Computational Methods Reveal a Series of Cyclic and Linear Lichenysins and Surfactins from the Vietnamese Marine Sediment-Derived Streptomyces Strain G222.

The Streptomyces strain G222, isolated from a Vietnamese marine sediment, was confidently identified by 16S rRNA gene sequencing. Its AcOEt crude extract was successfully analyzed using non-targeted LC-MS/MS analysis, and molecular networking, leading to a putative annotation of its chemical diversity thanks to spectral libraries from GNPS and in silico metabolite structure prediction obtained from SIRIUS combined with the bioinformatics tool conCISE (Consensus Annotation Propagation of in silico Elucidations). This dereplication strategy allowed the identification of an interesting cluster of a series of putative cyclic and linear lipopeptides of the lichenysin and surfactin families. Lichenysins (3-7) were isolated from the sub-fraction, which showed significant anti-biofilm activity against Pseudomonas aeruginosa MUC-N1. Their structures were confirmed by detailed 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC, TOCSY, ROESY) recorded in CD3OH, and their absolute configurations were determined using the modified Marfey's method. The isolated lichenysins showed anti-biofilm activity at a minimum concentration of 100 µM. When evaluated for antibacterial activity against a panel of Gram-positive and Gram-negative strains, two isolated lichenysins exhibited selective activity against the MRSA strain without affecting its growth curve and without membranotropic activity. This study highlights the power of the MS/MS spectral similarity strategy using computational methods to obtain a cross-validation of the annotated molecules from the complex metabolic profile of a marine sediment-derived Streptomyces extract. This work provides the first report from a Streptomyces strain of combined cyclic and linear lichenysins and surfactins, known to be characteristic compounds of the genus Bacillus.

Insights from the Shell Proteome: Biomineralization to Adaptation.

Bivalves have evolved a range of complex shell forming mechanisms that are reflected by their incredible diversity in shell mineralogy and microstructures. A suite of proteins exported to the shell matrix space plays a significant role in controlling these features, in addition to underpinning some of the physical properties of the shell itself. Although, there is a general consensus that a minimum basic protein tool kit is required for shell construction, to date, this remains undefined. In this study, the shell matrix proteins (SMPs) of four highly divergent bivalves (The Pacific oyster, Crassostrea gigas; the blue mussel, Mytilus edulis; the clam, Mya truncata, and the king scallop, Pecten maximus) were analyzed in an identical fashion using proteomics pipeline. This enabled us to identify the critical elements of a "basic tool kit" for calcification processes, which were conserved across the taxa irrespective of the shell morphology and arrangement of the crystal surfaces. In addition, protein domains controlling the crystal layers specific to aragonite and calcite were also identified. Intriguingly, a significant number of the identified SMPs contained domains related to immune functions. These were often are unique to each species implying their involvement not only in immunity, but also environmental adaptation. This suggests that the SMPs are selectively exported in a complex mix to endow the shell with both mechanical protection and biochemical defense.

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex Microcystis aeruginosa Extracts on Adult Medaka Fish.

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.

Shell proteome of rhynchonelliform brachiopods.

Brachiopods are a phylum of marine invertebrates that have an external bivalved shell to protect their living tissues. With few exceptions, this biomineralized structure is composed of calcite, mixed together with a minor organic fraction, comprising secreted proteins that become occluded in the shell structure, once formed. This organic matrix is thought to display several functions, in particular, to control mineral deposition and to regulate crystallite shapes. Thus, identifying the primary structure of matrix proteins is a prerequisite for generating bioinspired materials with tailored properties. In this study, we employed a proteomic approach to identify numerous peptides that constitute the shell proteins, in three rhynchonellid brachiopods from different localities. Our results suggest that the shell protein repertoires identified thus far, differ from that of better known calcifying metazoans, such as molluscs.

Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales.

Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium.

Proteomics of CaCO3 biomineral-associated proteins: how to properly address their analysis.

In a recent editorial (Proc. Natl. Acad. Sci., 2013 110, E2144-E2146) and elsewhere, questions have been raised regarding the experimental practices in relation to the proteomic analysis of organic matrices associated to the biomineralized CaCO3 skeletons of metazoans such as molluscan shells and coral skeletons. Indeed, although the use of new high sensitivity MS technology potentially allows to identify a greater number of proteins, it is also equally (or even more) sensitive to contamination of residual proteins from soft tissues, which are in close contact with the biomineral. Based on our own past and present experimental know-how-observations that are reproducible and coherent with the current understanding of extracellular biomineralization processes-we are convinced that a careful and appropriate cleaning of biominerals prior to any analysis is crucial for accurate proteomic investigations and subsequent pertinent interpretation of the results. Our goal is to alert the scientific community about the associated bias that definitely should be avoided, and to provide critical recommendations on sample preparation and experimental design, in order to better take advantage of the aptness of proteomic approaches aiming at improving our understanding of the molecular mechanisms in biomineralization.

