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Bacteria induce pigment formation in the basidiomycete Serpula lacrymans

Abstract (Expand)

Basidiomycete fungi are characterized ecologically for their vital functional role in ecosystem carbon recycling and chemically for their capacity to produce a diverse array of small molecules. … Chromophoric natural products derived from the quinone precursor atromentin, such as variegatic acid and involutin, have been shown to function in redox cycling. Yet, in the context of an inter-kingdom natural system these pigments are still elusive. Here, we co-cultured the model saprotrophic basidiomycete Serpula lacrymans with an ubiquitous terrestrial bacterium, either Bacillus subtilis, Pseudomonas putida, or Streptomyces iranensis. For each, there was induction of the gene cluster encoding a non-ribosomal peptide synthetase-like enzyme (atromentin synthetase) and an aminotransferase which together produce atromentin. Correspondingly during co-culturing there was an increase in secreted atromentin-derived pigments, i.e., variegatic, xerocomic, isoxerocomic and atromentic acid. Bioinformatic analyses from 14 quinone synthetase genes, twelve of which are encoded in a cluster, identified a common promoter motif indicating a general regulatory mechanism for numerous basidiomycetes. This article is protected by copyright. All rights reserved.

Editor:

Date Published: 5th Oct 2016

Publication Type: Not specified

Bakterien-induzierte Morphogenese der marinen Makroalge Ulva

Abstract (Expand)

The morphogenesis of the marine green algae Ulva mutabilis depends on bacteria that release diffusible morphogenetic compounds. Axenic U. mutabilis gametes develop into callus-like colonies without … normal cell walls. From the accompanying microbial flora two specific bacteria were isolated, which form a symbiotic tripartite community and induce readily algal morphogenesis. We use axenic algal cultures as a powerful tool to investigate the multiple cross-kingdom interactions on a molecular level.

Author: Thomas Wichard

Date Published: 2nd Oct 2016

Publication Type: Not specified

Siderophores as molecular tools in medical and environmental applications

Abstract (Expand)

Almost all life forms depend on iron as an essential micronutrient that is needed for electron transport and metabolic processes. Siderophores are low-molecular-weight iron chelators that safeguard the … supply of this important metal to bacteria, fungi and graminaceous plants. Although animals and the majority of plants do not utilise siderophores and have alternative means of iron acquisition, siderophores have found important clinical and agricultural applications. In this review, we will highlight the different uses of these iron-chelating molecules.

Authors: , H. Kage,

Date Published: 6th Aug 2016

Publication Type: Not specified

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors.

Abstract (Expand)

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) … that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3mug/200mug protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20muM AA resulted in strong LTC4 formation (~250ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50=3.1-3.5muM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50=17 and 300nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.

Authors: S. Liening, G. K. Scriba, S. Rummler, C. Weinigel, T. K. Kleinschmidt, J. Z. Haeggstrom, O. Werz, U. Garscha

Date Published: 2nd Aug 2016

Publication Type: Not specified

Identification of Metallophores and Organic Ligands in the Chemosphere of the Marine Macroalga Ulva (Chlorophyta) and at Land-Sea Interfaces

Abstract (Expand)

The roles of organic matter in seawater have often been discussed from the aspect of metal toxicity and bioavailability in seawater. In fact, organic ligands, as part of the organic matter, can work … as a trace metal ion buffer system. At the same time, however, the release of well-defined metal chelators as exudates by, for example, marine bacteria is necessary to compete with natural metal complexes and sustain the metal acquisition required for several processes including nitrogen fixation. The identification, isolation, and structure elucidation of chelators is, thus, essential to our understanding of metal stress management in the natural habitat and role of these chelators on cellular process. The isolation of an organic ligand from its chemosphere is a challenging task. The purpose of this paper is, therefore, to give an additional perspective on how the effective application of stable isotope pairs of a metal of interest (both cations and oxoanions) combined with mass spectrometric analyses can pave the way to discovering new organic ligands (i.e., metallophores) and the chelating characteristics of dissolved organic matter (DOM): Pairs of isotopes, such as 54Fe and 58Fe (or any other pair of available isotopes of a given metal), can be used to create easily detectable unique isotopic signatures in mass spectra when they are bound by chelators. The identification of organic ligands is outlined for a proposed model system of mutualistic interactions between the green macroalga Ulva (Chlorophyta) and associated bacteria, as well as discussed briefly for DOM along land-sea gradients. Overall, the characterization of a broader spectrum of chelators in aquatic systems will open a new window to decipher the eco-physiological functions of organic ligands as a metal ion buffer and metallophores in metal cycling in marine ecosystems.

Author: Thomas Wichard

Date Published: 29th Jul 2016

Publication Type: Not specified

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

Abstract (Expand)

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase … FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.

Authors: D. Kalb, T. Heinekamp, S. Schieferdecker, M. Nett, A. A. Brakhage, D. Hoffmeister

Date Published: 22nd Jul 2016

Publication Type: Not specified

Biodiversity and Activity of the Gut Microbiota across the Life History of the Insect Herbivore Spodoptera littoralis

Abstract (Expand)

Microbes that live inside insects play critical roles in host nutrition, physiology, and behavior. Although Lepidoptera (butterflies and moths) are one of the most diverse insect taxa, their microbial … symbionts are little-studied, particularly during metamorphosis. Here, using ribosomal tag pyrosequencing of DNA and RNA, we investigated biodiversity and activity of gut microbiotas across the holometabolous life cycle of Spodoptera littoralis, a notorious agricultural pest worldwide. Proteobacteria and Firmicutes dominate but undergo a structural "metamorphosis" in tandem with its host. Enterococcus, Pantoea and Citrobacter were abundant and active in early-instar, while Clostridia increased in late-instar. Interestingly, only enterococci persisted through metamorphosis. Female adults harbored high proportions of Enterococcus, Klebsiella and Pantoea, whereas males largely shifted to Klebsiella. Comparative functional analysis with PICRUSt indicated that early-instar larval microbiome was more enriched for genes involved in cell motility and carbohydrate metabolism, whereas in late-instar amino acid, cofactor and vitamin metabolism increased. Genes involved in energy and nucleotide metabolism were abundant in pupae. Female adult microbiome was enriched for genes relevant to energy metabolism, while an increase in the replication and repair pathway was observed in male. Understanding the metabolic activity of these herbivore-associated microbial symbionts may assist the development of novel pest-management strategies.

Authors: B. Chen, , C. Sun, S. Hu, X. Lu, , Y. Shao

Date Published: 9th Jul 2016

Publication Type: Not specified

Colonization of the Intestinal Tract of the Polyphagous Pest Spodoptera littoralis with the GFP-Tagged Indigenous Gut Bacterium Enterococcus mundtii

Abstract (Expand)

The alkaline gut of Lepidopterans plays a crucial role in shaping communities of bacteria. Enterococcus mundtii has emerged as one of the predominant gut microorganisms in the gastrointestinal tract … of the major agricultural pest, Spodoptera littoralis. Therefore, it was selected as a model bacterium to study its adaptation to harsh alkaline gut conditions in its host insect throughout different stages of development (larvae, pupae, adults, and eggs). To date, the mechanism of bacterial survival in insects' intestinal tract has been unknown. Therefore, we have engineered a GFP-tagged species of bacteria, E. mundtii, to track how it colonizes the intestine of S. littoralis. Three promoters of different strengths were used to control the expression of GFP in E. mundtii. The promoter ermB was the most effective, exhibiting the highest GFP fluorescence intensity, and hence was chosen as our main construct. Our data show that the engineered fluorescent bacteria survived and proliferated in the intestinal tract of the insect at all life stages for up to the second generation following ingestion.

