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New inhibitors of the bacterial transferase MraY from

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

New inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraY

Purpose: The PBP4* is a Penicillin Binding Protein belonging to the class C of AmpH type whose function remains poorly understood. This study aimed to evaluate the biophysical and enzymatic properties of the Bacillus subtilis PBP4* to gain insights into its role in the context of bacterial cell wall recycling. Methods: To characterize the PBP4*, the full-length PBP4* and its N-terminal penicillin-binding domain have been produced in Escherichia coli and purified. Results: A comparison of biophysical properties has shown that both recombinant proteins are monomeric in solution and retain the same thermal stability. On the other hand, the D-alanine methyl esterase activity detected with the full-length PBP4* is impeded by the cleavage of the 92 amino acid C-terminal domain. The esterase activity of the full-length PBP4* strates a clear D-stereospecificity. The PBP4* is also active on B. subtilis cell walls bearing teichoic acids, compounds commonly substituted with D-alanine residues. Conclusions: Our results are in agreement with the hypothesis that PBP4* could play a role in recycling cell wall components, as previously suggested.

Understanding the molecular mechanism by which the signal of the presence of an antibiotic is transduced from outside to inside the bacterial cell is of fundamental interest for the beta-lactam antibiotic resistance problem, but remains difficult to accomplish. No approach has ever addressed entire penicillin receptors in a membrane environment. Here we describe a method to investigate the purified Bacillus licheniformis BlaR1 receptor -a membrane-bound penicillin receptor involved in beta-lactam resistance- embedded into a lipid bilayer in absence or presence of penicillin. By selecting a mutated receptor blocked in its signal transduction pathway just after its activation by penicillin, we revealed the very first step of receptor signalling by unfolding the receptor from its C-terminal end by AFM-based single-molecule force spectroscopy. We showed that the presence of the antibiotic entails significant conformational changes within the receptor. Our approach opens an avenue to study signal-transduction pathways mediated by membrane-bound proteins in a membrane environment.

Monocyclic β-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-β-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically.

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the β1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.

The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background. Fil: Di Gregorio, Sabrina Noelia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fernandez, Silvina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina Fil: Cuirolo, Arabela Ximena. Université de Liège; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Verlaine, Olivier. Université de Liège; Bélgica Fil: Amoroso, Ana. Université de Liège; Bélgica Fil: Mengin Lecreulx, Dominique. Université Paris Sud; Francia Fil: Famiglietti, Angela María Rosa. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Joris, Bernard. Université de Liège; Bélgica Fil: Mollerach, Marta Eugenia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Herein, we describe the development of an efficient solid-supported methodology for the stereoselective synthesis of two peptides containing (2 S ,6 R )-diaminopimelic acid, ( S )-Ala-γ-( R )-Glu-(2 S ,6 R )-A 2 pm-( R )-Ala 1 and γ-( R )-Glu-(2 S ,6 R )-A 2 pm 2 . The platform consists of a Wang resin anchored by an amino acid chain including allylglycine. By olefin cross metathesis with vinylglycine, unsaturated protected (2 S ,6 R )-A 2 pm was fixed on solid support. Peptides were achieved by cleavage of cross metathesis products from resin, followed by reduction of double bonds along removing of protecting groups. Furthermore, this efficient solid phase approach will lead to peptide and muropeptide libraries.

Microalgae constitute a phylogenetically diverse group of eukaryotic photosynthetic microorganisms that thrive in various environments. They are the subject of increasing interest from both the scientific community and the industrial world due, on the one hand, to their ecological significance at the global scale and, on the other hand, to their large potential as sources of economically important products, from biofuels to new pharmaceutical drugs. Some species (such as Chlorella sp., Haematococcus pluvialis or Dunaliella salina) are already cultivated on large scales, mainly in the photoautotrophic mode. Due to the production cost of microalgal biomass, relatively high-value compounds are targeted for economically viable processes. Among them, fatty acids and pigments are common products for which relevant species and cultivation processes are studied. Antibacterial substances appear as promising but less studied targets. This chapter makes a review about the production of fatty acids, pigments and antibacterial/antifungal compounds, with a special emphasis on their production in the heterotrophic mode. Compared to phototrophic growth, heterotrophic growth may result in higher biomass productivity by avoiding light limitations. In addition, it decreases area footprint and installation costs. The heterotrophic metabolism of some model microalgae is also described as well as the productivities that could be encountered in heterotrophy.

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.

