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Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive in fections in populations at risk. There is at present an unmet need for preventive vaccines. Methods: IRTA GN-3728 genome was sequenced by Illumina and D-glutamate and D-glutamate/D-alanine knockout-auxotrophs were constructed. They were characterized using electron mi croscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. Results: The D-glutamate auxotroph, IRTA DmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA DmurI Dalr DdadX double auxotroph, con structed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effec tive against systemic disease. In the intestinal model, it proved to be safe, yielding a low degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homol ogous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. Conclusion: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans. This study was supported by a grant from SERGAS (The Galician Healthcare Service) (Programs “InnovaSaude” and “InnovaMicrolab”), the Spanish Network for Research in Infectious Diseases (RD16/0016/0006), CIBERINFEC, project PI18/00501, funded by Instituto de Salud Carlos III and co funded by European Union (ERDF, “A way to make Europe”), and PI21/00704, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, awarded to GB, and by CZ Vaccines. VFV is funded by a predoctoral fellowship from Xunta de Galicia (IN606A-2019/012). Sí

International audience; As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers' design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a] 2-SHa and [G10a] 3-SHa, and [G10a] 2-SHa conjugated with a polymer molecule, i.e., Jeff-[G10a] 2-SHa, were synthesized, purified on RP-HPLC and UPLC and fully characterized by mass, NMR spectroscopic techniques, circular dichroism, ultraviolet, infrared, dynamic light scattering, and atomic force microscopic studies. In pH-and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40-60 • C and pH values in the range of 6-12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs (breast, lung, prostate, pancreas, and liver). The high hemolytic activity of [G10a] 2-SHa and [G10a] 3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a] 2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.

The resistance of Lactobacillus plantarum to vancomycin depends on its peptidoglycan composition. Vancomycin has poor binding affinity with peptidoglycan precursors ending in D-alanyl-D-lactate (D-Ala-D-Lac) but binds strongly to peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala), resulting in resistance and sensitivity, respectively. The ligase Ddl, which generates D-Ala-D-Lac or D-Ala-D-Ala incorporated into the peptidoglycan precursor chain, is responsible for this specificity. To study the effect of peptidoglycan precursors on immunity, we constructed several strains of L. plantarum expressing the ddl gene of Lactococcus lactis to change their peptidoglycan precursors. The change in the termini of the peptidoglycan precursors was determined by the sensitivity of the strains to vancomycin. The overexpression of ddl increased the susceptibility of the strains to vancomycin. We further explored the regulation of the macrophage inflammatory response pathway by the wild-type and constructed strains, and found that these strains induced the MyD88-dependent TRAF6/MAPK pathway, and the increase in D-Ala L. plantarum peptidoglycan precursors increased the secretion of the inflammatory factors IL-6, IL-1β and TNF-α. These results indicate that D-Ala-ended peptidoglycan precursors play a central role in the variable immunomodulatory ability of L. plantarum.

Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present study evaluated the mechanism of actions of highly potent AMP temporin-SHa analogs, i.e., [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa, against methicillin-resistant Staphylococcus aureus (MRSA) NCTC (13277) with minimum inhibitory concentrations (MICs) of 14.35, 7.16, and 3.58 µM, respectively. These analogs exhibited significant anti-MRSA activity at physiological salt concentration, 30% fetal bovine serum, and 30% human serum. [G4a]-SHa and [G7a]-SHa were non-hemolytic and non-cytotoxic to normal mouse fibroblast 3T3 cell and human Caco-2 cell line. Atomic force microscopy revealed that these analogs have profound effect on the morphological changes in MRSA surface with significant leakage of cell cytoplasmic content. Propidium iodide uptake kinetic assay and (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) DiBAC 4 (3) membrane depolarization assay demonstrated that these analogs display a membrane disrupting property, characterized by elevation of plasma membrane permeability and rapid transmembrane potential depolarization. [G10a]-SHa showed a significant anti-biofilm activity against biofilm forming S. aureus (ATCC 6538). Acute in vivo toxicity studies revealed that [G10a]-SHa possesses some toxic effect at 100-mg/kg dose. While [G4a]-SHa at 100 mg/ kg, i.p. has no toxic effect even after 48 h, [G7a]-SHa also did not show any toxic effect at the dose of 100 mg/kg, i.p. during 24-h observation of animals. In conclusion, [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa show improved activity against MRSA and stability compared to SHa peptide. Although highly potent, [G10a]-SHa, due to its hemolytic activity, might be more suitable for topical application, whereas [G4a]-SHa and [G7a]-SHa have potential to be used for systemic application.

