Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D‐proline reductase (PR), a Stickland reaction that regenerates NAD⁺ from NADH. Many Clostridium spp. use Stickland metabolism (co‐fermentation of pairs of amino acids) to generate ATP and NAD⁺. Synthesis of PR is activated by PrdR, a proline‐responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD⁺‐generating pathways, such as the glycine reductase and succinate‐acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox‐sensing regulator that responds to the NAD⁺/NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD⁺ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR‐regulated Stickland metabolism in the virulence of C. difficile.     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-alpha-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

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.

     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-α-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress

Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.     

Site-directed mutational analysis of the novel catalytic domains of <Emphasis Type="Italic">α</Emphasis>-aminoadipate reductase (Lys2p) from <Emphasis Type="Italic"> Candida albicans</Emphasis>

The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.     

Amino acid requirements of Eikenella corrodens

The amino acid requirements of asaccharolytic Eikenella corrodens strains were investigated and a minimal amino acid medium was developed. Single amino acid deletions performed in a chemically defined medium indicated that these strains required arginine, cysteine, histidine, lysine, and proline, and partially required tyrosine. These six amino acids plus aspartic acid, glutamic acid, and glycine supported growth of E. corrodens in a medium containing only inorganic salts and vitamins.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

Mapping interactions between germinants and Clostridium difficile spores

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.     

Influence of growth conditions on glycine reductase of Clostridium sporogenes.

Cells of Clostridium sporogenes were deficient in glycine reductase activity when grown in a rich medium containing 40 mM each of exogenously added pyruvate and proline or hydroxyproline. These cells lacked the selenoprotein and at least one more protein of the glycine reductase system. Proline or hydroxyproline in the medium also influenced the uptake of glycine by the cells.     

Transport of glycine,serine, and proline into spinach leaf mitochondria

The transport of radioactive glycine, serine, and proline into the matrix of spinach leaf mitochondria was studied using the silicone oil centrifugation technique. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mm, an apparent diffusion process dominated. The uptake was not saturable at increasing amino acid concentrations, and there was no accumulation of amino acid in the matrix (i.e., concentration was similar to that in the medium). At concentrations lower than 0.5 mm, in addition to the diffusion process, an active uptake system that accumulated amino acid in the matrix became apparent. This system was partially inhibited by rotenone, antimycin A, and carbonylcyanide-m-chlorophenyl hydrazone. Also, uptake of glycine and serine was mutually inhibitory. These two amino acids exhibited comparatively less inhibitory effect on proline uptake, and proline also did not inhibit glycine or serine uptake. The results suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity. The diffusion process dominates at amino acid concentrations of 0.5 mm or higher, whereas the active uptake system becomes more prominent as the amino acid concentration decreases.     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.     

δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ¹-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase

The activity of dye-linked d-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicum JCM 9189. The dye-linked d-proline dehydrogenase was a membrane associated enzyme and was solubilized from the membrane fractions by treatment with Tween 20. The solubilized enzyme was purified 34-fold in the presence of 0.1% Tween 20 by four sequential chromatographies. The enzyme has a molecular mass of about 145 kDa and consisted of homotetrameric subunits with a molecular mass of about 42 kDa. The N-terminal amino acid sequence of the subunit was MKVAIVGGGIIGLFTAYHLRQQGADVVI. The enzyme retained its full activity both after incubation at 80 degrees C for 10 min and after incubation in the range of pH 4.0-10.0 at 50 degrees C for 10 min. The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, and d-proline was the most preferred substrate among the d-amino acids. The Michaelis constants for d-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively. Delta(1)-Pyrroline-2-carboxylate was identified as the reaction product from d-proline by thin layer chromatography. The prosthetic group of the enzyme was identified to be FAD by high-performance liquid chromatography. The gene encoding the enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the dye-linked d-proline dehydrogenase gene was determined and encoded a peptide of 363 amino acids with a calculated molecular weight of 40,341. The amino acid sequence of the Pb. islandicum enzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those of d-alanine dehydrogenases from the mesophiles so far reported. This shows that the membrane-bound d-proline dehydrogenase from Pb. islandicum is a novel FAD-dependent amino acid dehydrogenase.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.     

Identification and characterization of an extracellular protease activity produced by the marine Vibriosp 60

Abstract The marine fish pathogen Vibrio sp. 60 has been used as a host for heterologous expression of the Escherichia coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension. In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-cooH from the small subunit of herpes simplex virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain. Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor. Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity. The above observation is relevant for the biotechnological exploitation of Vibrio sp. 60.