International audience; The straightforward synthesis of 5'-methylene-[1,4]-triazole-substituted aminoribosyl uridines is described. Two families of compounds were synthesized from a unique epoxide which was regioselectively opened by acetylide ions (for compounds II) or azide ions (for compounds III). Sequential diastereoselective glycosylation with a ribosyl fluoride derivative, Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) with various complementary azide and alkyne partners afforded the targeted compounds after final deprotection. The biological activity of the 16 resulting compounds together with that of 14 previously reported compounds I, lacking the 5' methylene group, was evaluated on the MraY transferase activity. Out of the 30 tested compounds, 18 compounds revealed MraY inhibition with IC50 ranging from 15 to 150 μM. A molecular modeling study was performed to rationalize the observed structure-activity relationships (SAR), which allowed us to correlate the activity of the most potent compounds with an interaction involving Leu191 of MraYAA. The antibacterial activity was also evaluated and seven compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 μg mL(-1), including the methicillin resistant Staphylococcus aureus (MRSA).

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by 31P and 1H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal amino acid.

The partial genome sequencing of Bacillus amyloliquefaciens GA1 led to the identification of the aml gene cluster involved in the synthesis of the novel lantibiotic named amylolysin. Pure amylolysin was shown to have an antibacterial activity toward Gram-positive bacteria including methicillin resistant Staphylococcus aureus. The lantibiotic was also found efficient to inhibit the growth of Listeria monocytogenes strains on poultry meat upon a long storage at 4 ° C. In silico analyses of the aml gene cluster revealed the presence of a characteristic motif involved in interaction with peptidoglycan precursor lipid II. In the present work, this interaction was further investigated using the LiaRS based reporter gene that is able to sense specifically antibiotics that interfere with lipid II cycle. Beside this, the pore-forming ability of amylolysin was evidenced by means of membrane depolarization measurements and cell leaking experiments.

The photosensitive 4-azido-2,3,5,6-tetrafluoro-1-benzoyl core has been connected to maleimide-, chloroacetyloxy-, or succinimidyl carbonate motifs through various spacers to furnish a series of bifunctional linkers. One linker (7a) has been used for the construction of a biosensor for β-lactam antibiotic detection by attenuated total reflectance FTIR (ATR-FTIR) spectroscopy. Linkers featuring a photosensitive aryl azide motif at one end and a function reactive toward nucleophiles at the other end are described. One linker is used for the construction of a biosensor for β-lactam antibiotic detection by attenuated total reflectance FTIR spectroscopy. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nitroveratrole esters (i.e. 4,5-dimethoxy-2-nitrobenzyl (NV) esters) of penicillin and cephalosporin derivatives have been prepared in the course of a program dedicated to photosensitive protein microarrays. Surprisingly, some molecules revealed to be acylating inhibitors of R39 and BlaR-CTD proteins (two representative Penicillin Binding Proteins (PBPs)), while being bad substrates of beta-lactamases (bacterial defense enzymes). Electrostatic potential maps and docking experiments showed that the 2-nitro group of NV esters could play the role of a carboxylate in the penam series.

We report the first identification of a gene cluster involved in d -tagatose catabolism in Bacillus licheniformis . The pathway is closely related to the d -tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca , in contrast to the d -tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus .

The endospore-forming Bacillus subtilis has been used as probiotics over the last 50 years. However, little is known on how Bacillus spp act in the gut compared to other well-characterized probiotics such as lactic acid bacteria. It is believed that the competitive exclusion of pathogens results from different mode of action notably the production of antimicrobial compounds such as bacteriocins. Here, we report the characterization of the unexpected ability of a gut-associated B. subtilis BSP1 to synthetize the cyclic bacteriocin subtilosin A at high level. Our findings suggest that the BSP1 phenotype could be related, at least in part, to a subsequent increased expression level of the subtilosin A biosynthetic gene cluster sbo-alb in response to a higher activity of the stationery and sporulation master regulator Spo0A. ©2013 Science Publication.

Phormidesmis priestleyi ULC007 is an Antarctic freshwater cyanobacterium. Its draft genome is 5,684,389 bp long. It contains a total of 5,604 protein-encoding genes, of which 22.2% have no clear homologues in known genomes. To date, this draft genome is the first one ever determined for an axenic cyanobacterium from Antarctica.

6-APA derivatives were considered as selective labels for the construction of bifunctional linkers dedicated to the oriented immobilization of proteins on materials. Sulbactam-like compounds (i.e., 6-β-sulfonamido-penam sulfones) and penicillin G - like compounds (i.e., para-substituted 6-β- phenylacetamido-penams) were prepared and tested as irreversible inhibitors of representative β-lactamases and D,D-peptidases, respectively. The activity of the modified antibiotics was preserved despite their substitution with various anchoring arms. The (2-nitro-4,5-dimethoxy)-benzyl esters revealed of particular interest due to their capacity to acylate BlaR-CTD without deprotection. © 2012 Elsevier Ltd. All rights reserved.

