Validation and Application of UPLC-MS/MS Method to Analysis of Glyphosate and its Metabolites in Water.

A method was developed to determine glyphosate and their metabolites in water. The widespread use of this herbicide in agricultural activities worldwide, despite the reported adverse effects on both the environment and health, is a cause for concern and makes it necessary to monitor its presence through a method that guarantees the determination at trace levels. A direct extraction of the analytes with phosphate buffer was performed with subsequent derivatization with 9-fluorenylmethyl chloroformate. The quantification was determined by Ultra Performance Liquid Chromatography-tandem mass spectrometer. The method was validated through the following parameters: selectivity, detection and quantification limits, linearity, accuracy, precision and uncertainty. The average recoveries ranged between 94.08 and 103.31%. Additionally, detection limits from 0.396 to 0.433 µg/L, and the quantification limit was 5.0 µg/L for all the analytes evaluated. In terms of linearity and precision, the results obtained were in the ranges considered adequate (R2 ≥ 0.99 and CV ≤ 20%), the estimated expanded uncertainty was 12.95, 11.15 and 13.83% for glyphosate, aminomethylphosphonic acid and glufosinate, respectively. This method was successfully applied for the determination of the target analytes in irrigation water samples, detecting concentrations of aminomethylphosphonic acid over limit detection for some sampling sites.

Proteomic analysis reveals the metabolic versatility of Amycolatopsis sp. BX17: A strain native from milpa agroecosystem soil.

Amycolatopsis sp. BX17 is an actinobacterium isolated from milpa soils, which antagonizes the phytopathogenic fungus Fusarium graminearum. Metabolites secreted by the actinobacterium cultured in glucose-free medium inhibited 100% of the mycelial growth of F. graminearum RH1, while the inhibition rate was 65% in medium supplemented with 20 g/L glucose. With the aim of studying how the metabolism of strain BX17 is modulated by glucose as the main carbon source, media with 0 and 20 g/L glucose were selected to analyze the intracellular proteins by quantitative label-free proteomic analysis. Data are available via ProteomeXchange with identifier PXD028644. Proteins identified in bacteria cultured in medium without glucose were involved in glutamate metabolism, the Krebs cycle and the shikimate pathway, suggesting that amino acids are metabolized to synthesize antifungal compounds. In glucose-containing medium, carbon flux was directed mainly toward the synthesis of energy and cell growth. This study shows the metabolic versatility of Amycolatopsis BX17, and strengthens its potential use in designing biotechnological strategies for phytopathogen control. SIGNIFICANCE: Amycolatopsis BX17 is a bacterium isolated from milpa agroecosystems that antagonizes the phytopathogenic fungus Fusarium graminearum. Currently, there is scarce information about the metabolism involved in the biosynthesis of antifungal agents by this genus. We used a label-free proteomic approach to identify the differences in metabolic routes for antifungal biosynthesis in Amycolatopsis BX17 grown in media with 0 and 20 g/L glucose. Taken together the results suggest that the BX17 strain could be synthesizing the antifungal metabolite(s) from the Shikimate pathway through the synthesis and degradation of the amino acid tyrosine, which is a known precursor of glycopeptides with antibiotic and antifungal activity. While the lower antifungal activity of the metabolites secreted by Amycolatopsis BX17 when grown in a medium with glucose as the main carbon source, may be correlated with a lower synthesis of antifungal compounds, due to the directing of carbon flux toward metabolic pathways involved with energy synthesis and cell growth. Likewise, it is possible that the bacteria synthesize other compounds with biological activity, such as glycopeptides with antibiotic activity. These findings are relevant because they represent the first stage to understand the metabolic regulation involved in the biosynthesis of antifungal metabolites by the genus Amycolatopsis. Finally, improving our understanding of the metabolic regulation involved in the biosynthesis of antifungal metabolites is essential to design of strategies in agricultural biotechnology for phytopathogen control.

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin.

BACKGROUND: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. OBJECTIVE: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. METHODS: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. RESULTS: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25°C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45°C and exhibited one transition state with a melting temperature of 76.8°C. CONCLUSION: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

Bioavailability of Compounds Susceptible to Enzymatic Oxidation Enhances Growth of Shiitake Medicinal Mushroom (Lentinus edodes) in Solid-State Fermentation with Vineyard Prunings.