Authors: , J. Apel, Y. Shao,

Date Published: 6th Jul 2016

Publication Type: Not specified

Benefits of jasmonate-dependent defenses against vertebrate herbivores in nature.

Abstract (Expand)

Endogenous jasmonates are important regulators of plant defenses. If and how they enable plants to maintain their reproductive output when facing community-level herbivory under natural conditions, … however, remains unknown. We demonstrate that jasmonate-deficient Nicotiana attenuata plants suffer more damage by arthropod and vertebrate herbivores than jasmonate-producing plants in nature. However, only damage by vertebrate herbivores translates into a significant reduction in flower production. Vertebrate stem peeling has the strongest negative impact on plant flower production. Stems are defended by jasmonate-dependent nicotine, and the native cottontail rabbit Sylvilagus nuttallii avoids jasmonate-producing N. attenuata shoots because of their high levels of nicotine. Thus, endogenous jasmonates enable plants to resist different types of herbivores in nature, and jasmonate-dependent defenses are important for plants to maintain their reproductive potential when facing vertebrate herbivory. Ecological and evolutionary models on plant defense signaling should aim at integrating arthropod and vertebrate herbivory at the community level.

Authors: R. A. Machado, M. McClure, M. R. Herve, I. T. Baldwin, M. Erb

Date Published: 30th Jun 2016

Publication Type: Not specified

Pseudoxylallemycins A-F, Cyclic Tetrapeptides with Rare Allenyl Modifications Isolated from Pseudoxylaria sp. X802: A Competitor of Fungus-Growing Termite Cultivars.

Abstract (Expand)

Based on fungus-fungus pairing assays and HRMS-based dereplication strategy, six new cyclic tetrapeptides, pseudoxylallemycins A-F (1-6), were isolated from the termite-associated fungus Pseudoxylaria … sp. X802. Structures were characterized using NMR spectroscopy, HRMS, and Marfey's reaction. Pseudoxylallemycins B-D (2-4) possess a rare and chemically accessible allene moiety amenable for synthetic modifications, and derivatives A-D showed antimicrobial activity against Gram-negative human-pathogenic Pseudomonas aeruginosa and antiproliferative activity against human umbilical vein endothelial cells and K-562 cell lines.

Authors: H. Guo, N. B. Kreuzenbeck, S. Otani, M. Garcia-Altares, H. M. Dahse, C. Weigel, D. K. Aanen, C. Hertweck, M. Poulsen, C. Beemelmanns

Date Published: 25th Jun 2016

Publication Type: Not specified

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

Abstract (Expand)

Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed … production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE: The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.

Authors: , , M. A. Gregory, M. Flak, M. K. Krespach, P. F. Leadlay,

Date Published: 15th Jun 2016

Publication Type: Not specified

Searching for a Mate: Pheromone-Directed Movement of the Benthic Diatom Seminavis robusta

Abstract (Expand)

Diatoms are species-rich microalgae that often have a unique life cycle with vegetative cell size reduction followed by size restoration through sexual reproduction of two mating types (MT(+) and MT(-)). … In the marine benthic diatom Seminavis robusta, mate-finding is mediated by an L-proline-derived diketopiperazine, a pheromone produced by the attracting mating type (MT(-)). Here, we investigate the movement patterns of cells of the opposite mating type (MT(+)) exposed to a pheromone gradient, using video monitoring and statistical modeling. We report that cells of the migrating mating type (MT(+)) respond to pheromone gradients by simultaneous chemotaxis and chemokinesis. Changes in movement behavior enable MT(+) cells to locate the direction of the pheromone source and to maximize their encounter rate towards it.

Authors: K. G. Bondoc, C. Lembke, W. Vyverman,

Date Published: 5th Jun 2016

Publication Type: Not specified

High-throughput quantification of more than 100 primary- and secondary-metabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS

Abstract (Expand)

BACKGROUND: Plant metabolites are commonly functionally classified, as defense- or growth-related phytohormones, primary and specialized metabolites, and so forth. Analytical procedures for the … quantifications of these metabolites are challenging because the metabolites can vary over several orders of magnitude in concentrations in the same tissues and have very different chemical characteristics. Plants clearly adjust their metabolism to respond to their prevailing circumstances in very sophisticated ways that blur the boundaries among these functional or chemically defined classifications. But if plant biologists want to better understand the processes that are important for a plant's adaptation to its environment, procedures are needed that can provide simultaneous quantifications of the large range of metabolites that have the potential to play central roles in these adjustments in a cost and time effective way and with a low sample consumption. RESULTS: Here we present a method that combines well-established methods for the targeted analysis of phytohormones, including jasmonates, salicylic acid, abscisic acid, gibberellins, auxins and cytokinins, and extends it to the analysis of inducible and constitutive defense compounds, as well as the primary metabolites involved in the biosynthesis of specialized metabolites and responsible for nutritional quality (e.g., sugars and amino acids). The method is based on a single extraction of 10-100 mg of tissue and allows a broad quantitative screening of metabolites optimized by their chemical characteristics and concentrations, thereby providing a high throughput analysis unbiased by the putative functional attributes of the metabolites. The tissues of Nicotiana attenuata which accumulate high levels of nicotine and diterpene glycosides, provide a challenging matrix that thwarts quantitative analysis; the analysis of various tissues of this plant are used to illustrate the robustness of the procedure. CONCLUSIONS: The method described has the potential to unravel various, until now overlooked interactions among different sectors of plant metabolism in a high throughput manner. Additionally, the method could be particularly beneficial as screening method in forward genetic approaches, as well as for the investigation of plants from natural populations that likely differ in metabolic traits.

Authors: , C. Brutting, , M. Kallenbach

Date Published: 31st May 2016

Publication Type: Not specified

Plant-like biosynthesis of isoquinoline alkaloids in Aspergillus fumigatus

Abstract (Expand)

Natural product discovery efforts have focused primarily on microbial biosynthetic gene clusters (BGCs) containing large multimodular polyketide synthases and nonribosomal peptide synthetases; however, … sequencing of fungal genomes has revealed a vast number of BGCs containing smaller NRPS-like genes of unknown biosynthetic function. Using comparative metabolomics, we show that a BGC in the human pathogen Aspergillus fumigatus named fsq, which contains an NRPS-like gene lacking a condensation domain, produces several new isoquinoline alkaloids known as the fumisoquins. These compounds derive from carbon-carbon bond formation between two amino acid-derived moieties followed by a sequence that is directly analogous to isoquinoline alkaloid biosynthesis in plants. Fumisoquin biosynthesis requires the N-methyltransferase FsqC and the FAD-dependent oxidase FsqB, which represent functional analogs of coclaurine N-methyltransferase and berberine bridge enzyme in plants. Our results show that BGCs containing incomplete NRPS modules may reveal new biosynthetic paradigms and suggest that plant-like isoquinoline biosynthesis occurs in diverse fungi.

