://itol.embl.de/ Thermo-Finnigan Xcalibur version 2.0.7 ThermoFisher https://www.thermofisher.com/order/catalog/product/OPTON-30965 Rfam 14.10 ELIXIR https://rfam.org/ Agilent OpenLab CDS Agilent https://www.agilent.com/en/product/software-informatics/analytical-software-suite/chromatography-data-systems/openlab-cds ChemDraw 23.0 PerkinElmer NA Other Anaerobic Chamber Coy Laboratory Products NA C18 5 μm (250 × 2.1 mm) column Phenomenex Cat#50521-U C18 UPLC column (2.1 × 100 mm; 1.8 μm) Waters Cat#186002352 Security Guard C18 Cartridge (3.2 mm × 8 mm) Phenomenex Cat#AJ0-4287 Open in a new tab Highlights. Pyrimidine nucleotide synthesis is critical for Clostridioides difficile infection A thiouracil desulfurase (TudS) enables C. difficile to utilize 4-thiouracil (4-TU) TudS prevents 4-TU toxicity and incorporation into RNA by conversion to uracil 4-TU is present in the human gut and confers a fitness advantage to C. difficile Acknowledgements We thank members of the Skaar laboratory for critical review of this manuscript. We are grateful for the laboratories of Dr. Manuel Ascano and Dr. John Karijolich for providing methodology and reagents to assess s 4 U incorporation. This research is supported by the following National Institutes of Health (NIH) grants: R01 AI164587 (E.P.S.), U19 AI174999 (E.P.S.), R01 GM70641 (V. dC.-L.), T32 ES007028 (M.J.M.), F31 AI172352 (M.J.M.), K23 No. 1K23AI156132 (M.R.N.), U19 AI157981 (R.A.B.). Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. 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Investigating Colonization of the Healthy Adult Gastrointestinal Tract by Fungi. mSphere 3. 10.1128/mSphere.00092-18. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials 1 Document S1. Figures S1 – S6 and Tables S1 – S2 , S4 – S5 NIHMS2063643-supplement-1.pdf (11.3MB, pdf) 2 Table S3. Genomic information of select DUF523 features, related to Figure S1 . NIHMS2063643-supplement-2.xlsx (66.6KB, xlsx) 3 Table S6. Strains and primers used in this study, related to STAR Methods. NIHMS2063643-supplement-3.xlsx (16.5KB, xlsx) Data Availability Statement Raw sequencing files obtained from SLAM-seq experiments are available in the National Center for Biotechnology Information (NCBI) sequence read archive (SRA) under BioProject: PRJNA1219594. Whole genome sequencing data of E. coli 4-TU suppressor strains are available under BioProject: PRJNA1219637. Whole genome sequencing data of C. difficile 4-TU suppressor strains are available under BioProject: PRJNA1219607. This paper does not report original code. 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