d-Proline Reductase Underlies Proline-Dependent Growth of Clostridioides difficile.

Clostridioides difficile is a nosocomial pathogen that colonizes the gut and causes diarrhea, colitis, and severe inflammation. Recently, C. difficile has been shown to use toxin-mediated inflammation to promote host collagen degradation, which releases several amino acids into the environment. Amino acids act as electron donors and acceptors in Stickland metabolism, an anaerobic process involving redox reactions between pairs of amino acids. Proline, glycine, and hydroxyproline are the three main constituents of collagen and are assumed to act as electron acceptors, but their exact effects on the growth and physiology of C. difficile are still unclear. Using three standard culture media (supplemented brain heart infusion [BHIS], tryptone-yeast [TY], and C. difficile minimal medium [CDMM]) supplemented with proline, glycine, or hydroxyproline, we grew C. difficile strains R20291, JIR8094, and a panel of mutants unable to express the Stickland selenoenzymes d-proline reductase and glycine reductase. In the wild-type strains, growth yields in rich media (BHIS and TY) were higher with proline and hydroxyproline but not glycine; moreover, proline-stimulated growth yields required the activity of d-proline reductase, whereas hydroxyproline-stimulated growth yields were independent of its activity. While assumed to be a proline auxotroph, C. difficile could surprisingly grow in a defined medium (CDMM) without proline but only if d-proline reductase was absent. We believe the mere presence of this enzyme ultimately determines the organism's strict dependence on proline and likely defines the bioenergetic priorities for thriving in the host. Finally, we demonstrated that addition of proline and hydroxyproline to the culture medium could reduce toxin production but not in cells lacking selenoproteins. IMPORTANCE Stickland metabolism is a core facet of C. difficile physiology that likely plays a major role in host colonization. Here, we carefully delineate the effects of each amino acid on the growth of C. difficile with respect to the selenoenzymes d-proline reductase and glycine reductase. Moreover, we report that d-proline reductase forces C. difficile to strictly depend on proline for growth. Finally, we provide evidence that proline and hydroxyproline suppress toxin production and that selenoproteins are involved in this mechanism. Our findings highlight the significance of selenium-dependent Stickland reactions and may provide insight on what occurs during host infection, especially as it relates to the decision to colonize based on proline as a nutrient.

The Rv2633c protein of Mycobacterium tuberculosis is a non-heme di-iron catalase with a possible role in defenses against oxidative stress.

The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis. However, the activity and role of the Rv2633c protein in host colonization is unknown. Here, we analyzed the Rv2633c protein sequence, which revealed the presence of an HHE cation-binding domain common in hemerythrin-like proteins. Phylogenetic analysis indicated that Rv2633c is a member of a distinct subset of hemerythrin-like proteins exclusive to mycobacteria. The Rv2633c sequence was significantly similar to protein sequences from other pathogenic strains within that subset, suggesting that these proteins are involved in mycobacteria virulence. We expressed and purified the Rv2633c protein in Escherichia coli and found that it contains two iron atoms, but does not behave like a hemerythrin. It migrated as a dimeric protein during size-exclusion chromatography. It was not possible to reduce the protein or observe any evidence for its interaction with O2 However, Rv2633c did exhibit catalase activity with a kcat of 1475 s-1 and Km of 10.1 ± 1.7 mm Cyanide and azide inhibited the catalase activity with Ki values of 3.8 µm and 37.7 µm, respectively. Rv2633c's activity was consistent with a role in defenses against oxidative stress generated during host immune responses after M. tuberculosis infection of macrophages. We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses.

Proline-dependent regulation of Clostridium difficile Stickland metabolism.

Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the d-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions.

Age-moderated effects of consequence and intent information on punishment: an intuitive prosecutorial interpretation.

In responding to wrongdoings, people simultaneously pursue the goals of social control and fairness to the wrongdoer. Social control necessitates stronger weighting of consequences than causes; fairness entails the opposite. The authors hypothesized that the developmental shift from overweighting consequence to overweighting intent when determining levels of punishment illustrates a shift from a default defender of the normative order to a motivated crusader of fairness to the wrongdoer. Thus, punishment should increase slightly for intentional wrongdoings but decrease substantially for accidental wrongdoings as people age. In an experiment on disciplinary action in Singapore, 9-, 13-, and 17-year-olds learned about the consequences of and intentions behind wrongdoings by peers and predicted consistency of the same act in the future, assigned blame to the wrongdoers, and recommended punishment for them. Results supported hypotheses derived from a fair-but-biased-yet-correctible model of intuitive prosecutors.

Inhibition of selenoprotein synthesis is not the mechanism by which auranofin inhibits growth of Clostridioides difficile.

