Evaluating the biocontrol potential of Canadian strain Bacillus velezensis 1B-23 via its surfactin production at various pHs and temperatures.

BACKGROUND: Microorganisms, including Bacillus species are used to help control plant pathogens, thereby reducing reliance on synthetic pesticides in agriculture. Bacillus velezensis strain 1B-23 has been shown to reduce symptoms of bacterial disease caused by Clavibacter michiganensis subsp. michiganensis in greenhouse-grown tomatoes, with in vitro studies implicating the lipopeptide surfactin as a key antimicrobial. While surfactin is known to be effective against many bacterial pathogens, it is inhibitory to a smaller proportion of fungi which nonetheless cause the majority of crop diseases. In addition, knowledge of optimal conditions for surfactin production in B. velezensis is lacking. RESULTS: Here, B. velezensis 1B-23 was shown to inhibit in vitro growth of 10 fungal strains including Candida albicans, Cochliobolus carbonum, Cryptococcus neoformans, Cylindrocarpon destructans Fusarium oxysporum, Fusarium solani, Monilinia fructicola, and Rhizoctonia solani, as well as two strains of C. michiganensis michiganensis. Three of the fungal strains (C. carbonum, C. neoformans, and M. fructicola) and the bacterial strains were also inhibited by purified surfactin (surfactin C, or [Leu7] surfactin C15) from B. velezensis 1B-23. Optimal surfactin production occurred in vitro at a relatively low temperature (16 °C) and a slightly acidic pH of 6.0. In addition to surfactin, B. velenzensis also produced macrolactins, cyclic dipeptides and minor amounts of iturins which could be responsible for the bioactivity against fungal strains which were not inhibited by purified surfactin C. CONCLUSIONS: Our study indicates that B. velezensis 1B-23 has potential as a biocontrol agent against both bacterial and fungal pathogens, and may be particularly useful in slightly acidic soils of cooler climates.

Structure Activity Relationship for Fumonisin Phytotoxicity.

Fumonisins are mycotoxins produced by a number of species of Fusarium and Aspergillus. They are polyketides that possess a linear polyol structure with two tricarballylic acid side chains and an amine moiety. Toxicity results from their inhibition of Ceramide Synthase (CerS), which perturbs sphingolipid concentrations. The tricarballylic side chains and amine group of fumonisins are key molecular features responsible for inhibiting CerS, however their individual contributions toward overall toxicity are not fully understood. We have recently reported novel, deaminated fumonisins produced by A. niger and have identified an enzyme (AnFAO) responsible for their synthesis. Here we performed a structure/function activity assay to investigate the individual contributions of the tricarballylic acid and amine toward overall fumonisin toxicity. Lemna minor was treated at 40 µM against FB1, hydrolyzed FB1 (hFB1), deaminated FB1 (FPy1), or hydrolyzed/deaminated (hFPy1). Four end points were monitored: plant dry weight, frond surface area, lipidomics, and metabolomics. Overall, hFB1 was less toxic than FB1 and FPy1 was less toxic than hFB1. hFPy1 which lacks both the amine group and tricarballylic side chains was also less toxic than FB1 and hFB1, however it was not significantly less toxic than FPy1. Lipidomic analysis showed that FB1 treatment significantly increased levels of phosphotidylcholines, ceramides, and pheophorbide A, while significantly decreasing the levels of diacylglycerides, sulfoquinovosyl diacylglycerides, and chlorophyll. Metabolomic profiling revealed a number of significantly increased compounds that were unique to FB1 treatment including phenylalanine, asymmetric dimethylarginine (ADMA), S-methylmethionine, saccharopine, and tyrosine. Conversely, citrulline, N-acetylornithine and ornithine were significantly elevated in the presence of hFB1 but not any of the other fumonisin analogues. These data provide evidence that although removal of the tricarballylic side chains significantly reduces toxicity of fumonisins, the amine functional group is a key contributor to fumonisin toxicity in L. minor and justify future toxicity studies in mammalian systems.