Authors: J. A. Baccile, J. E. Spraker, H. H. Le, E. Brandenburger, C. Gomez, J. W. Bok, J. Macheleidt, A. A. Brakhage, D. Hoffmeister, N. P. Keller, F. C. Schroeder

Date Published: 12th Apr 2016

Publication Type: Not specified

Variochelins, Lipopeptide Siderophores from Variovorax boronicumulans Discovered by Genome Mining

Abstract (Expand)

Photoreactive siderophores have a major impact on the growth of planktonic organisms. To date, these molecules have mainly been reported from marine bacteria, although evidence is now accumulating that … some terrestrial bacteria also harbor the biosynthetic potential for their production. In this paper, we describe the genomics-driven discovery and characterization of variochelins, lipopeptide siderophores from the bacterium Variovorax boronicumulans, which thrives in soil and freshwater habitats. Variochelins are different from most other lipopeptide siderophores in that their biosynthesis involves a polyketide synthase. We demonstrate that the ferric iron complex of variochelin A possesses photoreactive properties and present the MS-derived structures of two degradation products that emerge upon light exposure.

Authors: , S. Schieferdecker, K. Athanasopoulou, I. Seccareccia,

Date Published: 29th Mar 2016

Publication Type: Not specified

Humudifucol and Bioactive Prenylated Polyphenols from Hops (Humulus lupulus cv. "Cascade")

Abstract (Expand)

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant … extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low muM concentrations.

Authors: M. Forino, , G. Chianese, L. Santagostini, , C. Weinigel, S. Rummler, G. Fico, , O. Taglialatela-Scafati

Date Published: 27th Feb 2016

Publication Type: Not specified

The hallucinogenic diterpene salvinorin A inhibits leukotriene synthesis in experimental models of inflammation

Abstract (Expand)

Leukotrienes (LTs) are lipid mediators derived from arachidonic acid (AA) involved in a number of autoimmune/inflammatory disorders including asthma, allergic rhinitis and cardiovascular diseases. … Salvinorin A (SA), a diterpene isolated from the hallucinogenic plant Salvia divinorum, is a well-established analgesic compound, but its anti-inflammatory properties are under-researched and its effects on LT production is unknown to date. Here, we studied the possible effect of SA on LT production and verified its actions on experimental models of inflammation in which LTs play a prominent role. Peritoneal macrophages (PM) stimulated by calcium ionophore A23187 were chosen as in vitro system to evaluate the effect of SA on LT production. Zymosan-induced peritonitis in mice and carrageenan-induced pleurisy in rats were selected as LT-related models to evaluate the effect of SA on inflammation as well as on LT biosynthesis. SA inhibited, in a concentration-dependent manner, A23187-induced LTB4 biosynthesis in isolated PM. In zymosan-induced peritonitis, SA inhibited cell infiltration, myeloperoxidase activity, vascular permeability and LTC4 production in the peritoneal cavity without decreasing the production of prostaglandin E2. In carrageenan-induced pleurisy in rats, a more sophisticated model of acute inflammation related to LTs, SA significantly inhibited LTB4 production in the inflammatory exudates, along with reducing the phlogistic process in the lung. In conclusion, SA inhibited LT production and it was effective in experimental models of inflammation in which LTs play a pivotal role. SA might be considered as a lead compound for the development of drugs useful in LTs-related diseases.

Authors: A. Rossi, , F. Tedesco, E. Pagano, G. Guerra, F. Troisi, , F. Roviezzo, J. K. Zjawiony, , A. A. Izzo, R. Capasso

Date Published: 10th Feb 2016

Publication Type: Not specified

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

Abstract (Expand)

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE (5(S)-hydroperoxyeicosatetraenoic … acid) and its subsequent transformation to LTA4. 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants ( approximately 60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.

Authors: J. Gerstmeier, M. E. Newcomer, S. Dennhardt, E. Romp, J. Fischer, , U. Garscha

Date Published: 5th Feb 2016

Publication Type: Not specified

Using the knowns to discover the unknowns: MS-based dereplication uncovers structural diversity in 17-hydroxygeranyllinalool diterpene glycoside production in the Solanaceae

Abstract (Expand)

Exploring the diversity of plant secondary metabolism requires efficient methods to obtain sufficient structural insights to discriminate previously known from unknown metabolites. De novo structure … elucidation and confirmation of known metabolites (dereplication) remain a major bottleneck for mass spectrometry-based metabolomic workflows, and few systematic dereplication strategies have been developed for the analysis of entire compound classes across plant families, partly due to the complexity of plant metabolic profiles that complicates cross-species comparisons. 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) are abundant defensive secondary metabolites whose malonyl and glycosyl decorations are induced by jasmonate signaling in the ecological model plant Nicotiana attenuata. The multiple labile glycosidic bonds of HGL-DTGs result in extensive in-source fragmentation (IS-CID) during ionization. To reconstruct these IS-CID clusters from profiling data and identify precursor ions, we applied a deconvolution algorithm and created an MS/MS library from positive-ion spectra of purified HGL-DTGs. From this library, 251 non-redundant fragments were annotated, and a workflow to characterize leaf, flower and fruit extracts of 35 solanaceous species was established. These analyses predicted 105 novel HGL-DTGs that were restricted to Nicotiana, Capsicum and Lycium species. Interestingly, malonylation is a highly conserved step in HGL-DTG metabolism, but is differentially affected by jasmonate signaling among Nicotiana species. This MS-based workflow is readily applicable for cross-species re-identification/annotation of other compound classes with sufficient fragmentation knowledge, and therefore has the potential to support hypotheses regarding secondary metabolism diversification.

