Dieter Spiteller

Candicidin-producing Streptomyces support leaf-cutting ants to protect their fungus garden against the pathogenic fungus Escovopsis

Leaf-cutting ants such as Acromyrmex octospinosus live in obligate symbiosis with fungi of the genus Leucoagaricus, which they grow with harvested leaf material. The symbiotic fungi, in turn, serve as a major food source for the ants. This mutualistic relation is disturbed by the specialized pathogenic fungus Escovopsis sp., which can overcome Leucoagaricus sp. and thus destroy the ant colony. Microbial symbionts of leaf-cutting ants have been suggested to protect the fungus garden against Escovopsis by producing antifungal compounds [Currie CR, Scott JA, Summerbell RC, Malloch D (1999) Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398:701–704.]. To date, however, the chemical nature of these compounds has remained elusive. We characterized 19 leaf-cutting ant–associated microorganisms (5 Pseudonocardia, 1 Dermacoccus, and 13 Streptomyces) from 3 Acromyrmex species, A. octospinosus, A. echinatior, and A. volcanus, using 16S-rDNA analysis. Because the strain Streptomyces sp. Ao10 proved highly active against the pathogen Escovopsis, we identified the molecular basis of its antifungal activity. Using bioassay-guided fractionation, high-resolution electrospray mass spectrometry (HR-ESI-MS), and UV spectroscopy, and comparing the results with an authentic standard, we were able identify candicidin macrolides. Candicidin macrolides are highly active against Escovopsis but do not significantly affect the growth of the symbiotic fungus. At least one of the microbial isolates from each of the 3 leaf-cutting ant species analyzed produced candicidin macrolides. This suggests that candicidins play an important role in protecting the fungus gardens of leaf-cutting ants against pathogenic fungi.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves : I. Membrane Potentials, Intracellular Calcium Variations, Oral Secretions, and Regurgitate Components

Membrane potentials (Vm) and intracellular calcium variations were studied in Lima bean (Phaseolus lunatus) leaves when the Mediterranean climbing cutworm (Spodoptera littoralis) was attacking the plants. In addition to the effect of the feeding insect the impact of several N-acyl Glns (volicitin, N-palmitoyl-Gln, N-linolenoyl-Gln) from the larval oral secretion was studied. The results showed that the early events upon herbivore attack were: a) a strong Vm depolarization at the bite zone and an isotropic wave of Vm depolarization spreading throughout the entire attacked leaf; b) a Vm depolarization observed for the regurgitant but not with volicitin {N-(17-hydroxy-linolenoyl)-Gln} alone; c) an enhanced influx of Ca2+ at the very edge of the bite, which is halved, if the Ca2+ channel blocker Verapamil is used. Furthermore, the dose-dependence effects of N-acyl Gln conjugates-triggered influx of Ca2+ studied in transgenic aequorin-expressing soybean (Glycine max) cells, showed: a) a concentration-dependent influx of Ca2+; b) a configuration-independent effect concerning the stereochemistry of the amino acid moiety; c) a slightly reduced influx of Ca2+ after modification of the fatty acid backbone by functionalization with oxygen and; d) a comparable effect with the detergent SDS. Finally, the herbivore wounding causes a response in the plant cells that cannot be mimicked by mechanical wounding. The involvement of Ca2+ in signaling after herbivore wounding is discussed.

Gut bacteria may be involved in interactions between plants, herbivores and their predators: Microbial biosynthesis of N- acylglutamine surfactants as elicitors of plant volatiles

Abstract N-Acylamino acids are dominant and widespread constituents of insect oral secretions (regurgitants), serving the insect as biosurfactants in the digestive process. During feeding the conjugates may be introduced into damaged leaves and contribute there to the elicitation of plant defenses such as the induction of volatile biosynthesis. From gut segments of Spodoptera exigua, Mamestra brassicae and Agrotis segetum 23 bacterial strains were isolated, ten of which were able to synthesise typical lepidopteran N-acylamino acids from externally added precursors. Four strains, Providencia rettgeri, Ochrobactrum spec., Myroides odoratus and Acinetobacter sp. genospecies 11 were identified on the basis of their 16 S rDNA. The organisms displayed a very broad substrate tolerance, since fatty acids of different chain length and different degree of saturation were converted into N-acylamino acids. Moreover, most of the proteinogenic amino acids, but not glutamic and aspartic acid, were used as substrates. The dominant occurrence of fatty acids conjugated with glutamine may result from a preferred transport of glutamine from the hemolymph into the gut of the insects. The involvement of bacteria in the biosynthesis of compounds which play a pivotal role in the interaction of plants, herbivores and their predators adds a new trophic level to this complex network of interactions. Due to their short generation cycle and the ease of adaptation endosymbiontic bacteria may have an outstanding importance for the coevolution of plant-insect interactions.

Chemical basis of the synergism and antagonism in microbial communities in the nests of leaf-cutting ants.