d-amino acids produced by Lactobacillus are thought to contribute to the taste quality and health functions; however, no studies have comprehensively evaluated the concentrations of the D- and L-forms of amino acids separately in individual Lactobacillus strains. To gain insight into amino acid concentrations in Lactobacillus, we evaluated amino acid concentrations in culture broth of Lactobacillus separately for the D- and L-forms. Lactobacillus strains were cultured in culture broth, and the amino acid concentrations in supernatant were assessed. The amino acid concentrations obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were subjected to cluster analysis based on Bray-Curtis distance with Ward's minimum variance method. In the analysis of amino acid concentrations under culture with different monosaccharides, the distances among strains cultured with the same monosaccharide were significantly greater than those among cultures of the same strain under different monosaccharides (p

Life in a three-dimensional biofilm is typical for many bacteria, yet little is known about how strains interact in this context. Here, we created essential gene CRISPR interference knockdown libraries in biofilm-forming Bacillus subtilis and measured competitive fitness during colony co-culture with wild type. Partial knockdown of some translation-related genes reduced growth rates and led to out-competition. Media composition led some knockdowns to compete differentially as biofilm versus non-biofilm colonies. Cells depleted for the alanine racemase AlrA died in monoculture but survived in a biofilm colony co-culture via nutrient sharing. Rescue was enhanced in biofilm colony co-culture with a matrix-deficient parent due to a mutualism involving nutrient and matrix sharing. We identified several examples of mutualism involving matrix sharing that occurred in three-dimensional biofilm colonies but not when cultured in two dimensions. Thus, growth in a three-dimensional colony can promote genetic diversity through sharing of secreted factors and may drive evolution of mutualistic behavior.

Listeria monocytogenes is a Gram‐positive, intracellular pathogen harboring the surface‐associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane‐anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface‐associated InlB non‐covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen‐defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor., Molecular Microbiology, 113 (3), ISSN:0950-382x, ISSN:1365-2958

