Antibacterial activity against enterovirulent Escherichia coli strains from Bacillus amyloliquefaciens B31 and Bacillus subtilis subsp. subtilis C4: MALDI-TOF MS profiling and MALDI TOF/TOF MS structural analysis on lipopeptides mixtures.

In this work, two Bacillus strains isolated from honey (Bacillus subtilis subsp. subtilis C4; access code HQ828992) and from a waste of an artisanal tannery (Bacillus amyloliquefaciens B31; access code KP893752) were evaluated in order to determine their antibacterial activity against five enteropathogenic Escherichia coli strains. The number of viable cultivable cells of the different strains of E. coli analyzed was determined by plate count. The crude cell-free supernatants of both Bacillus strains exerted anti-E. coli activities, whereas only the lipopeptide fraction of B31 had significant E. coli inhibition. The lipopeptides produced by the Bacillus were analyzed using matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS). The analysis was conducted combining the profiles (fingerprints) of the lipopeptides mixture and the individual lipopeptide fragmentation (tandem mass spectrometry [MS/MS] mode), both obtained from the same lipopeptides mixture sample, for higher output. Data obtained from C4 and B31 revealed that surfactin homologues were the most abundant lipopeptides produced by both strains studied. Additionally, kurstakin, iturin, and fengycin homologues were detected. Using the MS/MS mode, it was demonstrated that isobar compounds belonging to different families were produced by each Bacillus strain (e.g., C-16 bacillomycin D was detected in B31 samples, meanwhile C-15 iturin C was detected in C4). MS/MS analysis contributed with relevant information about the type of lipopeptides synthesized by Bacillus strains studied in this work.

Primary Metabolism Is Distinctly Modulated by Plant Resistance Inducers in Coffea arabica Leaves Infected by Hemileia vastatrix.

Epidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.

Caracterización molecular de los microorganismos presentes durante el proceso fermentativo de los granos de cacao (Theobroma cacao) / Molecular characterization of microorganisms during cocoa-bean fermentation process (Theobroma cacao)

La fermentación de granos de cacao es un proceso espontáneo de post cosecha muy importante para el desarrollo de aroma y sabor a chocolate el cual involucra un sin número de actividades microbianas complejas. En el presente estudio se identifican los microorganismos presentes en granos de cacao antes, durante y después del proceso de fermentación aplicando dos métodos: el análisis de secuenciamiento de ADN y la espectrometría de masas MALDI TOF/TOF. Dentro del grupo de bacterias y levaduras predominantes identificadas por el primer método se encontro a Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), y Pichia kudriavzevii (100%). Asimismo se caracterizó por huella de masas las secuencias peptídicas más importantes de cada cepa identificada. Por otro lado, aplicando el segundo método, se identificaron 57 especies de microorganismos, siendo el 73.7% especies bacterianas y el 26.3% especies de levaduras. Adicionalmente se detectaron secuencias peptídicas de la proteína vicilina responsable del aroma característico de los granos de cacao fermentados y a la proteína albumina de 21KDa.

Cocoa beans fermentation is a spontaneous process of post-harvest very important for the development of chocolate aroma and flavor, which involves a number of complex microbial activities. In this work, we identify the microorganisms present in cocoa beans before, during and after the fermentation process, applying DNA sequencing analysis and MALDI TOF / TOF mass spectrometry. With the first method, the predominant bacteria and yeast identified were Lactobacillus plantarum (29%), L. brevis (18%), Bacillus cereus (15%), Pediococcus acidilactici (12%), and Pichia kudriavzevii (100%). The most important peptide sequences of each identified strain by mass fingerprint were characterized too. By the second method, 51 species of microorganisms being 73.7% bacterial species and 26.3% yeast species were identified. Additionally peptide sequences responsible Vicilin protein characteristic aroma of the fermented cocoa beans and the albumin protein of 21KDa were detected.

Proteomic analysis of the regenerating liver following 2/3 partial hepatectomy in rats

BACKGROUND: Liver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism. RESULTS: Protein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR. CONCLUSIONS: For the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.