The Effects of Ditch Management in Agroecosystems on Embryonic and Tadpole Survival, Growth, and Development of Northern Leopard Frogs (Lithobates pipiens).

Agricultural drainage ditches help remove excess water from fields and provide habitat for wildlife. Drainage ditch management, which includes various forms of vegetation clearing and sediment dredging, can variably affect the ecological function of these systems. To determine whether ditch conditions following dredging/vegetation clearing management affected the survival, growth, and development of embryos and tadpoles of northern leopard frogs (Lithobates pipiens), we conducted three field studies using in situ cages over 2 years. We measured nutrients, pesticides, and other water quality properties in vegetated/unmanaged (i.e., no clearing or dredging) and newly cleared/dredged (i.e., treeless, then dredged), clay-bottomed drainage ditches in a river basin in Eastern Ontario, Canada. Nutrients, atrazine, and total neonicotinoid concentrations were generally lower at the cleared/dredged sites, whereas glyphosate was at higher concentrations. In contrast, water-quality variables measured in situ, particularly temperature, dissolved oxygen, and turbidity, tended to be higher in the cleared/dredged sites. Total phosphorous and total organic carbon concentrations at all sites were above the recommended limits for amphibian assays. No significant differences were detected in the survival, hatching success, or development of embryos among the ditch management treatments, but premature hatching was observed at one vegetated/unmanaged site where high specific conductivity may have been formative. We found the cleared/dredged sites supported earlier tadpole growth and development, likely as a result of the higher water temperatures. Increased temperature may have offset other growth/development stressors, such as those related to water chemistry. However, the long-term consequences of these differences on amphibian populations requires further study.

Simultaneous quantification of five pharmaceuticals and personal care products in biosolids and their fate in thermo-alkaline treatment.

The presence of pharmaceuticals and personal care products (PPCPs) in biosolids applied to farmland is of concern due to their potential accumulation in the environment and the subsequent effects on humans. Thermo-alkaline hydrolysis (TAH) is a method used for greater stabilization of biosolids after anaerobic digestion. In this work, the effect of TAH on five selected PPCPs including fluoroquinolone antibiotics, ciprofloxacin (CIP), and ofloxacin (OFLX), and three commonly used antimicrobial agents, miconazole (MIC), triclosan (TCS) and triclocarban (TCC) was evaluated. At the onset, extraction and analytical methods were optimized for maximum simultaneous recovery and LC-MS quantification of the target PPCPs from both water and biosolids for improved accuracy. The compounds were detected in the range of 54 ± 3 to 6166 ± 532 ng/g in raw biosoilds collected from a local WWTP. Next, batch control adsorption experiments of the selected PPCPs were conducted in various sludges, which indicated about 89%-98% sorption of the PPCPs onto solid phase due to their high octanol-water coefficients. Subsequently, thermo-alkaline (pH 9.5, 75 °C, 45 min) hydrolysis (TAH) was conducted to determine the extent of degradation of these compounds in deionized (DI) water and biosolids due to treatment. The degradation of these compounds due to TAH ranged from 42% to 99% and 37%-41% in pure water and biosolids, respectively, potentially lowering their risk in the environment due to land application. A list of compounds for which the optimized analytical method potentially can be used for detection and quantification in environmental samples is provided in the supporting document.

Deciphering S-methylcysteine biosynthesis in common bean by isotopic tracking with mass spectrometry.