The relevance of the homochirality of proteinogenic amino acids for life is undisputed, but a growing number of biological functions could be assigned to the non-proteogenic D-enantiomers. When it comes to D-AAs in plants, information was relatively scarce for a long time. Recently, it has been shown that D-AAs are taken up by plants from soil, but could also be synthesised de novo, and that D-AAs cause physiological e ects in plants. These were indications that D-AAs are responsible for a variety of yet poorly understood or even undiscovered functions in plants. For this reasons, I performed experiments to analyse transport, metabolism and functions of these molecules in Arabidopsis thaliana. Previously, it was shown that D-AAs are taken up and utilised by plants, but how the plant reduces excessive amounts of them still remained unclear. Analyses of plant D-AA content after D-Ala and D-Glu treatment, opened the question if exudation of D-AAs into the rhizosphere takes place, and whether it plays a role in the reduction of D-AA content in plants. Exudation of D-Ala and D-Glu could be confi rmed by AA analyses of growth media from seedlings treated with these D-AAs. Further tests revealed that other D-AAs are also secreted. Exudation experiments with transport inhibitors revealed that root exudation of D-AAs is rather passive and comparable to that of L-AAs, whereas uptake of D-AAs was found to be active. It was also found that plants are able to reduce their D-AA contents by means other than exudation, and therefore metabolic processes were postulated. Analyses of the D-AA speci c transaminase (AtDAT1) revealed that this enzyme is responsible for the metabolisation of the majority of tested D-AAs in Arabidopsis thaliana. The results of our biochemical experiments revealed that D-Met is the preferred substrate for this enzyme, with D-Ala as the major product. Furthermore, the loss of this enzyme led to a triple response and increased ethylene synthesis after D-Met treatment, indicating a functional relation between regulation of D-AA metabolism and plant development. These results show that AtDAT1 is a crucial enzyme in the D-AA metabolism in Arabidopsis thaliana and that these molecules have a relevant function in higher plants, contrary to what was communally accepted. Die Bedeutung der Homochiralität proteinogener Aminosäuren ist unbestritten, jedoch konnte auch den nicht-proteinogenen D-Enantiomeren eine wachsende Anzahl biologischer Funktionen zugeordnet werden. Über die Rolle, die D-Aminos auren (D-AAs) in Pflanzen spielen, gibt es bislang relativ wenige Informationen. Kürzlich konnte gezeigt werden, dass Pflanzen D-AAs einerseits aus dem Boden aufnehmen, andererseits de novo synthetisieren k onnen und dass D-AAs physiologische Effekte in Pflanzen bewirken. Dies weist darauf hin, dass D-AAs für eine Vielzahl von Funktionen in Pflanzen verantwortlich sein können, die bislang wenig verstandenen oder sogar noch nicht beschrieben sind. Aus diesen Gründen habe ich Experimente durchgeführt, die den Transport, den Metabolismus und die Funktion dieser Moleküle in Arabidopsis thaliana analysieren. In vorherigen Untersuchungen wurde gezeigt, dass D-AAs aufgenommen und verwertet werden, aber wie Pflanzen überschüssige Mengen von D-AAs reduzieren, ist bislang unklar. Konzentrationsbestimmungen von D-AAs nach Verabreichung von D-Ala und D-Glu warfen die Frage auf, ob D-AAs in die Rhizosphere exsudiert werden und ob dieser Vorgang eine Rolle für die Konzentrationsabnahme innerhalb der Pflanze spielt. Bei Keimlingen in Flüssigkultur, die mit D-Ala und D-Glu behandelt wurden, konnten diese Aminosäuren im Außenmedium nachgewiesen werden, was eine Exsudation bestätigt. Weitere Experimente zeigten, dass auch andere D-AAs ins Medium sekretiert wurden. Experimente mit Transportinhibitoren zeigten, dass die Exsudation passiv erfolgt und vergleichbar mit der Exsudation von L-AAs ist, während die Aufnahme von D-AAs einen aktiven Vorgang darstellt. Pflanzen sind nicht nur in der Lage, die Konzentration von D-AAs durch Exsudation zu reduzieren, sondern können sie auch in metabolischen Prozessen verwenden. Die Analyse der D-AA-spezifischen Transaminase (AtDAT1) ergab, dass dieses Enzym für den größten Teil des Metabolismus der D-AAs in Arabidopsis zuständig ist. Unsere biochemische Charakterisierung von AtDAT1 ergab, dass D-Met das bevorzugte Substrat darstellt während D-Ala das Hauptprodukt ist. Ein Verlust der Enzymfunktion führt zu einer Triple Response und verstärkter Ethylen-Synthese nach D-Met-Behandlung, was auf eine funktionelle Verbindung zwischen der Regulation des D-AA-Metabolismus und der P anzenentwicklung hinweist. Diese Ergebnisse zeigen, dass AtDAT1 ein zentrales Enzym des D-AAMetabolismus in Arabidopsis ist und dass D-AAs, anders als bisher angenommen, eine wichtige Funktion in höheren Pflanzen ausüben.

Staphylococcus aureus causes various diseases including skin and soft tissue infections, pneumonia, gastroenteritis, and sepsis. Antibiotic-resistant S. aureus such as methicillin-resistant S. aureus (MRSA) and multidrug-resistant S. aureus is a serious threat in healthcare-associated settings and in the communities. In this study, we investigated the effects of short-chain fatty acids, metabolites produced by commensal bacteria, on the growth of S. aureus both in vitro and in vivo. Sodium propionate (NaP) most potently inhibited the growth of MRSA and multidrug-resistant clinical isolates. Of note, only NaP, but not sodium acetate (NaA) or sodium butyrate (NaB), ameliorated MRSA skin infection, significantly lowering bacterial load, excessive cytokine production, and the size and weight of abscesses approximately by twofold. In addition, interestingly, S. aureus deficient of lipoteichoic acids (LTA) or wall teichoic acids (WTA), which are important in bacterial physiology and antimicrobial susceptibility, was more susceptible to NaP than the wild-type. Furthermore, S. aureus deficient of D-alanine motifs common in LTA and WTA was more susceptible to NaP, its growth being almost completely inhibited. Concordantly, MRSA treated with an inhibitor of D-alanylation on LTA and WTA was more susceptible to NaP, and co-treatment of NaP and a D-alanylation inhibitor further decreased the pathology of MRSA skin infection. Collectively, these results demonstrate that NaP ameliorates MRSA skin infection by attenuating the growth of S. aureus, and suggest an alternative combination treatment strategy against S. aureus infection.

