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1. Reverse diauxie phenotype in Pseudomonas aeruginosa biofilm revealed by exometabolomics and label-free proteomics

   https://doi.org/10.1038/s41522-019-0104-7  

   Yung, Yeni P. ; McGill, S. Lee ; Chen, Hui ; Park, Heejoon ; Carlson, Ross P. ; Hanley, Luke ( October 2019 , npj Biofilms and Microbiomes)

   Microorganisms enhance fitness by prioritizing catabolism of available carbon sources using a process known as carbon catabolite repression (CCR). Planktonically grownPseudomonas aeruginosais known to prioritize the consumption of organic acids including lactic acid over catabolism of glucose using a CCR strategy termed “reverse diauxie.”P. aeruginosais an opportunistic pathogen with well-documented biofilm phenotypes that are distinct from its planktonic phenotypes. Reverse diauxie has been described in planktonic cultures, but it has not been documented explicitly inP. aeruginosabiofilms. Here a combination of exometabolomics and label-free proteomics was used to analyze planktonic and biofilm phenotypes for reverse diauxie.P. aeruginosabiofilm cultures preferentially consumed lactic acid over glucose, and in addition, the cultures catabolized the substrates completely and did not exhibit the acetate secreting “overflow” metabolism that is typical of many model microorganisms. The biofilm phenotype was enabled by changes in protein abundances, including lactate dehydrogenase, fumarate hydratase, GTP cyclohydrolase, L-ornithine N(5)-monooxygenase, and superoxide dismutase. These results are noteworthy because reverse diauxie-mediated catabolism of organic acids necessitates a terminal electron acceptor like O₂, which is typically in low supply in biofilms due to diffusion limitation. Label-free proteomics identified dozens of proteins associated with biofilm formation including 16 that have not been previously reported, highlightingmore »both the advantages of the methodology utilized here and the complexity of the proteomic adaptation forP. aeruginosabiofilms. Documenting the reverse diauxic phenotype inP. aeruginosabiofilms is foundational for understanding cellular nutrient and energy fluxes, which ultimately control growth and virulence.

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2. Identification of DIR1-Dependant Cellular Responses in Guard Cell Systemic Acquired Resistance

   https://doi.org/10.3389/fmolb.2021.746523  

   David, Lisa ; Kang, Jianing ; Nicklay, Josh ; Dufresne, Craig ; Chen, Sixue ( December 2021 , Frontiers in Molecular Biosciences)

   After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific molecular mechanisms remain elusive. The lipid transfer protein defective in induced resistance 1 (DIR1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Here, we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1-1 mutant during SAR. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were also changed in guard cells of dir1-1 compared to the wild type. Identification of guard cell-specific SAR-related molecules may lead to new avenues of genetic modification/molecular breeding for disease-resistant plants.
3. Metabolomic analysis of honey bee ( Apis mellifera L.) response to glyphosate exposure

   https://doi.org/10.1039/D2MO00046F  

   Wang, Bo ; Habermehl, Calypso ; Jiang, Lin ( May 2022 , Molecular Omics)

   Glyphosate is among the world's most commonly used herbicides in agriculture and weed control. The use of this agrochemical has unintended consequences on non-target organisms, such as honey bees ( Apis mellifera L. ), the Earth's most prominent insect pollinator. However, detailed understanding of the biological effects in bees in response to sub-lethal glyphosate exposure is still limited. In this study, 1 H NMR-based metabolomics was performed to investigate whether oral exposure to an environmentally realistic concentration (7.12 mg L −1 ) of glyphosate affects the regulation of honey bee metabolites in 2, 5, and 10 days. On Day 2 of glyphosate exposure, the honey bees showed significant downregulation of several essential amino acids, including leucine, lysine, valine, and isoleucine. This phenomenon indicates that glyphosate causes an obvious metabolic perturbation when the honey bees are subjected to the initial caging process. The mid-term (Day 5) results showed negligible metabolite-level perturbation, which indicated the low glyphosate impact on active honeybees. However, the long-term (Day 10) data showed evident separation between the control and experimental groups in the principal component analysis (PCA). This separation is the result of the combinatorial changes of essential amino acids such as threonine, histidine, and methionine, whilemore »the non-essential amino acids glutamine and proline as well as the carbohydrate sucrose were all downregulated. In summary, our study demonstrates that although no significant behavioral differences were observed in honey bees under sub-lethal doses of glyphosate, metabolomic level perturbation can be observed under short-term exposure when met with other environmental stressors or long-term exposure.« less
4. Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

   https://doi.org/https://doi.org/10.1104/pp.18.01195  

   Lucas Busta, Won Cheol ( December 2018 , Plant physiology)

   Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resultingmore »in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules. Many organisms implement specialized biochemical pathways to convert ubiquitous metabolites into bioactive chemical compounds. Since plants comprise the majority of the human diet, specialized plant metabolites play crucial roles not only in crop biology but also in human nutrition. Some asterids produce lipid compounds called polyacetylenes (for review, see Negri, 2015) that exhibit antifungal activity (Garrod et al., 1978; Kemp, 1978; Harding and Heale, 1980, 1981; Olsson and Svensson, 1996) and accumulate in response to fungal phytopathogen attack (De Wit and Kodde, 1981; Elgersma and Liem, 1989). These observations have led to the longstanding hypothesis that polyacetylenes are natural pesticides. These same lipid compounds exhibit cytotoxic activity against human cancer cell lines and slow tumor growth (Fujimoto and Satoh, 1988; Matsunaga et al., 1989, 1990; Cunsolo et al., 1993; Bernart et al., 1996; Kobaek-Larsen et al., 2005; Zidorn et al., 2005), making them important nutritional compounds. The major source of polyacetylenes in the human diet is carrot (Daucus carota L.). Carrot is one of the most important crop species in the Apiaceae, with rapidly increasing worldwide cultivation (Rubatzky et al., 1999; Dawid et al., 2015). The most common carrot polyacetylenes are C17 linear aliphatic compounds containing two conjugated carbon-carbon triple bonds, one or two carbon-carbon double bonds, and a diversity of additional in-chain oxygen-containing functional groups. In carrot, the most abundant of these compounds are falcarinol and falcarindiol (Dawid et al., 2015). Based on their structures, it has been hypothesized that these compounds (alias falcarin-type polyacetylenes) are derived from ubiquitous fatty acids. Indeed, biochemical investigations (Haigh et al., 1968; Bohlman, 1988), radio-chemical tracer studies (Barley et al., 1988), and the discovery of pathway intermediates (Jones et al., 1966; Kawazu et al., 1973) implicate a diversion of flux away from linolenate biosynthesis as the entry point into falcarin-type polyacetylene biosynthesis (for review, see Minto and Blacklock, 2008). The final steps of linolenate biosynthesis are the conversion of oleate to linoleate, mediated by fatty acid desaturase 2 (FAD2), and linoleate to linolenate, catalyzed by FAD3. Some plant species contain divergent forms of FAD2 that, instead of or in addition to converting oleate to linoleate, catalyze the installation of unusual in-chain functional groups such as hydroxyl groups, epoxy groups, conjugated double bonds, or carbon-carbon triple bonds into the acyl chain (Badami and Patil, 1980) and thus divert flux from linolenate production into the accumulation of unusual fatty acids. Previous work in parsley (Petroselinum crispum; Apiaceae) identified a divergent form of FAD2 that (1) was up-regulated in response to pathogen treatment and (2) when expressed in soybean embryos resulted in production of the monoyne crepenynate and, by the action of an unassigned enzyme, dehydrocrepenynate (Kirsch et al., 1997; Cahoon et al., 2003). The results of the parsley studies are consistent with a pathogen-responsive, divergent FAD2-mediated pathway that leads to acetylenic fatty acids. However, information regarding the branch point into acetylenic fatty acid production in agriculturally relevant carrot is still largely missing, in particular, the identification and functional characterization of enzymes that can divert carbon flux away from linolenate biosynthesis into the production of dehydrocrepenynate and ultimately falcarin-type polyacetylenes. Such genes, once identified, could be used in the future design of transgenic carrot lines with altered polyacetylene content, enabling direct testing of in planta polyacetylene function and potentially the engineering of pathogen-resistant, more nutritious carrots. These genes could also provide the foundation for further investigations of more basic aspects of plant biology, including the evolution of fatty acid-derived natural product biosynthesis pathways across the Asterid clade, as well as the role of these pathways and compounds in plant ecology and plant defense. Recently, a high-quality carrot genome assembly was released (Iorizzo et al., 2016), providing a foundation for genome-enabled studies of Apiaceous species. This study also provided publicly accessible RNA sequencing (RNA-Seq) data from diverse carrot tissues. Using these resources, this study aimed to provide a detailed gas chromatography-based quantification of polyacetylenes in carrot tissues for which RNA-Seq data are available, then combine this information with bioinformatics analysis and heterologous expression to identify and characterize biosynthetic genes that underlie the major entry point into carrot polyacetylene biosynthesis. To achieve these goals, thin-layer chromatography (TLC) was combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection to identify and quantify polyacetylenic metabolites in five different carrot tissues. Then the sequences and tissue expression profiles of potential FAD2 and FAD2-like genes annotated in the D. carota genome were compared with the metabolite data to identify candidate pathway genes, followed by biochemical functionality tests using yeast (Saccharomyces cerevisae) and Arabidopsis (Arabidopsis thaliana) as heterologous expression systems.« less
5. The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential

   https://doi.org/10.1093/jac/dky545  

   Maiden, Michael M. ; Zachos, Mitchell P. ; Waters, Christopher M. ( January 2019 , Journal of Antimicrobial Chemotherapy)

   To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action.

   Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively.

   Here we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms.

   Oxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing.more »As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.

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