Clostridioides difficile infections (CDIs) are responsible for a significant number of antibiotic-associated diarrheal cases. The standard-of-care antibiotics for C. difficile are limited to fidaxomicin and vancomycin, with the recently obsolete metronidazole recommended if both are unavailable. No new antimicrobials have been approved for CDI since fidaxomicin in 2011, despite varying rates of treatment failure among all standard-of-care drugs. Drug repurposing is a rational strategy to generate new antimicrobials out of existing therapeutics approved for other indications. Auranofin is a gold-containing anti-rheumatic drug with antimicrobial activity against C. difficile and other microbes. In a previous report, our group hypothesized that inhibition of selenoprotein biosynthesis was auranofin's primary mechanism of action against C. difficile. However, in this study, we discovered that C. difficile mutants lacking selenoproteins are still just as sensitive to auranofin as their respective wild-type strains. Moreover, we found that selenite supplementation dampens the activity of auranofin against C. difficile regardless of the presence of selenoproteins, suggesting that selenite's neutralization of auranofin is not because of compensation for a chemically induced selenium deficiency. Our results clarify the findings of our original study and may aid drug repurposing efforts in discovering the compound's true mechanism of action against C. difficile.

Evaluation of Derivatives of (+)-Puupehenone against Clostridioides difficile and Other Gram-Positive Bacteria.

Patients receiving healthcare are at higher risk of acquiring healthcare-associated infections, which cause a significant number of illnesses and deaths. Most pathogens responsible for these infections are highly resistant to multiple antibiotics, prompting the need for discovery of new therapeutics to combat these evolved threats. We synthesized structural derivatives of (+)-puupehenone, a marine natural product, and observed growth inhibition of several clinically relevant Gram-positive bacteria, particularly Clostridioides difficile. The most potent compounds-(+)-puupehenone, 1, 15, 19, and 20-all inhibited C. difficile in the range of 2.0-4.0 µg/mL. Additionally, when present in the range of 1-8 µg/mL, a subset of active compounds-(+)-puupehenone, 1, 6, 15, and 20-greatly reduced the ability of C. difficile to produce exotoxins, which are required for disease in infected hosts. Our findings showcase a promising class of compounds for potential drug development against Gram-positive pathogens, such as C. difficile.

A selenium-dependent xanthine dehydrogenase triggers biofilm proliferation in Enterococcus faecalis through oxidant production.

Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with (75)Se is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production.

Redox-active radical scavenging nanomaterials.

Reactive oxygen and nitrogen species play a critical role in many degenerative diseases and in aging. Nanomaterials, especially modified fullerenes and cerium oxide nanoparticles, have been shown to effectively protect mammalian cells against damage caused by increased reactive oxygen or nitrogen species, likely through their direct reaction with superoxide radical, since each of these materials has been shown to act as effective superoxide dismutase mimetics in vitro. This critical review discusses the chemistry of these nanomaterials and the context in which their radical scavenging activities have been studied in biological model systems. Current studies are focused on determining the uptake, metabolism, distribution, toxicity and fate of these nanomaterials in cell and animal model systems. Ultimately if shown to be safe, these nanomaterials have the potential to be used to reduce the damaging effects of radicals in biological systems (101 references).

Reacting to headline news: circumstances leading to causal explanations versus implicational concerns.

How do people react to the headline news they receive? According to the model of people as intuitive scientists (Kelley, 1972; Ross, 1977), people-like scientists-make causal explanations (i.e., why did an event take place?) and assign responsibility to the person, the situation, or both. However, a more recently proposed social-functionalist model (Tetlock, 2002) views people less as intuitive scientists trying to understand the world and more as intuitive prosecutors trying to protect a fragile social order. Thus, implicational concerns (i.e., how would it affect people's lives, properties, and liberties?) with the news can also be likely reactions. Given the prescriptions of these models, the present authors tested the hypotheses that news reports evoke both causal explanations and implicational concerns among viewers, and that the degree of the two reactions depends on the valence (positive vs. negative) and theme (whether it is unusual or social order-linked) of the news. Singaporeans (N = 80) read one piece of headline news that represented a crossed level of valence (negative vs. positive) and theme (unusual vs. social order), and indicated the likelihood of causal explanations and implicational concerns as their first response to it. As hypothesized, positive news led to a greater likelihood of showing implicational concerns than of making causal explanations, the difference being reversed in the case of negative news; unusual news led to a greater likelihood of making causal explanations than of showing implication concerns; the likelihood of having implicational concerns with news related to social order was higher than making that of causal explanations; and the two responses were equally likely in the case of negative news. Overall, these results support a view of people as intuitive prosecutors interested in both causal explanations of and implicational concerns with a news report.

Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.

Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological double-edged sword. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for l-selenocysteine over l-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue's potential role in the l-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards l-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, l-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than l-selenocysteine. Finally, we discovered that SclA exhibited low activity with l-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for l-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.

Tuning hydrated nanoceria surfaces: experimental/theoretical investigations of ion exchange and implications in organic and inorganic interactions.

Long-term stability and surface properties of colloidal nanoparticles have significance in many applications. Here, surface charge modified hydrated cerium oxide nanoparticles (CNPs, also known as nanoceria) are synthesized, and their dynamic ion exchange interactions with the surrounding medium are investigated in detail. Time-dependent zeta (zeta) potential (ZP) variations of CNPs are demonstrated as a useful characteristic for optimizing their surface properties. The surface charge reversal of CNPs observed with respect to time, concentration, temperature, and doping is correlated to the surface modification of CNPs in aqueous solution and the ion exchange reaction between the surface protons (H(+)) and the neighboring hydroxyls ions (OH(-)). Using density functional theory (DFT) calculations, we have demonstrated that the adsorption of H(+) ions on the CNP surface is kinetically more favorable while the adsorption of OH(-) ions on CNPs is thermodynamically more favorable. The importance of selecting CNPs with appropriate surface charges and the implications of dynamic surface charge variations are exemplified with applications in microelectronics and biomedical.

Inhibition of selenium metabolism in the oral pathogen Treponema denticola.

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.

PEGylated nanoceria as radical scavenger with tunable redox chemistry.

We report the direct synthesis of cerium oxide nanoparticles (CNPs) in polyethylene glycol (PEG) based solutions with efficient radical scavenging properties. Synthesis of CNPs in PEG demonstrated a concentration dependent (of PEG) redox activity characterized by UV-visible spectroscopy. PEGylated CNPs acted as efficient radical scavengers, and superoxide dismutase (SOD) activity of CNPs synthesized in various concentration of PEG did not reduce compared to bare nanoceria. In addition to superoxide, PEGylated nanoceria demonstrated quenching of peroxide radicals as well. It was observed that the reaction with hydrogen peroxide leads to the formation of a charge transfer complex governed by the concentration of PEG. The stability of the charge transfer complex provides the tunable oxidation state of CNPs. The stability of this complex influences the regenerative capacity of the active 3+ oxidation state of CNPs. The cell viability as well as SOD activity of PEGylated CNPs is compared to those of bare CNPs, and the differences are outlined.

Exposure to monomethylarsonous acid (MMA(III)) leads to altered selenoprotein synthesis in a primary human lung cell model.

Monomethylarsonous acid (MMA(III)), a trivalent metabolite of arsenic, is highly cytotoxic and recent cell culture studies suggest that it might act as a carcinogen. The general consensus of studies indicates that the cytotoxicity of MMA(III) is a result of increased levels of reactive oxygen species (ROS). A longstanding relationship between arsenic and selenium metabolism has led to the use of selenium as a supplement in arsenic exposed populations, however the impact of organic arsenicals (methylated metabolites) on selenium metabolism is still poorly understood. In this study we determined the impact of exposure to MMA(III) on the regulation of expression of TrxR1 and its activity using a primary lung fibroblast line, WI-38. The promoter region of the gene encoding the selenoprotein thioredoxin reductase 1 (TrxR1) contains an antioxidant responsive element (ARE) that has been shown to be activated in the presence of electrophilic compounds. Results from radiolabeled selenoproteins indicate that exposure to low concentrations of MMA(III) resulted in increased synthesis of TrxR1 in WI-38 cells, and lower incorporation of selenium into other selenoproteins. MMA(III) treatment led to increased mRNA encoding TrxR1 in WI-38 cells, while lower levels of mRNA coding for cellular glutathione peroxidase (cGpx) were detected in exposed cells. Luciferase activity of TrxR1 promoter fusions increased with addition of MMA(III), as did expression of a rat quinone reductase (QR) promoter fusion construct. However, MMA(III) induction of the TRX1 promoter fusion was abrogated when the ARE was mutated, suggesting that this regulation is mediated via the ARE. These results indicate that MMA(III) alters the expression of selenoproteins based on a selective induction of TrxR1, and this response to exposure to organic arsenicals that requires the ARE element.

High affinity selenium uptake in a keratinocyte model.