Glycine and related endogenous compounds (d-serine, d-alanine, sarcosine) serve critical roles in both excitatory and inhibitory neurotransmission and are influenced by a multitude of enzymes and transporters, including glycine transporter 1 and 2 (GlyT1 and GlyT2), d-amino acid oxidase (DAAO), serine racemase (SRR), alanine-serine-cysteine transporter 1 (Asc-1), and kynurenine aminotransferase (KAT). MEDLINE, Web of Science, and PsychINFO were searched for relevant human trials of compounds. Many studies utilizing exogenous administration of small molecule agonists of the glycineB site of n-methyl-d-aspartate receptor have been studied as have a growing number of glycine transporter type 1 (GlyT1) inhibitors. The clinical effects of these compounds are reviewed as are the potential effects of newer novel compounds.

D-alanine (D-Ala) is an essential amino acid that has a key role in bacterial cell wall synthesis. Alanine racemase (Alr) is a unique enzyme that interconverts L-alanine and D-alanine in most bacteria, making this enzyme a potential target for antimicrobial drug development. Streptococcus mutans is a major causative factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Alr in S. mutans. We constructed alr mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that alr deletion was lethal to S. mutans. A minimal supplement of D-Ala (150 μg·mL−1) was required for the optimal growth of the alr mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the alr mutant strain. We also determined the compromised competitiveness of the alr mutant strain relative to the wild-type S. mutans against other oral streptococci (S. sanguinis or S. gordonii), demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of alr to the growth and competitiveness of S. mutans, Alr may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries. An enzyme found to be crucial to the growth and survival of a bacterium found in the mouth could be targeted to prevent tooth decay. The Streptococcus mutans bacterium is a major cause of tooth decay; fed by high sugar intake, it generates acid that leads to tooth demineralization. Ming-Yun Li and co-workers at Sichuan University in Chengdu, China, have demonstrated that an enzyme called alanine racemase (Alr) is crucial to the growth and maintanance of S. mutans cell wall structure. The team created alr mutant strain of the bacterium, and discovered that without the enzyme the bacterium cell wall tore easily, leading to cell disintegration. They also found that deleting Alr significantly reduced the bacterium's ability to compete effectively with other oral bacteria. The manipulation of the enzyme in oral biofilm could help reduce tooth decay.

As a complement to the renowned bicyclic beta-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-beta-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of alpha-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four beta-lactamase classes was observed, while weak inhibition of class C beta-lactamase P99 was demonstrated.

Lantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction of d -alanine in the lantibiotic lacticin 3147. The conversion of l -serine into d -alanine requires dehydroalanine as the substrate, which is produced in vivo by the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ in Lactococcus lactis . We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of the ltnJ gene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.

Nitrogen incorporation into total particulate suspended matter, hydrolysable amino acids and bacterial biomarker d-Alanine was assessed seasonally in the coastal North Sea using 15N-labeled ammonium, nitrate, nitrite and 15N- and 13C-labeled urea, glycine, leucine, phenylalanine, and two complex pools of dissolved organic matter (DOM) derived from algal and bacterial cultures (A-DOM, B-DOM). We investigated: 1) uptake rates for the various substrates and their contribution to total N uptake; 2) microbial preferences for the different N sources; 3) the coupling of C and N uptake from organic substrates; 4) the contribution of bacteria to the total microbial uptake of these substrates, and 5) the role of a complex pool of organic matter for plankton nutrition. Seasonality in the preferences for N substrates was observed, with A-DOM and B-DOM being preferred in autumn and winter whereas NH4+ was preferentially taken up in spring and summer. C and N uptake was coupled for all the organic substrates, except urea that was mainly used as a nitrogen source in summer and spring. Bacterial contribution to the uptake of A-DOM and B-DOM was, on an annual average, the lowest among the N-substrates. This suggests an important role for phytoplankton in the incorporation of complex organic matter and the importance of DOM for phytoplankton nutrition.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses. Several studies have suggested that free D-Asp has a crucial role in N-methyl D-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free D-Asp, L-Asp and N-methyl D-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing L-Asp, D-Asp and N-methyl D-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for D-Asp, 0.46 pg/μl for L-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for D-Asp, 1.41 pg/μl for L-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of D-Asp, L-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free D-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Lanthipeptides are peptides that contain several post-translationally modified amino acid residues and commonly show considerable antimicrobial activity. After translation, the amino acid residues of these peptides are modified by a distinct set of modification enzymes. This process results in peptides containing one or more lanthionine rings and dehydrated Ser and Thr residues. Previously, an in vivo lanthipeptide production system based on the modification machinery of the model lantibiotic nisin was reported. Here, we present the addition of the modification enzymes LtnJ and GdrnD to this production system. With these enzymes we can now produce lanthipeptides that contain D-alanines or a C-terminal aminovinyl-cysteine. We show experimentally that the decarboxylase GdmD is responsible for the C-terminal decarboxylation. Our results demonstrate that different lanthipeptide modification enzymes can work together in an in vivo production system. This yields a plug-and-play system that can be used to select different sets of modification enzymes to work on diverse, specifically designed substrates.