In vitro cross-linking of Mycobacterium tuberculosis peptidoglycan by L,D-transpeptidases and inactivation of these enzymes by carbapenems.

The Mycobacterium tuberculosis peptidoglycan is cross-linked mainly by l,d-transpeptidases (LDTs), which are efficiently inactivated by a single ß-lactam class, the carbapenems. Development of carbapenems for tuberculosis treatment has recently raised considerable interest since these drugs, in association with the ß-lactamase inhibitor clavulanic acid, are uniformly active against extensively drug-resistant M. tuberculosis and kill both exponentially growing and dormant forms of the bacilli. We have purified the five l,d-transpeptidase paralogues of M. tuberculosis (Mt1 to -5) and compared their activities with those of peptidoglycan fragments and carbapenems. The five LDTs were functional in vitro since they were active in assays of peptidoglycan cross-linking (Mt5), ß-lactam acylation (Mt3), or both (Mt1, Mt2, and Mt4). Mt3 was the only LDT that was inactive in the cross-linking assay, suggesting that this enzyme might be involved in other cellular functions such as the anchoring of proteins to peptidoglycan, as shown in Escherichia coli. Inactivation of LDTs by carbapenems is a two-step reaction comprising reversible formation of a tetrahedral intermediate, the oxyanion, followed by irreversible rupture of the ß-lactam ring that leads to formation of a stable acyl enzyme. Determination of the rate constants for these two steps revealed important differences (up to 460-fold) between carbapenems, which affected the velocity of oxyanion and acyl enzyme formation. Imipenem inactivated LDTs more rapidly than ertapenem, and both drugs were more efficient than meropenem and doripenem, indicating that modification of the carbapenem side chain could be used to optimize their antimycobacterial activity.

Iridoid- and flavonoid-enriched fractions of Cornus sanguinea and Cornus mas exert antioxidant and anti-inflammatory effects and inhibit key enzymes in the treatment of metabolic disorders.

Background: Berry fruits are recognized as a "superfood" due to their high content of bioactive compounds and health benefits. Scope and approach: Herein, extracts of Cornus sanguinea and Cornus mas fresh and dried fruits obtained by different extraction procedures (ethanolic and hydroalcoholic maceration, ultrasound-assisted extraction, and Soxhlet apparatus) were analysed using liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) and compared to identify the main healthy compounds and their impact on the inhibition of key enzymes (pancreatic lipase, α-glucosidase, and α-amylase) associated with metabolic disorders. The antioxidant activity and inhibition of nitric oxide (NO) and NF-κB pathway were also investigated. Key findings and conclusions: Flavonoids, iridoids, and phenolic acids were the main classes of identified compounds. Herein, kaempferol 3-O-galactoside, kaempferol 3-O-glucoside, quercetin, quercetin 3-O-xyloside, and myricetin 3-O-galactoside were detected for the first time in C. sanguinea. Remarkable antioxidant effects and promising α-glucosidase and lipase inhibitory activity were observed with extracts obtained by hydroalcoholic maceration of both Cornus dried fruits. Consequently, these extracts were subjected to fractionation using Amberlite XAD-16 resin. The most promising biological activities, which are attributed to the presence of some flavonoids and iridoids, were detected with the C. sanguinea fractions, in particular SD2(II). The results of this study offer new insights into the potential development of functional foods, nutraceuticals, and food supplements using the Cornus species.

Analysis of low complex region peptides derived from mollusk shell matrix proteins using CID, high-energy collisional dissociation, and electron transfer dissociation on an LTQ-orbitrap: implications for peptide to spectrum match.

Identification of proteins involved in mollusk biomineralization by proteomics approach is gaining importance. These proteins are often characterized by low-complexity regions (LCRs) made of repeating motifs that are constituted by few amino acids (e.g. IGG, DD, KK, and GGG). In this work, we have analyzed the fragmentation of model LCR peptides under different fragmentation regimes (CID, high-energy collisional dissociation [HCD], and electron transfer dissociation [ETD]) and its consequences on peptide to spectrum matches (PSMs) using two search algorithms (Mascot and PEAKS DB). For both search tools, higher number of PSMs was obtained using CID spectra, followed by HCD and ETD. Intense fragment ions present in the lower m/z region of HCD led to lower PSM scores and absence of low mass cut off seems to offer little advantage for the identification of LCR peptides. Generally, doubly charged peptides under ETD conditions did not fragment to yield sequence information rich spectra. The spectral quality is affected by the nature of the repeating motifs in the peptide. The performance of both Mascot and PEAKS DB (de novo based search tool) vary according to the fragment regime employed to acquire MS/MS spectra.

