Correction: A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

[This corrects the article DOI: 10.1371/journal.pone.0166561.].

The Adaptation of Botrytis cinerea Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways.

Proteomic Approach to Anemonia sulcata and Its Symbiont Symbiodinium spp. as New Source of Potential Biotechnological Applications and Climate Change Biomarkers.

Marine ecosystems are among the richest in terms of biodiversity, and at present, still remain largely unknown today. In the molecular biology era, several analyses have been conducted to unravel the biological processes in this ecosystem. These systems have provided biotechnological solutions to current problems, including the treatment of diseases, as well as for the development of new biotechnological tools with applications in biomedicine and/or agri-food. In addition, in the context of climate change and global warming, these studies become even more necessary for the development of molecular tools that allow a reliable follow-up of this situation to anticipate alterations and responses of bioindicator species and to create a database to prevent and predict the environmental and climatic changes before the damage is irreversible. Proteomics approaches have revealed their potential use to obtain the set of biological effectors that lead to the real biological station on a specific stage, the proteins. In addition, proteomics-based algorithms have allowed the discovery of proteins with new potential biotechnological applications from proteome data through "applied proteomics". In this project, the first proteome analysis of the sea anemone, Anemonia sulcata, and its symbiont has been developed. These organisms present a wide distribution sea ecosystem. In Spain, it is accepted as a fishing and aquaculture species. Moreover, Anemonia sulcate has a symbiotic relation with autotroph Dinoflagellates, Symbiodinium spp., that allows the study of its relation at the molecular level. For the first characterization of A. sulcata proteome, three independent biological replicates were used, and proteins were extracted and analyzed by LC-MS/MS, allowing the quantification of 325 proteins, 81 from Symbiodinium spp. proteins and 244 from A. sulcata proteins. These proteins were subjected to gene ontology categorization by Cellular Component, Molecular Function and Biological Process. These analyzes have allowed the identification of biomarkers of gene expression as potential powerful emerging diagnostic tools to identify and characterize the molecular drivers of climate change stresses and improve monitoring techniques. In addition, through the application of novel algorithms for the detection of bioactive compounds based on the analysis of molecules of marine origin, the proteome has allowed the identification of proteins with potential applications in the fields of biomedicine and agri-food.

Unravelling the Initial Triggers of Botrytis cinerea Infection: First Description of Its Surfactome.

Botrytis cinerea is a critically important phytopathogenic fungus, causing devastating crop losses; signal transduction cascades mediate the "dialogue" among the fungus, plant, and environment. Surface proteins play important roles as front-line receptors. We report the first description of the surfactome of a filamentous fungus. To obtain a complete view of these cascades during infection of B. cinerea, its surfactome has been described by optimization of the "shaving" process and LC-MS/MS at two different infection stages, and with both rapid and late responses to environmental changes. The best results were obtained using PBS buffer in the "shaving" protocol. The surfactome obtained comprises 1010 identified proteins. These have been categorized by gene ontology and protein-protein interactions to reveal new potential pathogenicity/virulence factors. From these data, the percentage of total proteins predicted for the genome of the fungus represented by proteins identified in this and other proteomics studies is calculated at 54%, a big increase over the previous 12%. The new data may be crucial for understanding better its biological activity and pathogenicity. Given its extensive exposure to plants and environmental conditions, the surfactome presents innumerable opportunities for interactions between the fungus and external elements, which should offer the best targets for fungicide development.

Deciphering the Dynamics of Signaling Cascades and Virulence Factors of B. cinerea during Tomato Cell Wall Degradation.