Authors: S. Heiling, S. Khanal, A. Barsch, G. Zurek, , E. Gaquerel

Date Published: 11th Jan 2016

Publication Type: Not specified

Macroalgal Morphogenesis Induced by Waterborne Compounds and Bacteria in Coastal Seawater

Abstract (Expand)

Axenic gametes of the marine green macroalga Ulva mutabilis Foyn (Ria Formosa, locus typicus) exhibit abnormal development into slow-growing callus-like colonies with aberrant cell walls. Under laboratory … conditions, it was previously demonstrated that all defects in growth and thallus development can be completely abolished when axenic gametes are inoculated with a combination of two specific bacterial strains originally identified as Roseobacter sp. strain MS2 and Cytophaga sp. strain MS6. These bacteria release diffusible morphogenetic compounds (= morphogens), which act similar to cytokinin and auxin. To investigate the ecological relevance of the waterborne bacterial morphogens, seawater samples were collected in the Ria Formosa lagoon (Algarve, Southern Portugal) at 20 sampling sites and tidal pools to assess their morphogenetic effects on the axenic gametes of U. mutabilis. Specifically the survey revealed that sterile-filtered seawater samples can completely recover growth and morphogenesis of U. mutabilis under axenic conditions. Morphogenetic activities of free-living and epiphytic bacteria isolated from the locally very abundant Ulva species (i.e., U. rigida) were screened using a multiwell-based testing system. The most represented genera isolated from U. rigida were Alteromonas, Pseudoalteromonas and Sulfitobacter followed by Psychrobacter and Polaribacter. Several naturally occurring bacterial species could emulate MS2 activity (= induction of cell divisions) regardless of taxonomic affiliation, whereas the MS6 activity (= induction of cell differentiation and cell wall formation) was species-specific and is probably a feature of difficult-to-culture bacteria. Interestingly, isolated bacteroidetes such as Algoriphagus sp. and Polaribacter sp. could individually trigger complete Ulva morphogenesis and thus provide a novel mode of action for bacterial-induced algal development. This study also highlights that the accumulation of algal growth factors in a shallow water body separated from the open ocean by barrier islands might have strong implications to, for example, the wide usage of natural coastal seawater in algal (land based) aquacultures of Ulva.

Authors: J. Grueneberg, A. H. Engelen, R. Costa,

Date Published: 8th Jan 2016

Publication Type: Not specified

Rapid Identification of Pseudomonas spp. via Raman Spectroscopy Using Pyoverdine as Capture Probe

Abstract (Expand)

Pyoverdine is a substance which is excreted by fluorescent pseudomonads in order to scavenge iron from their environment. Due to specific receptors of the bacterial cell wall, the iron loaded pyoverdine … molecules are recognized and transported into the cell. This process can be exploited for developing efficient isolation and enrichment strategies for members of the Pseudomonas genus, which are capable of colonizing various environments and also include human pathogens like P. aeruginosa and the less virulent P. fluorescens. A significant advantage over antibody based systems is the fact that siderophores like pyoverdine can be considered as "immutable ligands," since the probability for mutations within the siderophore uptake systems of bacteria is very low. While each species of Pseudomonas usually produces structurally unique pyoverdines, which can be utilized only by the producer strain, cross reactivity does occur. In order to achieve a reliable identification of the captured pathogens, further investigations of the isolated cells are necessary. In this proof of concept study, we combine the advantages of an isolation strategy relying on "immutable ligands" with the high specificity and speed of Raman microspectroscopy. In order to isolate the bacterial cells, pyoverdine was immobilized covalently on planar aluminum chip substrates. After capturing, single cell Raman spectra of the isolated species were acquired. Due to the specific spectroscopic fingerprint of each species, the bacteria can be identified. This approach allows a very rapid detection of potential pathogens, since time-consuming culturing steps are unnecessary. We could prove that pyoverdine based isolation of bacteria is fully Raman compatible and further investigated the capability of this approach by isolating and identifying P. aeruginosa and P. fluorescens from tap water samples, which are both opportunistic pathogens and can pose a threat for immunocompromised patients.

Authors: S. Pahlow, S. Stockel, S. Pollok, D. Cialla-May, , K. Weber,

Date Published: 8th Jan 2016

Publication Type: Not specified

A fivefold parallelized biosynthetic process secures chlorination of Armillaria mellea (honey mushroom) toxins

Abstract (Expand)

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. … During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1-ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A fivefold secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share a common ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.

Authors: J. Wick, D. Heine, G. Lackner, M. Misiek, , H. Jagusch, ,

Date Published: 15th Dec 2015

Publication Type: Not specified

CASSIS and SMIPS: promoter-based prediction of secondary metabolite gene clusters in eukaryotic genomes

Abstract (Expand)

MOTIVATION: Secondary metabolites (SM) are structurally diverse natural products of high pharmaceutical importance. Genes involved in their biosynthesis are often organized in clusters, i.e., are … co-localized and co-expressed. In silico cluster prediction in eukaryotic genomes remains problematic mainly due to the high variability of the clusters' content and lack of other distinguishing sequence features. RESULTS: We present Cluster Assignment by Islands of Sites (CASSIS), a method for SM cluster prediction in eukaryotic genomes, and Secondary Metabolites by InterProScan (SMIPS), a tool for genome-wide detection of SM key enzymes ('anchor' genes): polyketide synthases, non-ribosomal peptide synthetases and dimethylallyl tryptophan synthases. Unlike other tools based on protein similarity, CASSIS exploits the idea of co-regulation of the cluster genes, which assumes the existence of common regulatory patterns in the cluster promoters. The method searches for 'islands' of enriched cluster-specific motifs in the vicinity of anchor genes. It was validated in a series of cross-validation experiments and showed high sensitivity and specificity. AVAILABILITY AND IMPLEMENTATION: CASSIS and SMIPS are freely available at https://sbi.hki-jena.de/cassis CONTACT: thomas.wolf@leibniz-hki.de or ekaterina.shelest@leibniz-hki.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Authors: T. Wolf, V. Shelest, N. Nath,

Date Published: 9th Dec 2015

Publication Type: Not specified

The application of Raman spectroscopy for the detection and identification of microorganisms

Abstract (Expand)

A fast and reliable detection and identification of microorganisms is crucial in environmental science, for food quality as well as medical diagnosis. In these fields, all types of Raman spectroscopy … are gaining more and more importance during the last years. The review provides an extensive overview of recent research, technical expertise, and scientific findings based on Raman spectroscopic detection and identification of microorganisms within the years 2010 and 2015, demonstrating the diverse capability of Raman spectroscopy as a modern analytical tool. Raman spectroscopy distinguishes itself from other currently applied techniques by its easy application at low cost, its high speed of analysis, and its broad information content on both the chemical composition and the structure of biomolecules within the microorganisms. Slight chances in the chemical composition of microorganisms can be monitored by means of Raman spectroscopy and used to differentiate genera, species, or even strains. Detection of pathogens is possible from complex matrices, such as soil, food, and body fluids. Further, spectroscopic studies of host–pathogen interactions are addressed as well as the effect of antibiotics on bacteria.

Authors: Stephan Stöckel, Johanna Kirchhoff, Ute Neugebauer, Petra Rösch, Jürgen Popp

Date Published: 7th Dec 2015

Publication Type: Not specified

Multimodular type I polyketide synthases in algae evolve by module duplications and displacement of AT domains in trans

Abstract (Expand)

BACKGROUND: Polyketide synthase (PKS) catalyzes the biosynthesis of polyketides, which are structurally and functionally diverse natural products in microorganisms and plants. Here, we have analyzed … available full genome sequences of microscopic and macroscopic algae for the presence of type I PKS genes. RESULTS: Type I PKS genes are present in 15 of 32 analyzed algal species. In chlorophytes, large proteins in the MDa range are predicted in most sequenced species, and PKSs with free-standing acyltransferase domains (trans-AT PKSs) predominate. In a phylogenetic tree, PKS sequences from different algal phyla form clades that are distinct from PKSs from other organisms such as non-photosynthetic protists or cyanobacteria. However, intermixing is observed in some cases, for example polyunsaturated fatty acid (PUFA) and glycolipid synthases of various origins. Close relationships between type I PKS modules from different species or between modules within the same multimodular enzyme were identified, suggesting module duplications during evolution of algal PKSs. In contrast to type I PKSs, nonribosomal peptide synthetases (NRPSs) are relatively rare in algae (occurrence in 7 of 32 species). CONCLUSIONS: Our phylogenetic analysis of type I PKSs in algae supports an evolutionary scenario whereby integrated AT domains were displaced to yield trans-AT PKSs. Together with module duplications, the displacement of AT domains may constitute a major mechanism of PKS evolution in algae. This study advances our understanding of the diversity of eukaryotic PKSs and their evolutionary trajectories.