Inactivation of Mycobacterium tuberculosis l,d-transpeptidase LdtMt1 by carbapenems and cephalosporins.

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase Ldt(Mt1) by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt(Mt1), which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt(Mt1), with a binding rate constant of 0.08 µM(-1) min(-1). The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min(-1)) in comparison to the acylation rate constant (3.1 min(-1)). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10(-3) min(-1). Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt(Mt1) inactivator. Cephalosporins also formed covalent adducts with Ldt(Mt1), although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

Molecular acclimation of Halobacterium salinarum to halite brine inclusions.

Halophilic microorganisms have long been known to survive within the brine inclusions of salt crystals, as evidenced by the change in color for salt crystals containing pigmented halophiles. However, the molecular mechanisms allowing this survival has remained an open question for decades. While protocols for the surface sterilization of halite (NaCl) have enabled isolation of cells and DNA from within halite brine inclusions, "-omics" based approaches have faced two main technical challenges: (1) removal of all contaminating organic biomolecules (including proteins) from halite surfaces, and (2) performing selective biomolecule extractions directly from cells contained within halite brine inclusions with sufficient speed to avoid modifications in gene expression during extraction. In this study, we tested different methods to resolve these two technical challenges. Following this method development, we then applied the optimized methods to perform the first examination of the early acclimation of a model haloarchaeon (Halobacterium salinarum NRC-1) to halite brine inclusions. Examinations of the proteome of Halobacterium cells two months post-evaporation revealed a high degree of similarity with stationary phase liquid cultures, but with a sharp down-regulation of ribosomal proteins. While proteins for central metabolism were part of the shared proteome between liquid cultures and halite brine inclusions, proteins involved in cell mobility (archaellum, gas vesicles) were either absent or less abundant in halite samples. Proteins unique to cells within brine inclusions included transporters, suggesting modified interactions between cells and the surrounding brine inclusion microenvironment. The methods and hypotheses presented here enable future studies of the survival of halophiles in both culture model and natural halite systems.

The peptidoglycan of Mycobacterium abscessus is predominantly cross-linked by L,D-transpeptidases.

Few therapeutic alternatives remain for the treatment of infections due to multiresistant Mycobacterium abscessus. Here we show that the peptidoglycans of the "rough" and "smooth" morphotypes contain predominantly 3→3 cross-links generated by l,d-transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.

iTRAQ-based proteomic study of the effects of microcystin-LR on medaka fish liver.

Microcystins are cyanotoxins that occur in ground water and thus pose a potential health risk. Microcystin-LR (microcystin-leucine-arginine) is a potent hepatotoxin, and is suspected of being a tumour promoter. Poisoning with this toxin causes several dysfunctions in hepatocytes by inhibiting protein phosphatases 1 and 2A, and notably produces oxidative stress, disrupts the cytoskeleton, and deregulates mitogen-activated protein kinase pathway. Medaka fish (Oryzias latipes) was chosen as a model for studying the effects of this cyanotoxin on liver proteins using a gel-free approach, iTRAQ. Fish were gavaged with microcystin-LR. Two hours later, 325 proteins could be identified by Scaffold Q+ and 32 proteins revealed statistically significant variations above a ∣0.2∣ threshold of log(2) ratio by comparison with control. These proteins are mostly involved in the translation and maturation of proteins, lipid metabolism and detoxification. Notably, apolipoproteins are deregulated which indicates a possible alteration of chylomicron-mediated transport.

Activation of the L,D-transpeptidation peptidoglycan cross-linking pathway by a metallo-D,D-carboxypeptidase in Enterococcus faecium.

Bypass of the penicillin-binding proteins by an L,D-transpeptidase (Ldt(fm)) confers cross-resistance to beta-lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt(fm) is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross-linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt(fm) is controlled by a two-component regulatory system (DdcRS) and a metallo-D,D-carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C-terminal D-Ala residue of the cytoplasmic peptidoglycan precursor UDP-MurNAc-pentapeptide. The T(161)A and T(161)M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP-MurNAc-pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E(127)K) and affected its interaction with the cell envelope (I(14)N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross-resistance to glycopeptides and beta-lactams in natural conditions.

Coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins: a study with P. margaritifera.