Objectives The naturally occurring polyphenol (−)-epicatechin gallate (ECg) increases oxacillin susceptibility in mecA-containing strains of Staphylococcus aureus. Decreased susceptibility to lysostaphin suggests alterations to the wall teichoic acid (WTA) content of ECg-grown bacteria. Changes in WTA structure in response to ECg were determined. Methods Nuclear magnetic resonance spectroscopy of purified monomers from S. aureus was used to elucidate WTA structures. Molecular modelling of WTA chains was employed to determine their spatial configuration. Results ECg-grown methicillin-resistant S. aureus (MRSA) strains BB568 and EMRSA-16 displayed markedly reduced resistance to oxacillin, had thickened cell walls and separated poorly. Growth in ECg-supplemented medium reduced the substitution of the WTA backbone by d-alanine (d-Ala); ratios of N-acetyl glucosamine to d-Ala were reduced from 0.6 and 0.49 (for BB568 and EMRSA-16) to 0.3 and 0.28, respectively. Molecular simulations indicated a decrease in the positive charge of the bacterial wall, confirmed by increased binding of cationized ferritin, and an increase in WTA chain flexibility to a random coil conformation. Conclusions Structural elucidation and molecular modelling of WTA indicated that conformational changes associated with reduced d-Ala substitution may contribute to the increased susceptibility of MRSA to β-lactam antibiotics and account for other elements of the ECg-induced phenotype.

Nisin is a post-translationally modified antimicrobial peptide produced by Lactococcus lactis which binds to lipid II in the membrane to form pores and inhibit cell-wall synthesis. A nisinresistant (NisR) strain of L. lactis, which is able to grow at a 75-fold higher nisin concentration than its parent strain, was investigated with respect to changes in the cell wall. Direct binding studies demonstrated that less nisin was able to bind to lipid II in the membranes of L. lactis NisR than in the parent strain. In contrast to vancomycin binding, which showed ring-like binding, nisin was observed to bind in patches close to cell-division sites in both the wild-type and the NisR strains. Comparison of modifications in lipoteichoic acid of the L. lactis strains revealed an increase in D-alanyl esters and galactose as substituents in L. lactis NisR, resulting in a less negatively charged cell wall. Moreover, the cell wall displays significantly increased thickness at the septum. These results indicate that shielding the membrane and thus the lipid II molecule, thereby decreasing abduction of lipid II and subsequent pore-formation, is a major defence mechanism of L. lactis against nisin., JRC.I.2-In-vitro Toxicology

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly.D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L- alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