Authors: , N. Heimerl, M. Fichtner,

Date Published: 26th Nov 2015

Publication Type: Not specified

Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions

Abstract (Expand)

Biological nitrogen fixation can be catalyzed by three isozymes of nitrogenase: Mo-nitrogenase, V-nitrogenase and Fe-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, … Mo, V and Fe, and their bioavailability in ecosystems. Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii. In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of Mo and V under cellular conditions that are usually considered not Mo limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth.

Authors: C. Jouogo Noumsi, N. Pourhassan, R. Darnajoux, , , V. Burrus, J. P. Bellenger

Date Published: 10th Nov 2015

Publication Type: Not specified

Plant pathogenic anaerobic bacteria use aromatic polyketides to access aerobic territory

Abstract (Expand)

Around 25% of vegetable food is lost worldwide because of infectious plant diseases, including microbe-induced decay of harvested crops. In wet seasons and under humid storage conditions, potato tubers … are readily infected and decomposed by anaerobic bacteria (Clostridium puniceum). We found that these anaerobic plant pathogens harbor a gene locus (type II polyketide synthase) to produce unusual polyketide metabolites (clostrubins) with dual functions. The clostrubins, which act as antibiotics against other microbial plant pathogens, enable the anaerobic bacteria to survive an oxygen-rich plant environment.

Authors: G. Shabuer, K. Ishida, S. J. Pidot, M. Roth, H. M. Dahse,

Date Published: 7th Nov 2015

Publication Type: Not specified

Raman spectroscopy towards clinical application: drug monitoring and pathogen identification

Abstract (Expand)

Raman spectroscopy is a label-free method that measures quickly and contactlessly, providing detailed information from the sample, and has proved to be an ideal tool for medical and life science research. … In this review, recent advances of the technique towards drug monitoring and pathogen identification by the Jena Research Groups are reviewed. Surface-enhanced Raman spectroscopy (SERS) and ultraviolet resonance Raman spectroscopy in hollow-core optical fibres enable the detection of drugs at low concentrations as shown for the metabolites of the immunosuppressive drug 6-mercaptopurine as well as antimalarial agents. Furthermore, Raman spectroscopy can be used to characterise pathogenic bacteria in infectious diseases directly from body fluids, making time-consuming cultivation processes dispensable. Using the example of urinary tract infection, it is shown how bacteria can be identified from patients' urine samples within <1h. The methods cover both single-cell analysis and dielectrophoretic capturing of bacteria in suspension. The latter method could also be used for fast (<3.5h) identification of antibiotic resistance as shown exemplarily for vancomycin-resistant enterococci.

Authors: U. Neugebauer, ,

Date Published: 6th Nov 2015

Publication Type: Not specified

A Chemical Perspective on Microalgal-Microbial Interactions

Abstract (Expand)

The exchange of chemical compounds is central to the interactions of microalgae with other microorganisms. Although foundational for many food webs, these interactions have been poorly studied compared … with higher plant-microbe interactions. Emerging insights have begun to reveal how these interactions and the participating chemical compounds shape microbial communities and broadly impact biogeochemical processes.

Authors: E. F. Hom, , , ,

Date Published: 28th Oct 2015

Publication Type: Not specified

Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete Paxillus involutus

Abstract (Expand)

The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone … pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer.

Authors: J. Braesel, S. Gotze, F. Shah, D. Heine, , , A. Tunlid, P. Stallforth,

Date Published: 27th Oct 2015

Publication Type: Not specified

Harnessing Enzymatic Promiscuity in Myxochelin Biosynthesis for the Production of 5-Lipoxygenase Inhibitors

Abstract (Expand)

The siderophore myxochelin A is a potent inhibitor of human 5-lipoxygenase (5-LO). To clarify whether the iron-chelating properties of myxochelin A are responsible for this activity, several analogues … of this compound were generated in the native producer Pyxidicoccus fallax by precursor-directed biosynthesis. Testing in a cell-free assay unveiled three derivatives with bioactivity comparable with that of myxochelin A. Furthermore, it became evident that inhibition of 5-LO by myxochelins does not correlate with their iron affinities.

Authors: J. Korp, S. Konig, S. Schieferdecker, H. M. Dahse, G. M. Konig, ,

Date Published: 13th Oct 2015

Publication Type: Not specified

Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes

Abstract (Expand)

5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred … by 5-LO-activating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca(2+)-ionophore A23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 microM) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.-Gerstmeier, J., Weinigel, C., Rummler, S., Radmark, O., Werz, O., Garscha, U. Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes.

Authors: J. Gerstmeier, C. Weinigel, S. Rummler, O. Radmark, , U. Garscha

Date Published: 22nd Sep 2015

Publication Type: Not specified

Raman spectroscopic monitoring of the growth of pigmented and non-pigmented mycobacteria

Abstract (Expand)

Raman microspectroscopy has increased in popularity in the field of microbiology because it allows a spectral fingerprinting of bacterial pathogens at an unrivaled speed, which is important for the … early treatment of infectious diseases such as tuberculosis. An indispensable prerequisite for the success of this method is a profound knowledge, how the spectral profiles depend on the age of the bacteria. We therefore followed the growth of two rapidly growing Mycobacterium tuberculosis relatives, the pigmented Mycobacteriumaurum, and the non-pigmented Mycobacteriumsmegmatis, by means of Raman microspectroscopy. Both species showed remarkable temporal changes in the single-bacteria Raman spectra: In the signatures of M.aurum, pigment-associated Raman signals could be detected not until 72 h of growth and also remained highly variable thereafter. The Raman spectra of M.smegmatis exhibited lipid signals presumably arising from mycolic acids, which are a hallmark feature of mycobacteria, but only after the bacteria reached the late stationary growth phase (>48 h). A principal component analysis thus classified the Raman spectra according to the cultivation age. In summary, these findings have to be reckoned with in future studies dealing with the identification of mycobacteria via Raman microspectroscopy. Graphical abstract Changes in the chemical composition of bacterial cells over growth time may influence the results of Raman spectroscopic studies of bacteria.