The distribution of selenium in mammals has been recently shown to be mediated primarily by selenoprotein P. Even in the absence of selenoprotein P, selenium is distributed from the liver into all organs and tissues when supplemented in the diet. The form of selenium that is actively taken up by mammalian cells at trace concentrations has yet to be determined. We used a human keratinocyte model to determine whether reduction of the oxyanion selenite (SeO(3)(2-)) to the more reduced form of selenide (HSe(-)) would affect uptake. Indeed a reduced form of selenium, presumably selenide, was actively transported into keratinocytes and displayed saturation kinetics with an apparent K(m) of 279 nM. ATPase inhibitors blocked the uptake of selenide, as did the competing anions molybdate and chromate, but not sulfate. These results suggest that the small molecule form of selenium that is distributed in tissues is hydrogen selenide, despite its sensitivity to oxygen and reactivity to thiols.

The role of cerium redox state in the SOD mimetic activity of nanoceria.

Cerium oxide nanoparticles (nanoceria) have recently been shown to protect cells against oxidative stress in both cell culture and animal models. Nanoceria has been shown to exhibit superoxide dismutase (SOD) activity using a ferricytochrome C assay, and this mimetic activity that has been postulated to be responsible for cellular protection by nanoceria. The nature of nanoceria's antioxidant properties, specifically what physical characteristics make nanoceria effective at scavenging superoxide anion, is poorly understood. In this study electron paramagnetic resonance (EPR) analysis confirms the reactivity of nanoceria as an SOD mimetic. X-ray photoelectron spectroscopy (XPS) and UV-visible analyses of nanoceria treated with hydrogen peroxide demonstrate that a decrease in the Ce 3(+)/4(+) ratio correlates directly with a loss of SOD mimetic activity. These results strongly suggest that the surface oxidation state of nanoceria plays an integral role in the SOD mimetic activity of nanoceria and that ability of nanoceria to scavenge superoxide is directly related to cerium(III) concentrations at the surface of the particle.

Inhibition of hydrogen uptake in Escherichia coli by expressing the hydrogenase from the cyanobacterium Synechocystis sp. PCC 6803.

BACKGROUND: Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- <--> H2 (g). RESULTS: Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. CONCLUSION: Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2.

Impact of trivalent arsenicals on selenoprotein synthesis.

BACKGROUND: Exposure to arsenic has been associated with development of skin, lung, bladder, liver, and kidney cancer. Recent evidence suggests that an increase in oxidative stress in cells treated with arsenicals represents the molecular mechanism behind arsenic-induced carcinogenesis. Selenium, in the form of selenocysteine, is necessary for the activity of several enzymes with a role in defense against reactive oxygen species. A mutual sparing effect between arsenic and selenium has been shown in animal studies when both metalloids are present in high concentrations. OBJECTIVES: To determine whether changes in selenoprotein synthesis may be an underlying mechanism behind arsenic-induced carcinogenesis, we analyzed the new synthesis of selenoproteins within cells after exposure to inorganic or methylated arsenicals using a human keratinocyte cell model. RESULTS: Addition of arsenite to culture medium blocked new synthesis of selenoproteins when selenium was present in the form of selenite, and appeared to stimulate the use of serum-derived selenium. Monomethylarsonous acid (MMA(III)) treatment of cells, in contrast, did not block all new synthesis of selenoproteins but did result in an increase in cytosolic thioredoxin reductase (TrxR1) at both the mRNA and protein levels. MMA(III) also reduced the new synthesis of cellular glutatione peroxidase (cGpx) and other smaller selenoproteins. Dimethylarsinous acid (DMA(III)) stimulated selenoprotein synthesis by an as yet unknown mechanism. CONCLUSIONS: These results suggest that arsenite and MMA(III) are key metabolites that trigger higher levels of TrxR1, and both lead to a reduction in the expression of cGpx. Together these effects certainly could lead to carcinogenesis given the knowledge that many cancers have higher levels of TrxR, and reduced Gpx levels will reduce the cell's ability to defend against reactive oxygen species. Based on these results, the impact of the trivalent arsenicals arsenite and MMA(III) on selenoprotein synthesis may indeed represent a potential molecular mechanism for the higher rates of cancer observed in populations exposed to high levels of arsenic.

Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles.

In this report ceria nanoparticles are shown to act as catalysts that mimic superoxide dismutase (SOD) with the catalytic rate constant exceeding that determined for the enzyme SOD.

Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis.

Clostridium difficile is a significant concern as a nosocomial pathogen, and genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology/pathogenesis of the organisms can be studied. Here, we used TargeTron to investigate the role of selenoproteins in C. difficile Stickland metabolism and found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. This system rapidly and efficiently introduces site-specific mutations into the C. difficile genome (20-50% mutation frequency). The selD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTron selD mutation. Our findings suggest that Stickland metabolism could be a target for future antibiotic therapies and that the CRISPR-Cas9 system can introduce rapid and efficient modifications into the C. difficile genome.