Nitrogen flows through bacteria and algae in aquatic microbial communities are an important part of the nitrogen cycle, which plays a central role in aquatic ecosystems. However, work on uptake and retention of nitrogen in bacteria versus algae in natural microbial communities has long been hampered by a lack of adequate methodology, especially in turbid waters and sediments. This thesis deals with the development, validation and application of a new method for analysis of nitrogen and carbon flows through bacteria and algae in aquatic microbial communities. The method concerns analysis of incorporation of the stable isotopes 15N and 13C in hydrolysable amino acids (HAAs), including the bacterial biomarker D-alanine (D-Ala) in combination with stable isotope labeling. After detailed presentation and assessment of the analytical aspects of the method, the method was applied in three different stable isotope labeling studies: In the first study, we investigated the short term (24 h) incorporation of nitrogen and carbon from urea and amino acids by incubating surface sediment samples from the Scheldt Estuary with dual labeled (15N and 13C) urea and amino acids. Analysis of 15N and 13C incorporation into THAAs yielded some interesting results concerning the uptake dynamics of the two substrates and the underlying mechanisms (including uncoupling of 15N and 13C uptake and incorporation) while analysis of 15N and 13C incorporation into D-Ala revealed a small to negligible bacterial contribution to total microbial 15N incorporation. The second study concerns an in situ 15N pulse chase experiment investigating the incorporation and retention of nitrogen in the sediment of the subtropical Brunswick Estuary (Australia). Analysis of 15N in HAAs over a 30 day period revealed very efficient recycling of 15N by the microbial community within the sediment with a major role for bacteria. In the third study, the method was used to investigate the fate of peptidoglycan, a bacterial cell wall component, in sediment. The bacterial community of an intertidal mudflat in the Scheldt Estuary was labeled with 13C and we subsequent traced the fate of the 13C in various bacterial cell components (D-Ala (specific for peptidoglycan), THAAs (representing bulk proteinaceous material) and bacteria-specific PLFAs (representing cell membranes of living bacteria)) over a 4.5-month period in situ. Results revealed a relatively strong retention of 13C in D-Ala providing direct in situ evidence for relative accumulation of peptidoglycan during reworking and degradation of bacterial biomass in sediments. Altogether, the applications of the method presented in this thesis as well as those in other 15N- and/or 13C-labeling studies served as a thorough validation of the method which proved to be very suitable for its originally intended application (analysis of 15N incorporation by bacteria). Together with the additional features like analysis of 13C in bacteria and 15N and 13C in total (microbial) biomass, this makes the method a very useful, versatile new tool for studies on flows of nitrogen and carbon through bacteria and algae in aquatic microbial communities.

Nitrogen flows through bacteria and algae in aquatic microbial communities are an important part of the nitrogen cycle, which plays a central role in aquatic ecosystems. However, work on uptake and retention of nitrogen in bacteria versus algae in natural microbial communities has long been hampered by a lack of adequate methodology, especially in turbid waters and sediments. This thesis deals with the development, validation and application of a new method for analysis of nitrogen and carbon flows through bacteria and algae in aquatic microbial communities. The method concerns analysis of incorporation of the stable isotopes 15N and 13C in hydrolysable amino acids (HAAs), including the bacterial biomarker D-alanine (D-Ala) in combination with stable isotope labeling. After detailed presentation and assessment of the analytical aspects of the method, the method was applied in three different stable isotope labeling studies: In the first study, we investigated the short term (24 h) incorporation of nitrogen and carbon from urea and amino acids by incubating surface sediment samples from the Scheldt Estuary with dual labeled (15N and 13C) urea and amino acids. Analysis of 15N and 13C incorporation into THAAs yielded some interesting results concerning the uptake dynamics of the two substrates and the underlying mechanisms (including uncoupling of 15N and 13C uptake and incorporation) while analysis of 15N and 13C incorporation into D-Ala revealed a small to negligible bacterial contribution to total microbial 15N incorporation. The second study concerns an in situ 15N pulse chase experiment investigating the incorporation and retention of nitrogen in the sediment of the subtropical Brunswick Estuary (Australia). Analysis of 15N in HAAs over a 30 day period revealed very efficient recycling of 15N by the microbial community within the sediment with a major role for bacteria. In the third study, the method was used to investigate the fate of peptidoglycan, a bacterial cell wall component, in sediment. The bacterial community of an intertidal mudflat in the Scheldt Estuary was labeled with 13C and we subsequent traced the fate of the 13C in various bacterial cell components (D-Ala (specific for peptidoglycan), THAAs (representing bulk proteinaceous material) and bacteria-specific PLFAs (representing cell membranes of living bacteria)) over a 4.5-month period in situ. Results revealed a relatively strong retention of 13C in D-Ala providing direct in situ evidence for relative accumulation of peptidoglycan during reworking and degradation of bacterial biomass in sediments. Altogether, the applications of the method presented in this thesis as well as those in other 15N- and/or 13C-labeling studies served as a thorough validation of the method which proved to be very suitable for its originally intended application (analysis of 15N incorporation by bacteria). Together with the additional features like analysis of 13C in bacteria and 15N and 13C in total (microbial) biomass, this makes the method a very useful, versatile new tool for studies on flows of nitrogen and carbon through bacteria and algae in aquatic microbial communities.