The suboptimal content of sulfur-containing amino acids methionine and cysteine prevents common bean (Phaseolus vulgaris) from being an excellent source of protein. Nutritional improvements to this significant crop require a better understanding of the biosynthesis of sulfur-containing compounds including the nonproteogenic amino acid S-methylcysteine and the dipeptide γ-glutamyl-S-methylcysteine, which accumulate in seed. In this study, seeds were incubated with isotopically labelled serine, cysteine or methionine and analyzed by reverse phase chromatography-high resolution mass spectrometry to track stable isotopes as they progressed through the sulfur metabolome. We determined that serine and methionine are the sole precursors of free S-methylcysteine in developing seeds, indicating that this compound is likely to be synthesized through the condensation of O-acetylserine and methanethiol. BSAS4;1, a cytosolic ß-substituted alanine synthase preferentially expressed in developing seeds, catalyzed the formation of S-methylcysteine in vitro. A higher flux of labelled serine or cysteine was observed in a sequential pathway involving γ-glutamyl-cysteine, homoglutathione and S-methylhomoglutathione, a likely precursor to γ-glutamyl-S-methylcysteine. Preferential incorporation of serine over cysteine supports a subcellular compartmentation of this pathway, likely to be in the chloroplast. The origin of the methyl group in S-methylhomoglutathione was traced to methionine. There was substantial incorporation of carbons from methionine into the ß-alanine portion of homoglutathione and S-methylhomoglutathione, suggesting the breakdown of methionine by methionine γ-lyase and conversion of α-ketobutyrate to ß-alanine via propanoate metabolism. These findings delineate the biosynthetic pathways of the sulfur metabolome of common bean and provide an insight that will aid future efforts to improve nutritional quality.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance.

BACKGROUND: We previously reported on the interplay between miR156/SPL13 and WD40-1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners. RESULT: Transcript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance. CONCLUSIONS: We conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

Label-free quantitative proteomic analysis of alfalfa in response to microRNA156 under high temperature.

BACKGROUND: Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level. RESULTS: In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P < 0.05; |log2FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors. CONCLUSIONS: These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

Fate of micropollutants in chemically enhanced primary treatment using recovered coagulants.

In this study, the fate of several micropollutants (MPs) in wastewater due to coagulation using both fresh and recovered aluminum and iron coagulants was determined. 18 MPs from different groups such as antibiotics, food additives, and surfactants were selected and spiked into the primary influent collected from a local wastewater plant. The distribution of MPs in the recovered coagulant and treated effluent after coagulation was determined for both fresh and recycled coagulants. The distribution of MPs in wastewater and the removal during coagulation were compound specific; MPs with log Kow < 2.5 were predominantly present in the effluent after coagulation, while MPs with log Kow > 2.5 were sorbed on the coagulated sludge. The distribution ratio (Kd) of all the MPs (diclofenac, clarithromycin, etc.) with log Kow > 2.5 was determined along with their extent of accumulation in sludge due to the recycling of coagulants. Compounds such as sulfamethoxazole, erythromycin and sulfathiazole, showed low removal during coagulation. The tetracycline group of compounds showed possible chelation with iron and aluminum. Only <10% of the initially spiked MPs with log Kow > 2.5 was being recycled with the recovered coagulant, thus alleviating the concern of accumulation of the MPs during recycle of the coagulants.

Isoflavonoid-specific prenyltransferase gene family in soybean: GmPT01, a pterocarpan 2-dimethylallyltransferase involved in glyceollin biosynthesis.

Phytoalexin glyceollins are soybean-specific antimicrobial compounds that are derived from the isoflavonoid pathway. They are synthesized by soybean in response to extrinsic stress such as pathogen attack or injury, thereby conferring partial resistance if synthesized rapidly at the site of infection and at the required concentration. Soybean produces multiple forms of glyceollins that result from the differential prenylation reaction catalyzed by prenyltransferases (PTs) on either the C-2 or C-4 carbon of a pterocarpan glycinol. The soybean genome contains 77 PT-encoding genes (GmPTs) where at least 11 are (iso)flavonoid-specific. Transcript accumulation of five candidates GmPTs was increased in response to Phytophthora sojae infection, suggesting their role in phytoalexin synthesis. The induced GmPTs localize to plastids and display tissue-specific expression. We have in this study identified two additional GmPTs: an isoflavone dimethylallyltransferase 3 (IDT3); and a glycinol 2-dimethylallyl transferase GmPT01. GmPT01 prenylates (-)-glycinol at the C-2 position, localizes in the plastid, and exhibits root-specific gene expression. Furthermore, its expression is induced rapidly in response to stress, and is associated with a quantitative trait loci linked with resistance to P. sojae. Based on these results, we conclude that GmPT01 are possibly one of the loci involved in conferring partial resistance against stem and root rot disease in soybean.

Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection.

Endocytosis and endosomal trafficking regulate the proteins targeted to the plasma membrane and play essential roles in diverse cellular processes, including responses to pathogen attack. Here, we report the identification of Glycine max (soybean) endocytosis dynamin-like protein 5A (GmSDL5A) associated with purified soybean mosaic virus (SMV) virions from soybean using a bottom-up proteomics approach. Knockdown of GmSDL5A and its homologous gene GmSDL12A inhibits SMV infection in soybean. The role of analogous dynamin-like proteins in potyvirus infection was further confirmed and investigated using the Arabidopsis/turnip mosaic virus (TuMV) pathosystem. We demonstrate that dynamin-related proteins 2A and 2B in Arabidopsis thaliana (AtDRP2A, AtDRP2B), homologs of GmSDL5A, are recruited to the virus replication complex (VRC) of TuMV. TuMV infection is inhibited in both A. thalianadrp2a (atdrp2a) and atdrp2b knockout mutants. Overexpression of AtDRP2 promotes TuMV replication and intercellular movement. AtRDP2 interacts with TuMV VPg, CP, CI, and 6K2. Of these viral proteins, VPg, CP, and CI are essential for viral intercellular movement, and 6K2, VPg, and CI are critical components of the VRC. We reveal that VPg and CI are present in the punctate structures labeled by the endocytic tracer FM4-64, suggesting that VPg and CI can be endocytosed. Treatment of plant leaves with a dynamin-specific inhibitor disrupts the delivery of VPg and CI to endocytic structures and suppresses TuMV replication and intercellular movement. Taken together, these data suggest that dynamin-like proteins are novel host factors of potyviruses and that endocytic processes are involved in potyvirus infection.IMPORTANCE It is well known that animal viruses enter host cells via endocytosis, whereas plant viruses require physical assistance, such as human and insect activities, to penetrate the host cell to establish their infection. In this study, we report that the endocytosis pathway is also involved in virus infection in plants. We show that plant potyviruses recruit endocytosis dynamin-like proteins to support their infection. Depletion of them by knockout of the corresponding genes suppresses virus replication, whereas overexpression of them enhances virus replication and intercellular movement. We also demonstrate that the dynamin-like proteins interact with several viral proteins that are essential for virus replication and cell-to-cell movement. We further show that treatment of a dynamin-specific inhibitor disrupts endocytosis and inhibits virus replication and intercellular movement. Therefore, the dynamin-like proteins are novel host factors of potyviruses. The corresponding genes may be manipulated using advanced biotechnology to control potyviral diseases.

Characterization and complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6.

BACKGROUND: Bacillus velezensis is an endospore-forming, free-living soil bacterium with potential as a biopesticide against a broad spectrum of microbial pathogens of plants. Its potential for commercial development is enhanced by rapid replication and resistance to adverse environmental conditions, typical of Bacillus species. However, the use of beneficial microbes against phytopathogens has not gained dominance due to limitations that may be overcome with new biopesticidal strains and/or new biological knowledge. RESULTS: Here, we isolated B. velezensis strain 9D-6 and showed that it inhibits the in vitro growth of prokaryotic and eukaryotic pathogens, including the bacteria Bacillus cereus , Clavibacter michiganensis, Pantoea agglomerans, Ralstonia solanacearum, Xanthomonas campestris, and Xanthomonas euvesicatoria; and the fungi Alternaria solani, Cochliobolus carbonum, Fusarium oxysporum, Fusarium solani, Gibberella pulicaris, Gibberella zeae, Monilinia fructicola, Pyrenochaeta terrestris and Rhizoctonia solani. Antimicrobial compounds with activity against Clavibacter michiganensis were isolated from B. velezensis 9D-6 and characterized by high resolution LC-MS/MS, yielding formulae of C52H91N7O13 and C53H93N7O13, which correspond to [Leu7] surfactins C14 and C15 (also called surfactin B and surfactin C), respectively. We further sequenced the B. velezensis 9D-6 genome which consists of a single circular chromosome and revealed 13 gene clusters expected to participate in antimicrobial metabolite production, including surfactin and two metabolites that have not typically been found in this species - ladderane and lantipeptide. Despite being unable to inhibit the growth of Pseudomonas syringae DC3000 in an in vitro plate assay, B. velezensis 9D-6 significantly reduced root colonization by DC3000, suggesting that 9D-6 uses methods other than antimicrobials to control phytopathogens in the environment. Finally, using in silico DNA-DNA hybridization (isDDH), we confirm previous findings that many strains currently classified as B. amyloliquefaciens are actually B. velezensis. CONCLUSIONS: The data presented here suggest B. velezensis 9D-6 as a candidate plant growth promoting bacterium (PGPB) and biopesticide, which uses a unique complement of antimicrobials, as well as other mechanisms, to protect plants against phytopathogens. Our results may contribute to future utilization of this strain, and will contribute to a knowledge base that will help to advance the field of microbial biocontrol.

