A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound.

Rapeseed meal (RSM) is a cheap, abundant and renewable feedstock, whose biorefinery is a current challenge for the sustainability of the oilseed sector. RSM is rich in sinapic acid (SA), a p-hydroxycinnamic acid that can be decarboxylated into canolol (2,6-dimethoxy-4-vinylphenol), a valuable bioactive compound. Microbial phenolic acid decarboxylases (PADs), mainly described for the non-oxidative decarboxylation of ferulic and p-coumaric acids, remain very poorly documented to date, for SA decarboxylation. The species Neolentinus lepideus has previously been shown to biotransform SA into canolol in vivo, but the enzyme responsible for bioconversion of the acid has never been characterized. In this study, we purified and characterized a new PAD from the canolol-overproducing strain N. lepideus BRFM15. Proteomic analysis highlighted a sole PAD-type protein sequence in the intracellular proteome of the strain. The native enzyme (NlePAD) displayed an unusual outstanding activity for decarboxylating SA (Vmax of 600 U.mg-1, kcat of 6.3 s-1 and kcat/KM of 1.6 s-1.mM-1). We showed that NlePAD (a homodimer of 2 × 22 kDa) is fully active in a pH range of 5.5-7.5 and a temperature range of 30-55 °C, with optima of pH 6-6.5 and 37-45 °C, and is highly stable at 4 °C and pH 6-8. Relative ratios of specific activities on ferulic, sinapic, p-coumaric and caffeic acids, respectively, were 100:24.9:13.4:3.9. The enzyme demonstrated in vitro effectiveness as a biocatalyst for the synthesis of canolol in aqueous medium from commercial SA, with a molar yield of 92%. Then, we developed processes to biotransform naturally-occurring SA from RSM into canolol by combining the complementary potentialities of an Aspergillus niger feruloyl esterase type-A, which is able to release free SA from the raw meal by hydrolyzing its conjugated forms, and NlePAD, in aqueous medium and mild conditions. NlePAD decarboxylation of biobased SA led to an overall yield of 1.6-3.8 mg canolol per gram of initial meal. Besides being the first characterization of a fungal PAD able to decarboxylate SA, this report shows that NlePAD is very promising as new biotechnological tool to generate biobased vinylphenols of industrial interest (especially canolol) as valuable platform chemicals for health, nutrition, cosmetics and green chemistry.

Structural landscape of the respiratory syncytial virus nucleocapsids.

Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

Benefits of Iterative Searches of Large Databases to Interpret Large Human Gut Metaproteomic Data Sets.

The gut microbiota are increasingly considered as a main partner of human health. Metaproteomics enables us to move from the functional potential revealed by metagenomics to the functions actually operating in the microbiome. However, metaproteome deciphering remains challenging. In particular, confident interpretation of a myriad of MS/MS spectra can only be pursued with smart database searches. Here, we compare the interpretation of MS/MS data sets from 48 individual human gut microbiomes using three interrogation strategies of the dedicated Integrated nonredundant Gene Catalog (IGC 9.9 million genes from 1267 individual fecal samples) together with the Homo sapiens database: the classical single-step interrogation strategy and two iterative strategies (in either two or three steps) aimed at preselecting a reduced-sized, more targeted search space for the final peptide spectrum matching. Both iterative searches outperformed the single-step classical search in terms of the number of peptides and protein clusters identified and the depth of taxonomic and functional knowledge, and this was the most convincing with the three-step approach. However, iterative searches do not help in reducing variability of repeated analyses, which is inherent to the traditional data-dependent acquisition mode, but this variability did not affect the hierarchical relationship between replicates and all other samples.

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.

AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes.

BACKGROUND: Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete Trichoderma reesei, but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the T. reesei enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity. RESULTS: In this study, the enzymatic cocktails secreted by five strains from the genus Aspergillus (Aspergillus japonicus strains BRFM 405, 1487, 1489, 1490 and Aspergillus niger strain BRFM 430) were tested for their ability to boost a T. reesei reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from Aspergillus aculeatus (AaAA16) was produced to high level in Pichia pastoris. LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of T. reesei CBHI on cellulosic substrates. CONCLUSIONS: Although Aspergillus spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.

Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens".

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

BACKGROUND: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. RESULTS: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners. CONCLUSION: As a peculiar feature of P. brumalis, we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.

Lavender- and lavandin-distilled straws: an untapped feedstock with great potential for the production of high-added value compounds and fungal enzymes.

BACKGROUND: Lavender (Lavandula angustifolia) and lavandin (a sterile hybrid of L. angustifolia × L. latifolia) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery. RESULTS: Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, ß-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Pycnoporus cinnabarinus. Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics. CONCLUSIONS: The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy.

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, TvDyP1 and TvVP2, Identified from the Secretome of Trametes versicolor.

Trametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase TvDyP1 and the versatile peroxidase TvVP2. Both peroxidases were heterologously produced in Escherichia coli, biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and TvDyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while TvVP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of TvVP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, TvDyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with TvVP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and TvVP2 presented a greater ability to oxidize and decolorize the dye substrates than TvDyP1.IMPORTANCETrametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.

Profiling of Campylobacter jejuni Proteome in Exponential and Stationary Phase of Growth.

Campylobacter jejuni has been reported as a major cause of bacterial food-borne enteritides in developed countries during the last decade. Despite its fastidious growth requirements, including low level of oxygen and high level of CO2, this pathogen is able to persist in the environment without permanent loss of its viability and virulence. As C. jejuni is not able to multiply outside a host, the cells spend significant amount of time in stationary phase of growth. The entry into the stationary phase is often correlated to resistance to various stresses in bacteria. The switching between exponential and stationary phases is frequently mediated by the regulator sigma S (RpoS). However, this factor is absent in C. jejuni and molecular mechanisms responsible for transition of cells to the stationary phase remain elusive. In this work, proteomic profiles of cells from exponential and stationary phases were compared using 2-D electrophoresis (2DE) fingerprinting combined with mass spectrometry analysis and qRT-PCR. The identified proteins, whose expression differed between the two phases, are mostly involved in protein biosynthesis, carbon metabolism, stress response and motility. Altered expression was observed also in the pleiotropic regulator CosR that was over-expressed during stationary phase. A shift between transcript and protein level evolution of CosR throughout the growth of C. jejuni was observed using qRT-PCR and (2DE). From these data, we hypothesized that CosR could undergo a negative autoregulation in stationary phase. A consensus sequence resulting from promoter sequence alignment of genes potentially regulated by CosR, including its own upstream region, among C. jejuni strains is proposed. To verify experimentally the potential autoregulation of CosR at the DNA level, electrophoretic mobility shift assay was performed with DNA fragments of CosR promoter region and rCosR. Different migration pattern of the promoter fragments indicates the binding capacity of CosR, suggesting its auto-regulation potential.

The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

Innovative green technologies are of importance for converting plant wastes into renewable sources for materials, chemicals and energy. However, recycling agricultural and forestry wastes is a challenge. A solution may be found in the forest. Saprotrophic white-rot fungi are able to convert dead plants into consumable carbon sources. Specialized fungal enzymes can be utilized for breaking down hard plant biopolymers. Thus, understanding the enzymatic machineries of such fungi gives us hints for the efficient decomposition of plant materials. Using the saprotrophic white-rot fungus Pycnoporus coccineus as a fungal model, we examined the dynamics of transcriptomic and secretomic responses to different types of lignocellulosic substrates at two time points. Our integrative omics pipeline (SHIN+GO) enabled us to compress layers of biological information into simple heatmaps, allowing for visual inspection of the data. We identified co-regulated genes with corresponding co-secreted enzymes, and the biological roles were extrapolated with the enriched Carbohydrate-Active Enzyme (CAZymes) and functional annotations. We observed the fungal early responses for the degradation of lignocellulosic substrates including; 1) simultaneous expression of CAZy genes and secretion of the enzymes acting on diverse glycosidic bonds in cellulose, hemicelluloses and their side chains or lignin (i.e. hydrolases, esterases and oxido-reductases); 2) the key role of lytic polysaccharide monooxygenases (LPMO); 3) the early transcriptional regulation of lignin active peroxidases; 4) the induction of detoxification processes dealing with biomass-derived compounds; and 5) the frequent attachments of the carbohydrate binding module 1 (CBM1) to enzymes from the lignocellulose-responsive genes. Our omics combining methods and related biological findings may contribute to the knowledge of fungal systems biology and facilitate the optimization of fungal enzyme cocktails for various industrial applications.