Crystal structure of a heterochiral peptide, viz. Bac-Val(1)-D-Ala(2)-Leu(3)-Ala(4)-OMe with a D chiral residue in the second position of a sequence, has been determined [a = 40.44(1), b = 4.887(5), c = 15.381(5) Angstrom, beta = 109.6(1)degrees, space group C2, Z = 4, R = 0.11]. The peptide is in a parallel beta-sheet structure terminated by a distinct local bend. The structure is stabilised by N-H ... O as well as C-alpha-N ... O hydrogen bonds,The contiguous C-alpha-H ... O hydrogen bond observed in this structure is an unique observation. (C) Munksgaard 1997.

oz ÇÖZÜNEN MADDELERİN HETEROJEN BUZ ÇEKİRDE?İ OLUŞUM KİNETİ?İ ÜZERİNDEKİ ETKİLERİNİN DTK KULLANILARAK MODEL GIDA EMÜLSYONLARINDA ÇALIŞILMASI BUDAK, (Özilgen) Z. Sibel Doktora, Gıda Mühendisliği Bölümü Tez Yöneticisi: Prof. Dr. Ali ESİN Ortak Tez Yöneticisi: Prof. Dr. David S. Reid Eylül, 1995, 91 sayfa Bu çalışmada sucrose, D-alaninve potasyum nitratın heterojen buz kristali oluşumunun kinetiği üzerindeki etkisi, buz kristali çekirdeği oluşturucuları (Agl veya Pseudamanas syringae) ve farklı derişimlerde çözünen madde içeren model gıda emülsyonlarmda Diff erensiyel Tarama Kalorimetresi (DTK) kullanılarak çalışıldı. Homojen ve heterojen buz çekirdeği oluşumunun ölçülebilir hıza ulaştığı sıcaklıklar soğutarak tarama termogramlan ile belirlendi. P. syringae buz çekirdeği vıoluşumunu 263 K civarında, Agl (gümüş iyodür) ise daha düşük sıcaklıklarda yaklaşık 252 K de başlatmaktadır. Heterojen çekirdeklenme kinetiği izotermal şartlarda çalışıldı ve homojen çekirdeklenme için önerilen matematiksel anlatımlarla açıklanmaya çalışıldı. Çekirdek oluşum hızına sıcaklık etkisi, indirgenmiş sıcaklıklarla ifade edilen parametrenin üssel fonksiyonu olarak açıklandı. Sonuçlar çekirdek oluşturucunun cinsinin, çekirdeklenme hızının sıcaklıkla değişmesine çözünür madde etkisini tayin etmekde önemli olduğunu göstermiştir. Agl ile yapılan araştırmalar sonunda elde edilen model parametrelerinin aynı şartlarda P. syringae ile yapılan deneylerden elde edilen parametrelerden daha büyük olduğu görüldü. AgI/% 30 sucrose çözeltisi ile yapılan çalışmalarda, emülsyon özelliklerinin yüksek şeker konsantrasyonuna bağlı olarak bozulması veya Agl mn katalitik kısmının değişime uğramasından kaynaklanabilecek çok yüksek parametre değerleri elde edildi. P. syringae/B-alamn ve Agl/D-alanin sistemlerinde aminoasit konsantrasyonu değiştirilerek yapılan deneyler sonunda, model parametrelerinde çok az değişiklikler olduğu gözlendi. P. syringae/KNO^ sistemi ile yapılan araştırmalar, yüksek tuz konsantrasyonun P. syringaeln katalitik kısmında bulunan proteinlerin doğal yapısını bozabileceğini ve bunun heterojen çekirdeklenme hızını etkileyebileceğini göstermiştir. vuOrtama çözünen madde eklenmesine bağlı olan donma noktası ve heterojen çekirdeklenme sıcaklıkları düşüşleri arasında doğrusal ilişki bulundu. DTK deneyleri sonunda elde edilen parametreler, donma işlemlerinin tasarımında çekirdeklenme hızını tahmin etmek amacı ile kullanılabilecek empirik telafi denklemi (İn A = aB+b) ile ifade edilmiştir. Tasarım parametreleri olan A ve B' nin aynı deney şartlan altında bulunmuş olması gerekir. Bunun mümkün olmadığı durumlarda telafi denklemleri deneysel verilerin interpolasyonu için kullanılabilir. Anahtar kelimeler: Differansiyel tarama kalorimetresi, heterojen çekirdeklenme, buz çekirdeği oluşturucuları, donma noktası düşüşü, çekirdeklenme kinetiği, telafi denklemleri. vuı ABSTRACT THE USE OF DSC TO STUDY THE EFFECTS OF SOLUTES ON HETEROGENEOUS ICE NUCLEATION KINETICS IN MODEL FOOD EMULSIONS BUDAK (Özilgen), Z. Sibel Ph. D., Food Engineering Department Supervisor: Prof. Dr. Ali ESİN Co-Supervisor: Prof. Dr. David S. Reid September, 1995, 91 pages Effects of sucrose, D - alanine and potassium nitrate, on heterogeneous ice nucleation kinetics were studied with a differential scanning calorimeter (DSC) in model food emulsions by employing different solute concentrations and ice nucleants, silver iodide (Agl), and Pseudomonas syringae. Temperatures at which the homogeneous and heterogeneous nucleation processes achieved a measurable rate were located by using the cooling scan thermograms. Agl induces nucleation at a lower temperature, around 252 K, while P. syringae induces nucleation at higher temperatures around 263 mK. The kinetics of heterogeneous nucleation in the same sample were studied by an isothermal hold procedure. Heterogeneous nucleation rates were described with a mathematical expression in the same form as suggested by the classical homogeneous nucleation theory. Temperature effects on these rates were an exponential function of a parameter defined in terms of the reduced temperatures. Our results have confirmed that the type of the nucleation template was important in determining the temperature effects of the solutes on the heterogeneous nucleation rates. In the experiments with Agl, values of the model parameters were generally larger than those of the corresponding experiments with P. syringae. Observations with 30 % sucrose/Agl solutions indicated extremely high parameter values which might be caused by the change of the catalytic site of Agl molecules and variation of the emulsion properties by highly concentrated sucrose. With the P. syringae/B - alanine and Agl/D - alanine systems only modest changes were observed in the model parameters with changing D - alanine concentrations. Experimental data with the P. syringae/KNO% system suggested that the heterogeneous ice nucleation rates might be affected by denaturation of catalytic site proteins of P. syringae at high salt concentration. A linear relationship between freezing point depression and the reduction of heterogeneous ice nucleation IVtemperature was observed in association with the addition of solutes. Parameters obtained in the DSC experiments were described with an empirical compensation Equation of the form In A = aB + b, which may be used to predict the nucleation rates in design of the freezing processes. In such a design parameters A and B are required to be evaluated under the exact experimental conditions, which may not be always possible. The compensation Equation may be helpful for the interpolation of experimental data when there is no information available for the specific design conditions. Key words: Differential Scanning Calorimeter (DSC), heterogeneous nucleation, ice nucleants, freezing point depression, nucleation kinetics, compensation relation. v 91