Leaf-cutting ants cultivate the fungus Leucoagaricus gongylophorus, which serves as a major food source. This symbiosis is threatened by microbial pathogens that can severely infect L. gongylophorus. Microbial symbionts of leaf-cutting ants, mainly Pseudonocardia and Streptomyces, support the ants in defending their fungus gardens against infections by supplying antimicrobial and antifungal compounds. The ecological role of microorganisms in the nests of leaf-cutting ants can only be addressed in detail if their secondary metabolites are known. Here, we use an approach for the rapid identification of established bioactive compounds from microorganisms in ecological contexts by combining phylogenetic data, database searches, and liquid chromatography electrospray ionisation high resolution mass spectrometry (LC-ESI-HR-MS) screening. Antimycins A1–A4, valinomycins, and actinomycins were identified in this manner from Streptomyces symbionts of leaf-cutting ants. Matrix-assisted laser desorption ionization (MALDI) imaging revealed the distribution of valinomycin directly on the integument of Acromyrmex echinatior workers. Valinomycins and actinomycins were also directly identified in samples from the waste of A. echinatior and A. niger leaf-cutting ants, suggesting that the compounds exert their antimicrobial and antifungal potential in the nests of leaf-cutting ants. Strong synergistic effects of the secondary meta-bolites produced by ant-associated Streptomyces were observed in the agar diffusion assay against Escovopsis weberi. Actinomycins strongly inhibit soil bacteria as well as other Streptomyces and Pseudonocardia symbionts. The antifungal antimycins are not only active against pathogenic fungi but also the garden fungus L. gongylophorus itself. In conclusion, secondary metabolites of microbial symbionts of leaf-cutting ants contribute to shaping the microbial communities within the nests of leaf-cutting ants.

Absolute configuration of volicitin, an elicitor of plant volatile biosynthesis from lepidopteran larvae

The absolute configuration of the hydroxylinolenoyl moiety of volicitin (17-hydroxylinolenoyl-l-glutamine) 1a/b from four lepidopteran larvae was determined by methanolysis and derivatisation of the resulting ester with (1R)-1-phenyl-ethylisocyanate 3. The absolute configuration of the resulting (1′R)-17-(1′-phenyl-ethylcarbamoyloxy)-methyl linolenoates 4a/b followed from GLC comparison with synthetic references. Natural volicitin 1 from caterpillars of Spodoptera exigua, S. frugiperda, S. littoralis and Heliothis virescens (Noctuidae) exhibits a high ee (92–96%) and has a 17S configuration.

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Sulphoglycolysis in Escherichia coli K-12 closes a gap in the biogeochemical sulphur cycle

Sulphoquinovose (SQ, 6-deoxy-6-sulphoglucose) has been known for 50 years as the polar headgroup of the plant sulpholipid in the photosynthetic membranes of all higher plants, mosses, ferns, algae and most photosynthetic bacteria. It is also found in some non-photosynthetic bacteria, and SQ is part of the surface layer of some Archaea. The estimated annual production of SQ is 10,000,000,000 tonnes (10 petagrams), thus it comprises a major portion of the organo-sulphur in nature, where SQ is degraded by bacteria. However, despite evidence for at least three different degradative pathways in bacteria, no enzymic reaction or gene in any pathway has been defined, although a sulphoglycolytic pathway has been proposed. Here we show that Escherichia coli K-12, the most widely studied prokaryotic model organism, performs sulphoglycolysis, in addition to standard glycolysis. SQ is catabolised through four newly discovered reactions that we established using purified, heterologously expressed enzymes: SQ isomerase, 6-deoxy-6-sulphofructose (SF) kinase, 6-deoxy-6-sulphofructose-1-phosphate (SFP) aldolase, and 3-sulpholactaldehyde (SLA) reductase. The enzymes are encoded in a ten-gene cluster, which probably also encodes regulation, transport and degradation of the whole sulpholipid; the gene cluster is present in almost all (>91%) available E. coli genomes, and is widespread in Enterobacteriaceae. The pathway yields dihydroxyacetone phosphate (DHAP), which powers energy conservation and growth of E. coli, and the sulphonate product 2,3-dihydroxypropane-1-sulphonate (DHPS), which is excreted. DHPS is mineralized by other bacteria, thus closing the sulphur cycle within a bacterial community.

N-(15,16-Dihydroxylinoleoyl)-glutamine and N-(15,16- epoxylinoleoyl)-glutamine isolated from oral secretions of lepidopteran larvae

Abstract N-(15,16-Dihydroxylinoleoyl)-glutamine ( 1 ) and N-(15,16-epoxylinoleoyl)-glutamine ( 2 ) and were identified in the regurgitant of lepidopteran larvae (Spodoptera exigua and Spodoptera frugiperda) by LC–MS. After methanolysis and derivatisation with MSTFA, the positions of the hydroxy groups of 1 were identified by GC–MS. The structures of both conjugates were confirmed by synthesis.

Malonyl carba(dethia)- and Malonyl oxa(dethia)-coenzyme A as Tools for Trapping Polyketide Intermediates

Caught in a trap. In this study trapped polyketide species (see figure) were off‐loaded from a type III PKS by novel nonhydrolyzable malonyl coenzyme A analogues in which a methylene group or an oxygen atom replaces the sulfur atom of malonyl‐CoA. This strategy allows the straightforward characterisation of intermediates of polyketide biosynthesis by LC‐HR‐ESI‐MS/MS and provides valuable insights on the mechanism and timing of polyketide formation.

Elaboration of Neosamine Rings in the Biosynthesis of Neomycin and Butirosin

The proteins Neo‐11 and Neo‐18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl‐6′‐oxidase and 6′‐oxoglucosaminyl:L‐glutamate aminotransferase, respectively. The joint activity of Neo‐11 and Neo‐18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD‐dependent dehydrogenation followed by a pyridoxal‐5′‐phosphate‐mediated transamination. Neo‐18 is also shown to catalyze deamination at C‐6′′′ of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the btrB gene, a homologue of neo‐18 in the butirosin biosynthetic gene cluster (btr) in Bacillus circulans, exhibits the same activity as Neo‐18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.