The ascomycete Botrytis cinerea is one of the most relevant plant pathogenic fungi, affecting fruits, flowers, and greenhouse-grown crops. The infection strategy used by the fungus comprises a magnificent set of tools to penetrate and overcome plant defenses. In this context, the plant-pathogen communication through membrane receptors and signal transduction cascades is essential to trigger specific routes and the final success of the infection. In previous reports, proteomics approaches to B. cinerea signal transduction cascades changes in response to different carbon source and plant-based elicitors have been performed. Analyzing the secretome, membranome, phosphoproteome, and the phosphomembranome. Moreover, phenotypic changes in fungal biology was analyzed, specifically toxin production. To obtain the whole picture of the process and reveal the network from a system biology approach, this proteomic information has been merged with the phenotypic characterization, to be analyzed using several bioinformatics algorithms (GO, STRING, MCODE) in order to unravel key points in the signal transduction regulation crucial to overcome plant defenses, as well as new virulence/pathogenicity factors that could be used as therapeutic targets in the control of the gray mold rot disease. A total of 1721 and 663 exclusive or overexpressed proteins were identified under glucose (GLU) and deproteinized tomato cell walls (TCW), summarizing all of the protein identifications under phenotypic characterized stages. Under GO analysis, there are more biological process and molecular functions described in GLU, highlighting the increase in signaling related categories. These results agree with the high number of total identified proteins in GLU, probably indicating a more varied and active metabolism of the fungus. When analyzing only GO annotations related with signal transduction, it was revealed that there were proteins related to TOR signaling, the phosphorelay signal transduction system, and inositol lipid-mediated signaling, only under GLU conditions. On the contrary, calcium-mediated signaling GO annotation is only present between the proteins identified under TCW conditions. To establish a potential relationship between expressed proteins, cluster analyses showed 41 and 14 clusters under GLU and TCW conditions, confirming an increase in biological activity in GLU, where we identified a larger number of clusters related to transcription, translation, and cell division, between others. From these analyses, clusters related to signal transduction and clusters related to mycotoxin production were found, which correlated with the phenotypic characterization. The identification of the proteins encompassed in each condition and signal transduction cascade would provide the research community with new information about the B. cinerea infection process and potential candidates of pathogenicity/virulence factors, overcoming plant defenses, and new therapeutic targets.

An arsRB resistance operon confers tolerance to arsenite in the environmental isolate Terribacillus sp. AE2B 122.

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome.

Development of the "Applied Proteomics" Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana.

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the "omics" approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana.

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

An "omic" approach to Pyrocystis lunula: New insights related with this bioluminescent dinoflagellate.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an "omics" approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. SIGNIFICANCE: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described.

Proteomic study of the membrane components of signalling cascades of Botrytis cinerea controlled by phosphorylation.

Protein phosphorylation and membrane proteins play an important role in the infection of plants by phytopathogenic fungi, given their involvement in signal transduction cascades. Botrytis cinerea is a well-studied necrotrophic fungus taken as a model organism in fungal plant pathology, given its broad host range and adverse economic impact. To elucidate relevant events during infection, several proteomics analyses have been performed in B. cinerea, but they cover only 10% of the total proteins predicted in the genome database of this fungus. To increase coverage, we analysed by LC-MS/MS the first-reported overlapped proteome in phytopathogenic fungi, the "phosphomembranome" of B. cinerea, combining the two most important signal transduction subproteomes. Of the 1112 membrane-associated phosphoproteins identified, 64 and 243 were classified as exclusively identified or overexpressed under glucose and deproteinized tomato cell wall conditions, respectively. Seven proteins were found under both conditions, but these presented a specific phosphorylation pattern, so they were considered as exclusively identified or overexpressed proteins. From bioinformatics analysis, those differences in the membrane-associated phosphoproteins composition were associated with various processes, including pyruvate metabolism, unfolded protein response, oxidative stress response, autophagy and cell death. Our results suggest these proteins play a significant role in the B. cinerea pathogenic cycle.

A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria.

The chemical synthesis of new lipophilic polyphenols with improved properties presents technical difficulties. Here we describe the selection, isolation and identification of lipolytic bacteria from food-processing industrial wastes, and their use for tailoring a new set of compounds with great interest in the food industry. These bacteria were employed to produce lipolytic supernatants, which were applied without further purification as biocatalysts in the chemoselective and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols. The chemoselectivity of polyphenols acylation/deacylation was analyzed, revealing the preference of the lipases for phenolic hydroxyl groups and phenolic esters. In addition, the alcoholysis of peracetylated 3,4-dihydroxyphenylglycol resulted in a series of lipophilic 2-alkoxy-2-(3,4-dihydroxyphenyl)ethyl acetate through an unexpected lipase-mediated etherification at the benzylic position. These new compounds are more lipophilic and retained their antioxidant properties. This approach can provide access to unprecedented derivatives of 3,4-dihydroxyphenylglycol with improved properties.

Selection and characterization of biofuel-producing environmental bacteria isolated from vegetable oil-rich wastes.

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.