Grapes are widely produced in northwestern Mexico, generating many wood trimmings (vineyard prunings) that have no further local use. This makes vineyard prunings a very attractive alternative for the cultivation of white-rot medicinal mushrooms such as Lentinus edodes. This type of wood can also offer a model for the evaluation of oxidative enzyme production during the fermentation process. We tested the effect of wood from vineyard prunings on the vegetative growth of and production of ligninolytic enzymes in L. edodes in solid-state fermentation and with wheat straw as the control substrate. The specific growth rate of the fungus was 2-fold higher on vineyard pruning culture (µM = 0.95 day-1) than on wheat straw culture (µM = 0.47 day-1). Laccase-specific production was 4 times higher in the vineyard prunings culture than on wheat straw (0.34 and 0.08 mU · mg protein-1 · ppm CO2-1, respectively), and manganese peroxidase production was 3.7 times higher on wheat straw culture than on vineyard prunings (2.21 and 0.60 mU · mg protein-1 · ppm CO2-1, respectively). To explain accurately these differences in growth and ligninolytic enzyme activity, methanol extracts were obtained from each substrate and characterized. Resveratrol and catechins were the main compounds identified in vineyard prunings, whereas epigallocatechin was the only one detected in wheat straw. Compounds susceptible to enzymatic oxidation are more bioavailable in vineyard prunings than in wheat straw, and thus the highest L. edodes growth rate is associated with the presence of these compounds.

New insights into the interaction between the quorum-sensing receptor NprR and its DNA target, or the response regulator Spo0F.

The NprR protein and NprRB signaling peptide comprise a bifunctional quorum-sensing system from the Bacillus cereus group that is involved in transcriptional activation through DNA-binding and in sporulation initiation by binding to Spo0F. We characterized in vitro the direct interactions established by NprR that may be relevant for performing its two functions. Apo-NprR interacted with Spo0F, but not with the target DNA. The NprRB signaling peptide SSKPDIVG that binds strongly to Apo-NprR, failed to bind and disrupt the NprR-Spo0F complex. Finally, the NprR-NprRB complex bound both to Spo0F and the target DNA with similar affinity. Based on our findings, we propose that rather than a switch triggered by NprRB, the NprR/NprRB ratio and the availability of Spo0F binding sites define the function of NprR.

Regulation of sporulation initiation by NprR and its signaling peptide NprRB: molecular recognition and conformational changes.

NprR belongs to the RNPP family of quorum-sensing receptors, a group of intracellular regulators activated directly by signaling oligopeptides in Gram-positive bacteria. In Bacillus thuringiensis (Bt), nprR is located in a transcriptional cassette with nprRB that codes for the precursor of the signaling peptide NprRB. NprR is a transcriptional regulator activated by binding of reimported NprRB; however, several reports suggest that NprR also participates in sporulation but the mechanism is unknown. Our in silico results, based on the structural similarity between NprR from Bt and Spo0F-binding Rap proteins from Bacillus subtilis, suggested that NprR could bind Spo0F to modulate the sporulation phosphorelay in Bt. Deletion of nprR-nprRB cassette from Bt caused a delay in sporulation and defective trigger of the Spo0A∼P-activated genes spoIIA and spoIIIG. The DNA-binding domain of NprR was not necessary for this second function, since truncated NprRΔHTH together with nprRB gene was able to restore the sporulation wild type phenotype in the ΔnprR-nprRB mutant. Fluorescence assays showed direct binding between NprR and Spo0F, supporting that NprR is a bifunctional protein. To understand how the NprR activation by NprRB could result in two different functions, we studied the molecular recognition mechanism between the signaling peptide and the receptor. Using synthetic variants of NprRB, we found that SSKPDIVG displayed the highest affinity (Kd = 7.19 nM) toward the recombinant NprR and demonstrated that recognition involves conformational selection. We propose that the peptide concentration in the cell controls the oligomerization state of the NprR-NprRB complex for switching between its two functions.

Evolution and some functions of the NprR-NprRB quorum-sensing system in the Bacillus cereus group.

Quorum-sensing (QS) is a bacterial mechanism for regulation of gene expression in response to cell density. In Gram-positive bacteria, oligopeptides are the signaling molecules to elicit QS. The RNPP protein family (Rap, NprR, PlcR, and PrgX) are intracellular QS receptors that bind directly to their specific signaling peptide for regulating the transcription of several genes. NprR is the activator of a neutral protease in Bacillus subtilis, and it has been recently related to sporulation, cry genes transcription and extracellular protease activity in strains from the B. cereus group. In the B. thuringiensis genome, downstream nprR, a gene encoding a putative QS signaling propeptide (nprRB) was found. We hypothesized that the nprR and nprRB co-evolved because of their coordinated function in the B. cereus group. A phylogenetic tree of nucleotide sequences of nprR revealed six pherotypes, each corresponding to one putative mature NprRB sequence. The nprR tree does not match the current taxonomic grouping of the B. cereus group or the phylogenetic arrangement obtained when using MLST markers from the same strains. SKPDI and other synthetic peptides encoded in the nprRB gene from B. thuringiensis serovar thuringiensis strain 8741 had effect on temporal regulation of sporulation and expression of a cry1Aa'Z transcriptional fusion, but those peptides that stimulated earlier detection of spores decreased cry1Aa expression suggesting that NprR may either activate or repress the transcription of different genes.