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

Inactivation of Cellobiose Dehydrogenases Modifies the Cellulose Degradation Mechanism of Podospora anserina.

Conversion of biomass into high-value products, including biofuels, is of great interest to developing sustainable biorefineries. Fungi are an inexhaustible source of enzymes to degrade plant biomass. Cellobiose dehydrogenases (CDHs) play an important role in the breakdown through synergistic action with fungal lytic polysaccharide monooxygenases (LPMOs). The three CDH genes of the model fungus Podospora anserina were inactivated, resulting in single and multiple CDH mutants. We detected almost no difference in growth and fertility of the mutants on various lignocellulose sources, except on crystalline cellulose, on which a 2-fold decrease in fertility of the mutants lacking P. anserina CDH1 (PaCDH1) and PaCDH2 was observed. A striking difference between wild-type and mutant secretomes was observed. The secretome of the mutant lacking all CDHs contained five beta-glucosidases, whereas the wild type had only one. P. anserina seems to compensate for the lack of CDH with secretion of beta-glucosidases. The addition of P. anserina LPMO to either the wild-type or mutant secretome resulted in improvement of cellulose degradation in both cases, suggesting that other redox partners present in the mutant secretome provided electrons to LPMOs. Overall, the data showed that oxidative degradation of cellulosic biomass relies on different types of mechanisms in fungi. IMPORTANCE: Plant biomass degradation by fungi is a complex process involving dozens of enzymes. The roles of each enzyme or enzyme class are not fully understood, and utilization of a model amenable to genetic analysis should increase the comprehension of how fungi cope with highly recalcitrant biomass. Here, we report that the cellobiose dehydrogenases of the model fungus Podospora anserina enable it to consume crystalline cellulose yet seem to play a minor role on actual substrates, such as wood shavings or miscanthus. Analysis of secreted proteins suggests that Podospora anserina compensates for the lack of cellobiose dehydrogenase by increasing beta-glucosidase expression and using an alternate electron donor for LPMO.

Comparison of Proteomics Profiles of Campylobacter jejuni Strain Bf under Microaerobic and Aerobic Conditions.

Campylobacter jejuni accounts for one of the leading causes of foodborne bacterial enteritis in humans. Despite being considered an obligate microaerobic microorganism, C. jejuni is regularly exposed to oxidative stress. However, its adaptive strategies to survive the atmospheric oxygen level during transmission to humans remain unclear. Recently, the clinical C. jejuni strain Bf was singled out for its unexpected ability to grow under ambient atmosphere. Here, we aimed to understand better the biological mechanisms underlying its atypical aerotolerance trait using two-dimensional protein electrophoresis, gene expression, and enzymatic activities. Forty-seven proteins were identified with a significantly different abundance between cultivation under microaerobic and aerobic conditions. The over-expressed proteins in aerobiosis belonged mainly to the oxidative stress response, enzymes of the tricarboxylic acid cycle, iron uptake, and regulation, and amino acid uptake when compared to microaerobic conditions. The higher abundance of proteins related to oxidative stress was correlated to dramatically higher transcript levels of the corresponding encoding genes in aerobic conditions compared to microaerobic conditions. In addition, a higher catalase-equivalent activity in strain Bf was observed. Despite the restricted catabolic capacities of C. jejuni, this study reveals that strain Bf is equipped to withstand oxidative stress. This ability could contribute to emergence and persistence of particular strains of C. jejuni throughout food processing or macrophage attack during human infection.

Visual Comparative Omics of Fungi for Plant Biomass Deconstruction.