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Untangling the fibre ball: Proteomic characterization of South American camelid hair fibres by untargeted multivariate analysis and molecular networking.

The proteomic analysis of hairs, yarns or textiles has emerged as a powerful method to determine species of origin, mainly used in archaeozoological research and fraud control. Differentiation between the South American camelid (SAC) species (the wild guanaco and vicuña and their respective domesticates the llama and alpaca) is particularly challenging due to poor database information and significant hybridization between species. In this study, we analysed 41 modern and 4 archaeological samples from the four SACs species. Despite strong similarities with Old World Camelidae, we identified 7 peptides specific to SACs assigned to keratin K86 and the keratin-associated proteins KAP13-1 and KAP11-1. Untargeted multivariate analysis of the LC-MS data permitted to distinguish SAC species and propose discriminant features. MS/MS-based molecular networking combined with database-assisted de novo sequencing permitted to identify 5 new taxonomic peptides assigned to K33a, K81 and/or K83 keratins and KAP19-1. These peptides differentiate the two wild species, guanaco and vicuña. These results show the value of combining database search and untargeted metabolomic approaches for paleoproteomics, and reveal for the first time the potential of molecular networks to highlight deamidation related to diagenesis and cluster highly similar peptides related to interchain homologies or intra- or inter-specific polymorphism. SIGNIFICANCE: This study used an innovative approach combining multivariate analysis of LC-MS data together with molecular networking and database-assisted de novo sequencing to identify taxonomic peptides in palaeoproteomics. It constitutes the first attempt to differentiate between hair fibres from the four South American camelids (SACs) based on proteomic analysis of modern and archaeological samples. It provides different proteomic signatures for each of the four SAC species and proposes new SAC taxonomic peptides of interest in archaeozoology and fraud control. SACs have been extensively exploited since human colonization of South America but have not been studied to the extent of their economic, cultural and heritage importance. Applied to the analysis of ancient Andean textiles, our results should permit a better understanding of cultural and pastoral practices in South America. The wild SACs are endangered by poaching and black-market sale of their fibre. For the first time, our results provide discriminant features for the determination of species of origin of contraband fibre.

Optoproteomics elucidates the interactome of L-type amino acid transporter 3 (LAT3).

Membrane protein interactions are crucial for diverse biological processes. We report the application of genetic code expansion in combination with photo-crosslinking chemistry, as we termed "optoproteomics", to identify proteins interacting with the human L-type membrane amino acid transporter 3 (LAT3, also known as SLC43A1). The site-specifically incorporated photo-cross-linker p-azido-L-phenylalanine (AzF), which reacts with proteins in their proximity, enabled the capture of weak and transient partners of LAT3 in living cells. We identify 11 unique interacting proteins which are light-sensitive and 19 unique proteins that are site-specific, validating the approach and providing insights into the LAT3 protein-protein interaction network currently unavailable.

The beta-lactam-sensitive D,D-carboxypeptidase activity of Pbp4 controls the L,D and D,D transpeptidation pathways in Corynebacterium jeikeium.

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.

The differential spatial distribution of secondary metabolites in Arabidopsis leaves reacting hypersensitively to Pseudomonas syringae pv. tomato is dependent on the oxidative burst.

Secondary metabolites (SMs) play key roles in pathogen responses, although knowledge of their precise functions is limited by insufficient characterization of their spatial response. The present study addressed this issue in Arabidopsis leaves by non-targeted and targeted metabolite profiling of Pseudomonas syringae pv. tomato (Pst-AvrRpm1) infected and adjacent uninfected leaf tissues. While overlap was observed between infected and uninfected areas, the non-targeted metabolite profiles of these regions differed quantitatively and clustering analysis underscores a differential distribution of SMs within distinct metabolic pathways. Targeted metabolite profiling revealed that infected tissues accumulate more salicylic acid and the characteristic phytoalexin of Arabidopsis, camalexin, than uninfected adjacent areas. On the contrary, the antioxidant coumarin derivative, scopoletin, was induced in infected tissues while its glucoside scopolin predominated in adjacent tissues. To elucidate the still unclear relationship between the accumulation of SMs and reactive oxygen species (ROS) accumulation and signalling, a catalase-deficient line (cat2) in which ROS signalling is up-regulated, was used. Metabolic analysis of cat2 suggests that some SMs have important interactions with ROS in redox homeostasis during the hypersensitive response to Pst-AvrRpm1. Overall, the study demonstrates that ROS availability influences both the amount and the pattern of infection-induced SM accumulation.

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell.

BACKGROUND: The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. RESULTS: Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains. CONCLUSION: This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.