Authors: S. Stockel, A. S. Stanca, J. Helbig, ,

Date Published: 21st Sep 2015

Publication Type: Not specified

Transformation ofUlva mutabilis(Chlorophyta) by vector plasmids integrating into the genome

Abstract (Expand)

A method for the stable transformation of the green marine macroalga Ulva mutabilis was developed based on vector plasmids integrating into the genome. By combination of the expression signals (promoter, … enhancer, and transcriptional termination sequences) of a chromosomal rbcS gene from U. mutabilis with the bleomycin resistance gene (ble) from Streptoalloteichus hindustanus, a dominant selectable marker gene was constructed for the preparation of a series of E. coli—U. mutabilis shuttle vector plasmids. Special vectors were prepared for the introduction and expression of foreign genes in Ulva, for insertional mutagenesis and gene tagging by plasmid integration into the genome, and for protein tagging by the green fluorescent protein, as well as tools for posttranscriptional gene silencing and cosmid cloning to prepare genomic gene libraries for mutant gene complementation. The vectors were successfully tested in pilot experiments, where they were efficiently introduced into Ulva gametes, zoospores or protoplasts of somatic blade cells by treatment with Ca2+-ions and polyethylene glycol under isotonic conditions at low ionic strength. The parthenogenetically propagated phleomycin-resistant transformants of the mutant slender (sl) and the wildtype (wt) were demonstrated to be carrying the plasmids randomly integrated into the chromosomes often as tandem repeat clusters.

Authors: Wolfgang Oertel, Thomas Wichard, Adelheid Weissgerber

Date Published: 18th Sep 2015

Publication Type: Not specified

Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters

Abstract (Expand)

Microorganisms form diverse multispecies communities in various ecosystems. The high abundance of fungal and bacterial species in these consortia results in specific communication between the microorganisms. … A key role in this communication is played by secondary metabolites (SMs), which are also called natural products. Recently, it was shown that interspecies "talk" between microorganisms represents a physiological trigger to activate silent gene clusters leading to the formation of novel SMs by the involved species. This review focuses on mixed microbial cultivation, mainly between bacteria and fungi, with a special emphasis on the induced formation of fungal SMs in co-cultures. In addition, the role of chromatin remodeling in the induction is examined, and methodical perspectives for the analysis of natural products are presented. As an example for an intermicrobial interaction elucidated at the molecular level, we discuss the specific interaction between the filamentous fungi Aspergillus nidulans and Aspergillus fumigatus with the soil bacterium Streptomyces rapamycinicus, which provides an excellent model system to enlighten molecular concepts behind regulatory mechanisms and will pave the way to a novel avenue of drug discovery through targeted activation of silent SM gene clusters through co-cultivations of microorganisms.

Authors: , J. Fischer, J. Weber, D. J. Mattern, C. C. Konig, V. Valiante, ,

Date Published: 20th Apr 2015

Publication Type: Not specified

Exploring bacteria-induced growth and morphogenesis in the green macroalga order Ulvales (Chlorophyta)

Abstract (Expand)

Green macroalgae, such as Ulvales, lose their typical morphology completely when grown under axenic conditions or in the absence of the appropriate microbiome. As a result, slow growing aberrant … phenotypes or even callus-like morphotypes are observed in Ulvales. The cross-kingdom interactions between marine algae and microorganisms are hence not only restricted by the exchange of macronutrients, including vitamins and nutrients, but also by infochemicals such as bacterial morphogenetic compounds. The latter are a fundamental trait mediating the mutualism within the chemosphere where the organisms interact with each other via compounds in their surroundings. Approximately 60 years ago, pilot studies demonstrated that certain bacteria promote growth, whereas other bacteria induce morphogenesis; this is particularly true for the order of Ulvales. However, only slow progress was made towards the underlying mechanism due to the complexity of, for example, algal cultivation techniques, and the lack of standardized experiments in the laboratory. A breakthrough in this research was the discovery of the morphogenetic compound thallusin, which was isolated from an epiphytic bacterium and induces normal germination restoring the foliaceous morphotypes of Monostroma. Owing to the low concentration, the purification and structure elucidation of highly biologically active morphogenetic compounds are still challenging. Recently, it was found that only the combination of two specific bacteria from the Rhodobacteraceae and Flavobacteriaceae can completely recover the growth and morphogenesis of axenic Ulva mutabilis cultures forming a symbiotic tripartite community by chemical communication. This review combines literature detailing evidences of bacteria-induced morphogenesis in Ulvales. A set of standardized experimental approaches is further proposed for the preparation of axenic algal tissues, bacteria isolation, co-cultivation experiments, and the analysis of the chemosphere.

Editor:

Date Published: 3rd Mar 2015

Publication Type: Not specified

The green seaweed Ulva: a model system to study morphogenesis

Abstract (Expand)

Green macroalgae, mostly represented by the Ulvophyceae, the main multicellular branch of the Chlorophyceae, constitute important primary producers of marine and brackish coastal ecosystems. Ulva or … sea lettuce species are some of the most abundant representatives, being ubiquitous in coastal benthic communities around the world. Nonetheless the genus also remains largely understudied. This review highlights Ulva as an exciting novel model organism for studies of algal growth, development and morphogenesis as well as mutualistic interactions. The key reasons that Ulva is potentially such a good model system are: (i) patterns of Ulva development can drive ecologically important events, such as the increasing number of green tides observed worldwide as a result of eutrophication of coastal waters, (ii) Ulva growth is symbiotic, with proper development requiring close association with bacterial epiphytes, (iii) Ulva is extremely developmentally plastic, which can shed light on the transition from simple to complex multicellularity and (iv) Ulva will provide additional information about the evolution of the green lineage.

Authors: , B. Charrier, F. Mineur, J. H. Bothwell, O. D. Clerck, J. C. Coates

Date Published: 19th Feb 2015

Publication Type: Not specified

Myxochelins target human 5-lipoxygenase

Abstract (Expand)

Extracts of the predatory myxobacterium Pyxidicoccus fallax HKI 727 showed antiproliferative effects on leukemic K-562 cells. Bioactivity-guided fractionation led to the isolation of the bis-catechol … myxochelin A and two new congeners. The biosynthetic origin of myxochelins C and D was confirmed by feeding studies with isotopically labeled precursors. Pharmacological testing revealed human 5-lipoxygenase (5-LO) as a molecular target of the myxochelins. In particular, myxochelin A efficiently inhibited 5-LO activity with an IC50 of 1.9 muM and reduced the proliferation of K-562 cells at similar concentrations.

Authors: S. Schieferdecker, S. Konig, , H. M. Dahse, ,

Date Published: 16th Feb 2015

Publication Type: Not specified

Regulation of gametogenesis and zoosporogenesis in Ulva linza (Chlorophyta): comparison with Ulva mutabilis and potential for laboratory culture

Abstract (Expand)

Green Ulvophyte macroalgae represent attractive model systems for understanding growth, development, and evolution. They are untapped resources for food, fuel, and high-value compounds, but can also … form nuisance blooms. To fully analyze green seaweed morphogenesis, controlled laboratory-based culture of these organisms is required. To date, only a single Ulvophyte species, Ulva mutabilis Foyn, has been manipulated to complete its whole life cycle in laboratory culture and to grow continuously under axenic conditions. Such cultures are essential to address multiple key questions in Ulva development and in algal-bacterial interactions. Here we show that another Ulva species, U. linza, with a broad geographical distribution, has the potential to be grown in axenic culture similarly to U. mutabilis. U. linza can be reliably induced to sporulate (form gametes and zoospores) in the laboratory, by cutting the relevant thallus tissue into small pieces and removing extracellular inhibitors (sporulation and swarming inhibitors). The germ cells work as an ideal feed stock for standardized algae cultures. The requirement of U. linza for bacterial signals to induce its normal morphology (particularly of the rhizoids) appears to have a species-specific component. The axenic cultures of these two species pave the way for future comparative studies of algal-microbial interactions.