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen-producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster-specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster-specific primers, targeting the cluster-conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

International audience; Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

The bacterial cell wall maintains a cell's integrity while allowing growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multigigadalton bag-like structure, and, additionally in gram-positive bacteria, of covalently linked anionic polymers collectively called teichoic acids. These anionic polymers are thought to play important roles in host-cell adhesion, inflammation, and immune activation. In this Article, we compare the flexibility and the organization of peptidoglycans from gram-negative bacteria (E. coli) with its counterpart from different gram-positive bacteria using solid-state nuclear magnetic resonance spectroscopy (NMR) under magic-angle sample spinning (MAS). The NMR fingerprints suggest an identical local conformation of the PG in all of these bacterial species. Dynamics in the peptidoglycan network decreases from E. coli to B. subtilis and from B. subtilis to S. aureus and correlates mainly with the degree of peptide cross-linkage. For intact bacterial cells and isolated cell walls, we show that (31)P solid-state NMR is particularly well adapted to characterize and differentiate wall teichoic acids of different species. We have further observed complexation with divalent ions, highlighting an important structural aspect of gram-positive cell wall architecture. We propose a new model for the interaction of divalent cations with both wall teichoic acids and carbonyl groups of peptidoglycan.

We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP values that are averaged to -65+/-3 kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from -25 for Gly to -115 kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of -63.5+/-2.5 kcal/aa, i.e., only 1.5 kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.

This paper describes the production, the purification and the antilisterial activity of amylolysin, a novel bacteriocin from B. amyloliquefaciens GA1. The strain genome was first analysed using PCR techniques for the presence of gene clusters that direct the synthesis of characterised bacteriocins from B. amyloliquefaciens and the closely related B. subtilis. Our results suggest that amylolysin corresponds to a novel bacteriocin. The effect of amylolysin on the growth of different isolates of Listeria monocytogenes was evaluated in poultry meat during 21 days of storage at 4 °C. A potent antilisterial effect was observed for all the indicator strains tested, demonstrating that amylolysin is a novel bacteriocin that could be used as a food preservative.

6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.

In the eubacterial cell, the peptidoglycan is perpetually hydrolyzed throughout the cell cycle by different enzymes such as lytic transglycosylases, endopeptidases, and amidases. In Escherichia coli , four N -acetylmuramoyl- l -alanine amidases, AmiA, -B, -C, and -D, are present in the periplasm. AmiA, -B, and -C are soluble enzymes, whereas AmiD is a lipoprotein anchored in the outer membrane. To determine more precisely the specificity and the kinetic parameters of AmiD, we overproduced and purified the native His-tagged AmiD in the presence of detergent and a soluble truncated form of this enzyme by removing its signal peptide and the cysteine residue responsible for its lipidic anchorage. AmiD is a zinc metalloenzyme and is inactivated by a metal chelator such as EDTA. Native His-tagged and truncated AmiD hydrolyzes peptidoglycan fragments that have at least three amino acids in their peptide chains, and the presence of an anhydro function on the N -acetylmuramic acid is not essential for its activity. The soluble truncated AmiD exhibits a biphasic kinetic time course that can be explained by the inactivation of the enzyme by the substrate. This behavior highlights a new strategy to inhibit this class of enzymes.

Staphylococcus aureusis an opportunistic intracellular organism. Although they poorly accumulate in eukaryotic cells, β-lactams show activity against intracellular methicillin (meticillin)-susceptibleS. aureus(MSSA) if the exposure times and the drug concentrations are sufficient. Intraphagocytic methicillin-resistantS. aureus(MRSA) strains are susceptible to penicillins and carbapenems because the acidic pH favors the acylation of PBP 2a by these β-lactams through pH-induced conformational changes. The intracellular activity (THP-1 macrophages and keratinocytes) of ceftobiprole, which shows almost similar in vitro activities against MRSA and MSSA in broth, was examined against a panel of hospital-acquired and community-acquired MRSA strains (MICs, 0.5 to 2.0 mg/liter at pH 7.4 and 0.25 to 1.0 mg/liter at pH 5.5) and was compared with its activity against MSSA isolates. The key pharmacological descriptors {relative maximal efficacy (Emax), relative potency (the concentration causing a reduction of the inoculum halfway betweenE0andEmax[EC50]), and static concentration (Cs)} were measured. All strains showed sigmoidal dose-responses, withEmaxbeing about a 1 log10CFU decrease from the postphagocytosis inoculum, and EC50andCsbeing 0.2 to 0.3× and 0.6 to 0.9× the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and pH 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays, (ii) showed only partial restoration of the activity against MRSA in broth at acidic pH, and (iii) were collectively less effective against MRSA in THP-1 macrophages and were ineffective in keratinocytes. The improved activity of ceftobiprole toward intracellular MRSA compared with the activities of conventional cephalosporins can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.

Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29-kDa β-lactamase (cefuroximase) with an unusually high activity towards the β-lactamase-stable oximino-cephalosporins (e.g. cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA β-lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the β-lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39-kDa molecular-class-C β-lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC β-lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non-inducible P. vulgaris mutants to the inducible, wild-type phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC β-lactamase. Very different structural genes can thus be under the control of identical systems.

Most bacteria have multiple peptidoglycan hydrolases capable of cleaving covalent bonds in peptidoglycan sacculi or its fragments. An overview of the different classes of peptidoglycan hydrolases and their cleavage sites is provided. The physiological functions of these enzymes include the regulation of cell wall growth, the turnover of peptidoglycan during growth, the separation of daughter cells during cell division and autolysis. Specialized hydrolases enlarge the pores in the peptidoglycan for the assembly of large trans-envelope complexes (pili, flagella, secretion systems), or they specifically cleave peptidoglycan during sporulation or spore germination. Moreover, peptidoglycan hydrolases are involved in lysis phenomena such as fratricide or developmental lysis occurring in bacterial populations. We will also review the current view on the regulation of autolysins and on the role of cytoplasm hydrolases in peptidoglycan recycling and induction of beta-lactamase.

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided.

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php.

Summary Multiple strains of Bacillus spp. were demonstrated to stimulate plant defence responses. However, very little is known about the nature of molecular determi- nants secreted by these Gram-positive bacteria that are responsible for the elicitation of the induced systemic resistance (ISR) phenomenon. This study shows that the lipopeptides surfactins and fengycins may be involved in this elicitation process. In bean, pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants, similar to the one induced by living cells of the producing strain S499. Moreover, experiments conducted on bean and tomato plants showed that overexpression of both surfactin and fengycin biosynthetic genes in the naturally poor producer Bacillus subtilis strain 168 was associated with a significant increase in the potential of the derivatives to induce resistance. In tomato cells, key enzymes of the lipoxygenase pathway appeared to be activated in resistant plants following induction by lipopeptide overproducers. To our knowledge, such lipopeptides constitute a novel class of compounds from non-pathogenic bacteria that can be perceived by plant cells as signals to initiate defence mechanisms.

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.

SummaryTo study the properties of the BlaR penicillin­receptor involved in the induction of the Bacillus licheniformis β­lactamase, the water­soluble carboxy terminal domain of the protein (BlaR­CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various ­lactamβ antibiotics were studied. The second­order acylation rate constants K 2 /K' ranged from 0.0017 to more than 1 µM ­1 s ­1 and the deacylation rate constants were lower than 4 x 10 ­5 s ­1 . These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR­CTD is thus one of the most sensitive penicillin­binding proteins presently described. In the light of these results, the kinetics of ­lactamase induction in β Bacillus licheniformis were re­examined. When starting with a rather high cell density, a good ­lactamase substrate such asβ benzylpenicillin is too sensitive to ­lactamase­mediated hydrolysis to allow full induction. By contrast, a poorβ β β­lactamase substrate (7­aminocephalosporanic acid) can fully derepress ­lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction.IntroductionThe

In the absence of penicillin, the beta-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 microm by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the beta-lactamase locus (blaP-blaI-blaR1) were lower than (1.9 microm) or in the same range as (75 microm) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K(d)(OP1) = 1.7 +/- 0.5 10(-15) m(2), K(d)(OP2) = 3.3 +/- 0.9 10(-15) m(2), and K(d)(OP3) = 10.5 +/- 2.5 10(-15) m(2). The role of the DNA binding properties of BlaI on the beta-lactamase regulation is discussed.

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of b-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L. q 2003 Published by Elsevier Science Ltd

[en] Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels infuture transportation and energy production. However, current industrial hydrogen production processes, such assteam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, inparticular the use of fermentative microorganisms, have attracted scientific interest in recent years. However thelow overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailedunderstanding of the relationships between genome content, gene expression patterns, pathway utilisation andmetabolite synthesis is required to optimise the yield of biohydrogen production pathways.Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridiumbutyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when themetabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitoredin 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differenceswere observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and theywere pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated withconcurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation ofthe [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expressionlevels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, whilethe nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmentalfactors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/orthe nitrogenase.Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides amultidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism ofclostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways basedon genetic engineering techniques., info:eu-repo/semantics/published

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH2-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes. Mol. Reprod. Dev. 64: 199–206, 2003. © 2003 Wiley-Liss, Inc.

Summary The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP β-lactamases by β-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of BlaI during β-lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaR1 genes. In contrast to the situation in B. licheniformis 749/I, β-lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS–PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the BlaI isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of β-lactamase biosynthesis.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.

Background: The l-aminopeptidase d-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal l and/or d-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both d and l stereospecificity. The DmpA active form is an αβ heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. Results: The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an αββα sandwich in which two mixed β sheets are flanked on both sides by two α helices. Conclusions: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.