Diagnostic fragmentation filtering for the discovery of new chaetoglobosins and cytochalasins.

RATIONALE: Microbial natural products are often biosynthesized as classes of structurally related compounds that have similar tandem mass spectrometry (MS/MS) fragmentation patterns. Mining MS/MS datasets for precursor ions that share diagnostic or common features enables entire chemical classes to be identified, including novel derivatives that have previously been unreported. Analytical data analysis tools that can facilitate a class-targeted approach to rapidly dereplicate known compounds and identify structural variants within complex matrices would be useful for the discovery of new natural products. METHODS: A diagnostic fragmentation filtering (DFF) module was developed for MZmine to enable the efficient screening of MS/MS datasets for class-specific product ions(s) and/or neutral loss(es). This approach was applied to series of the structurally related chaetoglobosin and cytochalasin classes of compounds. These were identified from the culture filtrates of three fungal genera: Chaetomium globosum, a putative new species of Penicillium (called here P. cf. discolor: closely related to P. discolor), and Xylaria sp. Extracts were subjected to LC/MS/MS analysis under positive electrospray ionization and operating in a data-dependent acquisition mode, performed using a Thermo Q-Exactive mass spectrometer. All MS/MS datasets were processed using the DFF module and screened for diagnostic product ions at m/z 130.0648 and 185.0704 for chaetoglobosins, and m/z 120.0808 and 146.0598 for cytochalasins. RESULTS: Extracts of C. globosum and P. cf. discolor strains revealed different mixtures of chaetoglobosins, whereas the Xylaria sp. produced only cytochalasins; none of the strains studied produced both classes of compounds. The dominant chaetoglobosins produced by both C. globosum and P. cf. discolor were chaetoglobosins A, C, and F. Tetrahydrochaetoglobosin A was identified from P. cf. discolor extracts and is reported here for the first time as a natural product. The major cytochalasins produced by the Xylaria sp. were cytochalasin D and epoxy cytochalasin D. A larger unknown "cytochalasin-like" molecule with the molecular formula C38 H47 NO10 was detected from Xylaria sp. culture filtrate extracts and is a current target for isolation and structural characterization. CONCLUSIONS: DFF is an effective LC/MS data analysis approach for rapidly identifying entire classes of compounds from complex mixtures. DFF has proved useful in the identification of new natural products and allowing for their partial characterization without the need for isolation.

NbEXPA1, an α-expansin, is plasmodesmata-specific and a novel host factor for potyviral infection.

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD-enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2-fold increase) and 48 (≥2-fold reduction) are significantly differentially accumulated in the PD-enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α-expansin designated NbEXPA1, a cell wall loosening protein, is PD-specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA-dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD-proteome dataset that is useful in future studies to expound PD biology and PD-mediated virus-host interactions but also characterizes NbEXPA1 as the first PD-specific cell wall loosening protein and its essential role in potyviral infection.

Spectral Counting Approach to Measure Selectivity of High-Resolution LC-MS Methods for Environmental Analysis.