Authors: E. F. Vesty, R. W. Kessler, , J. C. Coates

Date Published: 26th Jan 2015

Publication Type: Not specified

Comments on the distribution and phylogeny of type I polyketide synthases and nonribosomal peptide synthetases in eukaryotes

Abstract

Not specified

Editor:

Date Published: 2nd Sep 2014

Publication Type: Not specified

Functional genomic profiling of Aspergillus fumigatus biofilm reveals enhanced production of the mycotoxin gliotoxin

Abstract (Expand)

The opportunistic pathogenic mold Aspergillus fumigatus is an increasing cause of morbidity and mortality in immunocompromised and in part immunocompetent patients. A. fumigatus can grow in multicellular … communities by the formation of a hyphal network encased in an extracellular matrix. Here, we describe the proteome and transcriptome of planktonic- and biofilm-grown A. fumigatus mycelium after 24 and 48 h. A biofilm- and time-dependent regulation of many proteins and genes of the primary metabolism indicates a developmental stage of the young biofilm at 24 h, which demands energy. At a matured biofilm phase, metabolic activity seems to be reduced. However, genes, which code for hydrophobins, and proteins involved in the biosynthesis of secondary metabolites were significantly upregulated. In particular, proteins of the gliotoxin secondary metabolite gene cluster were induced in biofilm cultures. This was confirmed by real-time PCR and by detection of this immunologically active mycotoxin in culture supernatants using HPLC analysis. The enhanced production of gliotoxin by in vitro formed biofilms reported here may also play a significant role under in vivo conditions. It may confer A. fumigatus protection from the host immune system and also enable its survival and persistence in chronic lung infections such as aspergilloma.

Authors: S. Bruns, M. Seidler, D. Albrecht, S. Salvenmoser, N. Remme, C. Hertweck, A. A. Brakhage, O. Kniemeyer, F. M. Muller

Date Published: 21st Jul 2010

Publication Type: Not specified

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga Ostreococcus tauri

Abstract (Expand)

INTRODUCTION: The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism … of this alga. OBJECTIVES: In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness. METHODS: Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling. RESULTS: Of 111 metabolites quantified over light-dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 muM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri. CONCLUSIONS: This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.

Authors: M. Hirth, S. Liverani, S. Mahlow, F. Y. Bouget, G. Pohnert, S. Sasso

Date Published: No date defined

Publication Type: Not specified

Morphogenesis of Ulva mutabilis (Chlorophyta) induced by Maribacter species (Bacteroidetes, Flavobacteriaceae)

Abstract (Expand)

Growth and morphogenesis of the sea lettuce Ulva (Chlorophyta) depends on the combination of regulative morphogenetic compounds released by specific associated bacteria. Axenic Ulva gametes develop … parthenogenetically into callus-like colonies consisting of undifferentiated cells without normal cell walls. In Ulva mutabilis Føyn, two bacterial strains, Maribacter sp. strain MS6 and Roseovarius strain MS2, can restore the complete algal morphogenesis forming a tripartite symbiotic community. Morphogenetic compounds (=morphogens) released by the MS6-strain induce rhizoid formation and cell wall development in U. mutabilis, while several bacteria of the Roseobacter clade, including the MS2-strain, promote blade cell division and thallus elongation. In this study, 12 type strains of the Flavobacteriaceae family, including six Maribacter strains, were examined for their morphogenetic activity in comparison to the original MS6-strain isolated from U. mutabilis. The bioassay is based on the functional complementation of the tested Flavobacteriaceae strain with the Roseovarius MS2-strain. If the test-strain possesses morphogenetic activity complementary to the factor of the MS2-strain, the complete morphogenesis of U. mutabilis can be restored. This bioassay revealed not only the stand-alone activity of certain bacteria, but also their essential capability to take part in the orchestrated bacteria-induced morphogenesis of U. mutabilis. All Maribacter type strains isolated from Ulva could phenocopy the MS6-strain, whereas some distantly related Flavobacteriaceae and a Maribacter strain isolated from a red alga did not possess any activity. Keywords: bacteroidetes; cell differentiation; green macroalga; morphogens; thallusin

Authors: Thomas Wichard, Anne Weiss, R. Costa

Date Published: No date defined

Publication Type: Not specified

Antimicrobial discovery inspired by ecological interactions

Abstract

Not specified

Author: C. Hertweck

Date Published: No date defined

Publication Type: Not specified

Facile carbohydrate-mimetic modifications of poly(ethylene imine) carriers for gene delivery applications

Abstract (Expand)

Commercially-available linear and branched PEIs (LPEI and BPEI) were chemically-modified with carbohydrates and carbohydrate-mimetics to improve biocompatibility. Hydroxyl moieties were installed in a … close proximity via reaction of PEI's amines with paraformaldehyde (pF) or glycidol. Mixing PEI with pF led to the formation of hemiaminal moieties as well as N-methylation of the backbone through an Eschweiler–Clarke-type rearrangement. The amount of attached hydroxyl groups depended on the initial amount of pF and the results were in agreement with NMR studies on model reactions with primary and secondary amines. The primary amines of BPEI triggered the ring-opening of glycidol and sugar-containing epoxides, in methanol and at room temperature. PEI chains modified with pF displayed the same cytotoxicity as the parent polymer, unless a sufficient amount of pF was added to trigger N-methylation of the backbone. In contrast, glycidol and sugar-functionalized BPEIs exhibited lower toxicity but similar (if not higher) transfection efficiency as compared to unmodified BPEI.

Authors: Christoph Englert, Mareva Fevre, Rudy J. Wojtecki, Wei Cheng, Qingxing Xu, Chuan Yang, Xiyu Ke, Matthias Hartlieb, Kristian Kempe, Jeannette M. García, Robert J. Ono, Ulrich S. Schubert, Yi Yan Yang, James L. Hedrick

Date Published: No date defined

Publication Type: Not specified

Human macrophages differentially produce specific resolvin or leukotriene signals that depend on bacterial pathogenicity.

