Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin.

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

The Selenophosphate Synthetase Gene, selD, Is Important for Clostridioides difficile Physiology.

The endospore-forming pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and health care. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis, and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using transcriptome sequencing (RNA-seq), we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. IMPORTANCE C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile life cycle, other nonantibiotic treatment options are in urgent need of development. Toward this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology: Stickland metabolism. Here, we use our CRISPR-Cas9 system to "knock in" a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology, from vegetative growth to spore formation and outgrowth postgermination.

Coupled laboratory and field investigations resolve microbial interactions that underpin persistence in hydraulically fractured shales.

Hydraulic fracturing is one of the industrial processes behind the surging natural gas output in the United States. This technology inadvertently creates an engineered microbial ecosystem thousands of meters below Earth's surface. Here, we used laboratory reactors to perform manipulations of persisting shale microbial communities that are currently not feasible in field scenarios. Metaproteomic and metabolite findings from the laboratory were then corroborated using regression-based modeling performed on metagenomic and metabolite data from more than 40 produced fluids from five hydraulically fractured shale wells. Collectively, our findings show that Halanaerobium, Geotoga, and Methanohalophilus strain abundances predict a significant fraction of nitrogen and carbon metabolites in the field. Our laboratory findings also exposed cryptic predatory, cooperative, and competitive interactions that impact microorganisms across fractured shales. Scaling these results from the laboratory to the field identified mechanisms underpinning biogeochemical reactions, yielding knowledge that can be harnessed to potentially increase energy yields and inform management practices in hydraulically fractured shales.

Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway.

Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO2 molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a ΔacsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD+ "acetobutyrogenesis," requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile, explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon.IMPORTANCEClostridium difficile is an anaerobic, multidrug-resistant, toxin-producing pathogen with major health impacts worldwide. It is the only widespread pathogen harboring a complete set of Wood-Ljungdahl pathway (WLP) genes; however, the role of the WLP in C. difficile is poorly understood. In other anaerobic bacteria and archaea, the WLP can operate in one direction to convert CO2 to acetic acid for biosynthesis or in either direction for energy conservation. Here, conditions are defined in which WLP levels in C. difficile increase markedly, functioning to support metabolism of carbohydrates. Amino acid nutritional requirements were better defined, with new insight into how the WLP and butyrate pathways act in concert, contributing significantly to energy metabolism by a mechanism that may have broad physiological significance within the group of nonclassical acetogens.

Growth-associated catabolic potential of Acetoanaerobium sticklandii DSM 519 on gelatin and amino acids.

Acetoanaerobium sticklandii DSM 519 is a hyperammonia-producing anaerobe that catabolizes proteins and amino acids into organic solvents and volatile acids via the Stickland reactions. However, the specific growth rate and metabolic capability of this organism on proteins and amino acids are not yet known. Therefore, the present study was intended to evaluate its specific growth rate and metabolic potential on gelatin and amino acids in the experimental media. We carried out metabolic assay experiments to calculate its ability to utilize pure gelatin, single amino acids, and amino acid pairs at different growth phases. The results of this study show that complete assimilation of gelatin was achieved by its log-phase culture. The subsequent fermentation of amino acids was much faster than gelatin hydrolysis. The rate of gelatin degradation was associated with the growth and catabolic rates of this organism. Many amino acids were not assimilated completely for its growth and energy conservation. A log-phase culture of this organism preferably utilized l-cysteine, l-arginine, and l-serine, and released more fraction of ammonia. As shown by our analysis, the catabolic rates of these amino acids were determined by the rates of respective enzymes involved in amino acid catabolic pathways and feedback repression of ammonia. The growth kinetic data indicated that at the initial growth stage, a metabolic shift in its solventogenesis and acidogenesis phases was associated with catabolism of certain amino acids. Thus, the results of this study provide a new insight to exploit its log-phase culture as a starter for the production of biofuel components from gelatin processing industries.

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519.

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.

Clostridioides difficile colonization is not mediated by bile salts and requires Stickland fermentation of proline in an in vitro model of infection.

Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.

Endopeptidase activities of Clostridium botulinum toxins in the development of this bacterium.

By-products like CO2 and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation.

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes.

Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for C. difficile infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in C. difficile cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic C. difficile, confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, via its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with C. difficile for proline and therefore restore colonization resistance against C. difficile in the gut.

The Stickland Reaction Precursor trans-4-Hydroxy-l-Proline Differentially Impacts the Metabolism of Clostridioides difficile and Commensal Clostridia.

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, trans-4-hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild-type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for energy metabolism. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important in determining their role in nutrient competition against C. difficile. IMPORTANCE Proline is an essential environmental amino acid that C. difficile uses to support growth and cause significant disease. A posttranslationally modified form, hydroxyproline, is highly abundant in collagen, which is degraded by host proteases in response to C. difficile toxin activity. The ability to dehydrate hydroxyproline via the HypD glycyl radical enzyme is widespread among gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. We found that C. difficile and three commensal Clostridia strains had significant, but different, alterations to their metabolic gene expression in the presence of hydroxyproline in vitro. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche. Further elucidation of the differences between them in the presence of hydroxyproline and other key nutrients will be important to determining their role in nutrient competition against C. difficile.