Advances in high-resolution mass spectrometers have allowed for the development of nontargeted screening methods, where data sets can be archived and retrospectively mined as new environmental contaminants are identified. We have developed a spectral counting approach to calculate the selectivities of LC-MS acquisition modes taking mass accuracy, sample matrix, and the analyte properties into account. The selectivities of high-resolution MS (HRMS) alone or in combination with all-ion-fragmentation (AIF), data-independent-acquisition (DIA), and data-dependent-acquisition (DDA) modes, performed on a Q-Exactive Orbitrap were compared by retrospectively screening surface water samples for 95 pharmaceuticals. Samples were reanalyzed using targeted LC-MS/MS to confirm the accuracy of each acquisition method and to quantitate the 29 putatively detected drugs. LC-HRMS provided the lowest calculated selectivities and accordingly produced the highest number of false positives (6). In contrast, DDA provided the highest selectivities, yielding only one false positive; however, it was bias toward the most intense signals resulting in the detection of only 10 compounds. AIF had lower selectivities than traditional LC-MS/MS, produced one false positive and did not detect 6 confirmed compounds. Because of the high-quality archived data, DIA selectivities were better than traditional LC-MS/MS, showed no bias toward the most intense signals, achieved low limits of detection, and confidently detected the greatest number of pharmaceuticals (22) with only one false positive. This spectral counting method can be used across different instrument platforms or samples and provides a robust and empirical estimation of selectivities to give more confident detection of trace analytes.

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming.

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are "hot spots" for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the "resistome" of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPPAZI 4, encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPPAZI 4 can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and "repurposed" by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.

Formation of oxidative and non-oxidative dimers in metallothioneins: Implications for charge-state analysis for structural determination.

RATIONALE: Metallothioneins (MTs) are a class of dynamic proteins that have been investigated extensively using mass spectrometric methods due to their amenability to ionization. Here we detect the formation of oxidative and non-oxidative MT dimers using high-resolution mass spectrometry (HRMS) which has previously been overlooked with lower-resolution techniques. METHODS: Recombinant human MT1a and its isolated domain fragments were analyzed by high-resolution Thermo Q-Exactive and Bruker time-of-flight (TOF) mass spectrometers. Covalent Cys modification was performed using N-ethylmalemide to probe the effect of Cys oxidation on dimer formation. RESULTS: Dimerization was detected in the analysis of select charge states of Zn7 MT and apo-ßMT. Specifically, high resolution (140 k) revealed the +6 dimer peaks overlapping with the +3 charge state, but not with the other charge states (+4, +5, +6). The proteins with covalently modified Cys did not show dimer formation in any of their charge states. Apo-α and apo-ßαMT also did not form dimers under the conditions tested. CONCLUSIONS: Dimerization of MT was detected for zinc metalated and certain apo-MT forms with HRMS, which was not seen with lower-resolution techniques. These dimers appear overlapped only with certain charge states, confounding their analysis for structural characterization of MTs. The Zn-MT dimers appeared to be non-oxidative; however, the formation of dimers in the apo-protein is likely dependent on Cys oxidation.

Metabolites of Trichoderma species isolated from damp building materials.

Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.

Structure and Stability of Carbohydrate-Lipid Interactions. Methylmannose Polysaccharide-Fatty Acid Complexes.

We report a detailed study of the structure and stability of carbohydrate-lipid interactions. Complexes of a methylmannose polysaccharide (MMP) derivative and fatty acids (FAs) served as model systems. The dependence of solution affinities and gas-phase dissociation activation energies (Ea ) on FA length indicates a dominant role of carbohydrate-lipid interactions in stabilizing (MMP+FA) complexes. Solution (1) H NMR results reveal weak interactions between MMP methyl groups and FA acyl chain; MD simulations suggest the complexes are disordered. The contribution of FA methylene groups to the Ea is similar to that of heats of transfer of n-alkanes from the gas phase to polar solvents, thus suggesting that MMP binds lipids through dipole-induced dipole interactions. The MD results point to hydrophobic interactions and H-bonds with the FA carboxyl group. Comparison of collision cross sections of deprotonated (MMP+FA) ions with MD structures suggests that the gaseous complexes are disordered.