The reactions of alkali hexanitrocobaltates(III) with DL-alanine, D-alanine, L-alanine, and β-alanine have been investigated. Thereby new classes of alkali dinitro-bis (aminoacido)cobaltates(III), M[CoAm 2 (NO 2 ) 2 ] were obtained. It was established that the products obtained contain two nitro groups in the cis position.

d-amino acid oxidase (DAO) is a peroxisomal enzyme that catalyzes the oxidative deamination of several neutral and basic d-amino acids to their corresponding alpha-keto acids. In most mammalian species studied, high DAO activity is found in the kidney, liver, brain and polymorphonuclear leukocytes, and its main function is to maintain low circulating d-amino acid levels. DAO expression and activity have been associated with acute and chronic kidney diseases and with several pathologies related to N-methyl-d-aspartate (NMDA) receptor hypo/hyper-function; however, its precise role is not completely understood. In the present study we show that DAO activity can be detected in vivo in the rat kidney using hyperpolarized d-[1-C-13]alanine. Following a bolus of hyperpolarized d-alanine, accumulation of pyruvate, lactate and bicarbonate was observed only when DAO activity was not inhibited. The measured lactate-to-d-alanine ratio was comparable to the values measured when the l-enantiomer was injected. Metabolites downstream of DAO were not observed when scanning the liver and brain. The conversion of hyperpolarized d-[1-C-13]alanine to lactate and pyruvate was detected in blood ex vivo, and lactate and bicarbonate were detected on scanning the blood pool in the heart in vivo; however, the bicarbonate-to-d-alanine ratio was significantly lower compared with the kidney. These results demonstrate that the specific metabolism of the two enantiomers of hyperpolarized [1-C-13]alanine in the kidney and in the blood can be distinguished, underscoring the potential of d-[1-C-13]alanine as a probe of d-amino acid metabolism.