Gut associated metabolites and their roles in Clostridioides difficile pathogenesis.

The nosocomial pathogen Clostridioides difficile is a burden to the healthcare system. Gut microbiome disruption, most commonly by broad-spectrum antibiotic treatment, is well established to generate a state that is susceptible to CDI. A variety of metabolites produced by the host and/or gut microbiota have been shown to interact with C. difficile. Certain bile acids promote/inhibit germination while other cholesterol-derived compounds and amino acids used in the Stickland metabolic pathway affect growth and CDI colonization. Short chain fatty acids maintain intestinal barrier integrity and a myriad of other metabolic compounds are used as nutritional sources or used by C. difficile to inhibit or outcompete other bacteria in the gut. As the move toward non-antibiotic CDI treatment takes place, a deeper understanding of interactions between C. difficile and the host's gut microbiome and metabolites becomes more relevant.

The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation.

The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.

Kinetic modeling of Stickland reactions-coupled methanogenesis for a methanogenic culture.

Studying amino acid catabolism-coupled methanogenesis is the important standpoints to decipher the metabolic behavior of a methanogenic culture. L-Glycine and L-alanine are acted as sole carbon and nitrogen sources for acidogenic bacteria. One amino acid is oxidized and another one is reduced for acetate production via pyruvate by oxidative deamination process in the Stickland reactions. Herein, we have developed a kinetic model for the Stickland reactions-coupled methanogenesis (SRCM) and simulated objectively to maximize the rate of methane production. We collected the metabolic information from enzyme kinetic parameters for amino acid catabolism of Clostridium acetobutylicum ATCC 824 and methanogenesis of Methanosarcina acetivorans C2A. The SRCM model of this study consisted of 18 reactions and 61 metabolites with enzyme kinetic parameters derived experimental data. The internal or external metabolic flux rate of this system found to control the acidogenesis and methanogenesis in a methanogenic culture. Using the SRCM model, flux distributions were calculated for each reaction and metabolite in order to maximize the methane production rate from the glycine-alanine pair. Results of this study, we demonstrated the metabolic behavior, metabolite pairing while mutually interact, and advantages of syntrophic metabolism of amino acid-directed methane production in a methanogenic starter culture.

Metabolism the Difficile Way: The Key to the Success of the Pathogen Clostridioides difficile.

Strains of Clostridioides difficile cause detrimental diarrheas with thousands of deaths worldwide. The infection process by the Gram-positive, strictly anaerobic gut bacterium is directly related to its unique metabolism, using multiple Stickland-type amino acid fermentation reactions coupled to Rnf complex-mediated sodium/proton gradient formation for ATP generation. Major pathways utilize phenylalanine, leucine, glycine and proline with the formation of 3-phenylproprionate, isocaproate, butyrate, 5-methylcaproate, valerate and 5-aminovalerate. In parallel a versatile sugar catabolism including pyruvate formate-lyase as a central enzyme and an incomplete tricarboxylic acid cycle to prevent unnecessary NADH formation completes the picture. However, a complex gene regulatory network that carefully mediates the continuous adaptation of this metabolism to changing environmental conditions is only partially elucidated. It involves the pleiotropic regulators CodY and SigH, the known carbon metabolism regulator CcpA, the proline regulator PrdR, the iron regulator Fur, the small regulatory RNA CsrA and potentially the NADH-responsive regulator Rex. Here, we describe the current knowledge of the metabolic principles of energy generation by C. difficile and the underlying gene regulatory scenarios.

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production.

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity.

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

Oxidative Stickland reactions in an obligate aerobic organism - amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus.

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Acidogenic fermentation characteristics of different types of protein-rich substrates in food waste to produce volatile fatty acids.

In this study, tofu and egg white, representing typical protein-rich substrates in food waste based on vegetable and animal protein, respectively, were investigated for producing volatile fatty acids (VFAs) by acidogenic fermentation. VFA production, composition, conversion pathways and microbial communities in acidogenesis from tofu and egg white with and without hydrothermal (HT) pretreatment were compared. The results showed HT pretreatment could improve the VFA production of tofu but not for egg white. The optimum VFA yields were 0.46g/gVS (tofu with HT) and 0.26g/gVS (egg white without HT), respectively. Tofu could directly produce VFAs through the Stickland reaction, while egg white was converted to lactate and VFAs simultaneously. About 30-40% of total protein remained in all groups after fermentation. Up to 50% of the unconverted soluble protein in the HT groups was protease. More lactate-producing bacteria, mainly Leuconostoc and Lactobacillus, were present during egg white fermentation.