Wood-decay fungi contain the cellular mechanisms to decompose such plant cell wall components as cellulose, hemicellulose, and lignin. A multi-omics approach to the comparative analysis of wood-decay fungi gives not only new insights into their strategies for decomposing recalcitrant plant biomass, but also an understanding of how to exploit these mechanisms for biotechnological applications. We have developed an analytical workflow, Applied Biomass Conversion Design for Efficient Fungal Green Technology (ABCDEFGT), to simplify the analysis and interpretation of transcriptomic and secretomic data. ABCDEFGT utilizes self-organizing maps for grouping genes with similar transcription patterns, and an overlay with secreted proteins. The key feature of ABCDEFGT is simple graphic outputs of genome-wide transcriptomic and secretomic topographies, which enables visual inspection without a priori of the omics data and facilitates discoveries of co-regulated genes and proteins. Genome-wide omics landscapes were built with the newly sequenced fungal species Pycnoporus coccineus, Pycnoporus sanguineus, and Pycnoporus cinnabarinus grown on various carbon sources. Integration of the post-genomic data revealed a global overlap, confirming the pertinence of the genome-wide approach. ABCDEFGT was evaluated by comparison with the latest clustering method for ease of output interpretation, and ABCDEFGT gave a better biological representation of fungal behaviors. The genome-wide multi-omics strategy allowed us to determine the potential synergy of particular enzymes decomposing cellulose, hemicellulose, and lignin such as Lytic Polysaccharide Monooxygenases, modular enzymes associated with a cellulose binding module1, and Class II Peroxidase isoforms co-regulated with oxido-reductases. Overall, ABCDEFGT was capable of visualizing genome-wide transcriptional and secretomic profiles for intuitive interpretations and is suitable for exploration of newly-sequenced organisms.

Interactions between genotype and environment drive the metabolic phenotype within Escherichia coli isolates.

To gain insights into the adaptation of the Escherichia coli species to different environments, we monitored protein abundances using quantitative proteomics and measurements of enzymatic activities of central metabolism in a set of five representative strains grown in four contrasted culture media including human urine. Two hundred and thirty seven proteins representative of the genome-scale metabolic network were identified and classified into pathway categories. We found that nutrient resources shape the general orientation of metabolism through coordinated changes in the average abundances of proteins and in enzymatic activities that all belong to the same pathway category. For example, each culture medium induces a specific oxidative response whatever the strain. On the contrary, differences between strains concern isolated proteins and enzymes within pathway categories in single environments. Our study confirms the predominance of genotype by environment interactions at the proteomic and enzyme activity levels. The buffering of genetic variation when considering life-history traits suggests a multiplicity of evolutionary strategies. For instance, the uropathogenic isolate CFT073 shows a deregulation of iron demand and increased oxidative stress response.

Enhanced degradation of softwood versus hardwood by the white-rot fungus Pycnoporus coccineus.

BACKGROUND: White-rot basidiomycete fungi are potent degraders of plant biomass, with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. Deeper functional analyses are however necessary to understand the enzymatic mechanisms leading to lignocellulose breakdown. The Polyporale fungus Pycnoporus coccineus BRFM310 grows well on both coniferous and deciduous wood. In the present study, we analyzed the early response of the fungus to softwood (pine) and hardwood (aspen) feedstocks and tested the effect of the secreted enzymes on lignocellulose deconstruction. RESULTS: Transcriptomic and proteomic analyses revealed that P. coccineus grown separately on pine and aspen displayed similar sets of transcripts and enzymes implicated in lignin and polysaccharide degradation. In particular, the expression of lignin-targeting oxidoreductases, such as manganese peroxidases, increased upon cultivation on both woods. The sets of enzymes secreted during growth on both pine and aspen were more efficient in saccharide release from pine than from aspen, and characterization of the residual solids revealed polysaccharide conversion on both pine and aspen fiber surfaces. CONCLUSION: The combined analysis of soluble sugars and solid residues showed the suitability of P. coccineus secreted enzymes for softwood degradation. Analyses of solubilized products and residual surface chemistries of enzyme-treated wood samples pointed to differences in fiber penetration by different P. coccineus secretomes. Accordingly, beyond the variety of CAZymes identified in P. coccineus genome, transcriptome and secretome, we discuss several parameters such as the abundance of manganese peroxidases and the potential role of cytochrome P450s and pectin degradation on the efficacy of fungi for softwood conversion.

Shotgun metaproteomic profiling of biomimetic anaerobic digestion processes treating sewage sludge.

Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

BACKGROUND: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.