Data independent acquisition-digital archiving mass spectrometry: application to single kernel mycotoxin analysis of Fusarium graminearum infected maize.

New and conjugated mycotoxins of concern to regulators are frequently being identified, necessitating the costly need for new method development and sample reanalysis. In response, we developed an LC-data independent acquisition (LC-DIA) method on a Q-Exactive Orbitrap mass spectrometer tailored for mycotoxins analysis. This method combines absolute quantification of targeted fungal metabolites with non-targeted digital archiving (DA) of data on all ionizable compounds for retrospective analysis. The quantitative power of this approach was assessed by spiking 23 mycotoxins at a range of concentrations into clean maize extracts. The linearity and limits of detection achieved were comparable to conventional LC-MS/MS and significantly better than 'all-ion-fragmentation' scanning mode. This method was applied to single kernel analysis of Fusarium infected maize, where we quantified nine Fusarium metabolites and three metabolites from unexpected contaminations by Alternaria and Penicillium species. Retrospective analysis of this data set allowed us to detect the recently reported 15-acetyldeoxynivalenol-3-O-ß-D-glucoside without requiring re-analysis of the samples. To our knowledge, this is the first reported occurrence of this conjugated mycotoxin in naturally contaminated maize, and led us to further study maize artificially inoculated with the 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol chemotypes of Fusarium graminearum. Analysis of these samples showed that the maize genotype tested glycosylates 15-acetyldeoxynivalenol but not 3-acetyldeoxynivalenol likely because the glycosylation site was blocked. In addition to confirming that these two F. graminearum chemotypes behave differently when infecting the host plant, it demonstrates the utility of using a single screening method to quantify known mycotoxins and archive a completely non-targeted dataset for future analysis.

Product ion filtering with rapid polarity switching for the detection of all fumonisins and AAL-toxins.

RATIONALE: Fumonisins and AAL-toxins are structurally similar mycotoxins that contaminate agricultural crops and foodstuffs. Traditional analytical screening methods are designed to target the known compounds for which standards are available but there is clear evidence that many other derivatives exist and could be toxic. A fast, semi-targeted method for the detection of all known fumonisins, AAL-toxins and related emerging toxins is required. METHODS: Strains of Fusarium verticillioides, Alternaria arborescens and Aspergillus welwitschiae were grown on their associated crops (maize, tomatoes, and grapes, respectively). Extracts were first analyzed in negative mode using product ion filtering to detect the tricarballylic ester product ion that is common to fumonisins and AAL-toxins (m/z 157.0142). During the same liquid chromatography (LC) run, rapid polarity switching was then used to collect positive mode tandem mass spectrometric (MS(2) ) data for characterization of the detected compounds. RESULTS: Fumonisin B1 , B2 , B3 and B4 were detected on Fusarium contaminated maize, AAL-toxins TA, TB, TD, TE were detected on Alternaria inoculated tomatoes and fumonisin B2 , B4 and B6 on Aspergillus contaminated grapes. Additionally, over 100 structurally related compounds possessing a tricarballylic ester were detected from the mould inoculated plant material. These included a hydroxyl-FB1 from F. verticillioides inoculated maize, keto derivatives of AAL-toxins from A. arborescens inoculated tomatoes, and two previously unreported classes of non-aminated fumonisins from Asp. welwitschiae contaminated grapes. CONCLUSIONS: A semi-targeted method for the detection of all fumonisins and AAL-toxins in foodstuffs was developed. The use of the distinctive tricarballylic ester product anion for detection combined with rapid polarity switching and positive mode MS(2) is an effective strategy for differentiating between known isomers such as FB1 and FB6 . This analytical tool is also effective for the identification of new compounds as evident from the discoveries of the previously unreported hydroxyl-FB1 , keto-AAL-toxins, and the two new families of non-aminated fumonisins.