Abstract (Expand)

Proinflammatory eicosanoids (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPM) are temporally regulated during infections. Here we show that human macrophage phenotypes … biosynthesize unique lipid mediator signatures when exposed to pathogenic bacteria. E. coli and S. aureus each stimulate predominantly proinflammatory 5-lipoxygenase (LOX) and cyclooxygenase pathways (i.e., leukotriene B4 and prostaglandin E2) in M1 macrophages. These pathogens stimulate M2 macrophages to produce SPMs including resolvin D2 (RvD2), RvD5, and maresin-1. E. coli activates M2 macrophages to translocate 5-LOX and 15-LOX-1 to different subcellular locales in a Ca(2+)-dependent manner. Neither attenuated nor non-pathogenic E. coli mobilize Ca(2+) or activate LOXs, rather these bacteria stimulate prostaglandin production. RvD5 is more potent than leukotriene B4 at enhancing macrophage phagocytosis. These results indicate that M1 and M2 macrophages respond to pathogenic bacteria differently, producing either leukotrienes or resolvins that further distinguish inflammatory or pro-resolving phenotypes.

Authors: O. Werz, J. Gerstmeier, S. Libreros, X. De la Rosa, M. Werner, P. C. Norris, N. Chiang, C. N. Serhan

Date Published: No date defined

Publication Type: Not specified

A poly(A) ribonuclease controls the cellotriose-based interaction between Piriformospora indica and its host Arabidopsis.

Abstract (Expand)

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. The fungal cell wall extract induces cytoplasmic … calcium [Ca2+]cyt elevation in host plant roots. Here, we show that an elici-tor-active cell wall moiety, released by P. indica into the medium, is cellotriose (CT). CT in-duces a mild defense-like response including the production of reactive oxygen species, changes in membrane potentials and the expression of genes involved in growth regulation and root development. CT based [Ca2+]cyt elevation in Arabidopsis roots does not require BAK1 coreceptor, or the putative Ca2+ channels TPC1, GLR3.3, -2.4 and -2.5 and operates synergistically with the elicitor chitin. We identified an ethylmethane-sulfonate-induced mu-tant ([Ca2+]cyt elevation mutant, cycam) impaired in response to CT, cellooligomers (n = 2, 4-7), but not to chitooligomers (n = 4-8) in roots. The mutant contains a single nucleotide ex-change in the gene encoding for a poly(A) ribonuclease (AtPARN, At1g55870) which de-grades poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our finding of cellotriose as a novel chemical mediator might help to understand the complex P. indica-plant mutual rela-tionship in beneficial symbiosis.

Authors: J. M. Johnson, J. Thurich, E. K. Petutschnig, L. Altschmied, D. Meichsner, I. Sherameti, J. Dindas, A. Mrozinska, C. Paetz, S. S. Scholz, A. C. Furch, V. Lipka, R. Hedrich, B. Schneider, A. Svatos, R. Oelmuller

Date Published: No date defined

Publication Type: Not specified

Synergistic activity of cosecreted natural products from amoebae-associated bacteria.

Abstract (Expand)

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular … rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Authors: J. Arp, S. Gotze, R. Mukherji, D. J. Mattern, M. Garcia-Altares, M. Klapper, D. A. Brock, A. A. Brakhage, J. E. Strassmann, D. C. Queller, B. Bardl, K. Willing, G. Peschel, P. Stallforth

Date Published: No date defined

Publication Type: Not specified

Photocontrolled Release of Chemicals from Nano- and Microparticle Containers.

Abstract (Expand)

A benzoin-derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker … unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well-controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano- and microparticle containers was performed by confocal laser scanning microscopy in a proof-of-principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV-sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self-healing processes.

Authors: C. Englert, I. Nischang, C. Bader, P. Borchers, J. Alex, M. Prohl, M. Hentschel, M. Hartlieb, A. Traeger, G. Pohnert, S. Schubert, M. Gottschaldt, U. S. Schubert

Date Published: No date defined

Publication Type: Not specified

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii.

Abstract (Expand)

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, … polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green-algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in two-day old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption of zygospore development. In agreement with a role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions. This article is protected by copyright. All rights reserved.

Authors: N. Heimerl, E. Hommel, M. Westermann, D. Meichsner, M. Lohr, C. Hertweck, A. R. Grossman, M. Mittag, S. Sasso

Date Published: No date defined

Publication Type: Not specified

Defining the transcriptomic landscape of Candida glabrata by RNA-Seq.

Abstract (Expand)

Candida glabrata is the second most common pathogenic Candida species and has emerged as a leading cause of nosocomial fungal infections. Its reduced susceptibility to antifungal drugs and its close … relationship to Saccharomyces cerevisiae make it an interesting research focus. Although its genome sequence was published in 2004, little is known about its transcriptional dynamics. Here, we provide a detailed RNA-Seq-based analysis of the transcriptomic landscape of C. glabrata in nutrient-rich media, as well as under nitrosative stress and during pH shift. Using RNA-Seq data together with state-of-the-art gene prediction tools, we refined the annotation of the C. glabrata genome and predicted 49 novel protein-coding genes. Of these novel genes, 14 have homologs in S. cerevisiae and six are shared with other Candida species. We experimentally validated four novel protein-coding genes of which two are differentially regulated during pH shift and interaction with human neutrophils, indicating a potential role in host-pathogen interaction. Furthermore, we identified 58 novel non-protein-coding genes, 38 new introns and condition-specific alternative splicing. Finally, our data suggest different patterns of adaptation to pH shift and nitrosative stress in C. glabrata, Candida albicans and S. cerevisiae and thus further underline a distinct evolution of virulence in yeast.

Authors: J. Linde, S. Duggan, M. Weber, F. Horn, P. Sieber, D. Hellwig, K. Riege, M. Marz, R. Martin, Reinhard Guthke, O. Kurzai

Date Published: No date defined

Publication Type: Not specified

Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling.

Abstract (Expand)

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for … microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Authors: J. F. Mori, N. Ueberschaar, S. Lu, R. E. Cooper, G. Pohnert, K. Kusel

Date Published: No date defined

Publication Type: Not specified

Algal Oxylipins Mediate the Resistance of Diatoms against Algicidal Bacteria.

Abstract (Expand)

Algicidal bacteria can lyse microalgal blooms and trigger shifts within plankton communities. Resistant algal species can escape lysis, and have the opportunity to dominate the phytoplankton after a … bacterial infection. Despite their important function in ecosystem regulation, little is known about mechanisms of resistance. Here, we show that the diatom Chaetoceros didymus releases eicosanoid oxylipins into the medium, and that the lytic algicidal bacterium, Kordia algicida, induces the production of several wound-activated oxylipins in this resistant diatom. Neither releases nor an induction occurs in the susceptible diatom Skeletonema costatum that is lysed by the bacterium within a few days. Among the upregulated oxylipins, hydroxylated eicosapentaenoic acids (HEPEs) dominate. However, also, resolvins, known lipid mediators in mammals, increase upon exposure of the algae to the algicidal bacteria. The prevailing hydroxylated fatty acid, 15-HEPE, significantly inhibits growth of K. algicida at a concentration of approximately 1 microM. The oxylipin production may represent an independent line of defense of the resistant alga, acting in addition to the previously reported upregulation of proteases.

Authors: N. Meyer, J. Rettner, M. Werner, O. Werz, G. Pohnert

Date Published: No date defined

Publication Type: Not specified

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