Biowissenschaften
Refine
Year of publication
Document Type
- Article (33)
Language
- English (33) (remove)
Has Fulltext
- yes (33)
Is part of the Bibliography
- no (33)
Keywords
- Photorhabdus (4)
- Natural products (2)
- Secondary metabolism (2)
- Xenorhabdus (2)
- natural products (2)
- quorum sensing (2)
- 4,4’-dihydroxy-nostoxanthin (1)
- 4,4’-diketo-nostoxanthin (1)
- Aspergillus nidulans (1)
- Autoinducer-2 (1)
Institute
- Biowissenschaften (33)
- Institut für Ökologie, Evolution und Diversität (6)
- Biodiversität und Klima Forschungszentrum (BiK-F) (5)
- Senckenbergische Naturforschende Gesellschaft (5)
- LOEWE-Schwerpunkt für Integrative Pilzforschung (2)
- Medizin (2)
- Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) (1)
- Exzellenzcluster Makromolekulare Komplexe (1)
- Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) (1)
Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
Non-ribosomal peptide synthetases (NRPSs) are large multienzyme machineries. They synthesize numerous important natural products starting from amino acids. For peptide synthesis functionally specialized NRPS modules interact in a defined manner. Individual modules are either located on a single or on multiple different polypeptide chains. The “peptide-antimicrobial-Xenorhabdus” (PAX) peptide producing NRPS PaxS from Xenorhabdus bacteria consists of the three proteins PaxA, PaxB and PaxC. Different docking domains (DDs) located at the N-termini of PaxB and PaxC and at the C-termini of PaxA and BaxB mediate specific non-covalent interactions between them. The N-terminal docking domains precede condensation domains while the C-terminal docking domains follow thiolation domains. The binding specificity of individual DDs is important for the correct assembly of multi-protein NRPS systems. In many multi-protein NRPS systems the docking domains are sufficient to mediate the necessary interactions between individual protein chains. However, it remains unclear if this is a general feature for all types of structurally different docking domains or if the neighboring domains in some cases support the function of the docking domains. Here, we report the 1H, 13C and 15 N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (CDD) from PaxA and for its binding partner – the N-terminal docking domain (NDD) from PaxB from the Gram-negative entomopathogenic bacterium Xenorhabdus cabanillasii JM26 in their free states and for a 1:1 complex formed by the two proteins. These NMR resonance assignments will facilitate further structural and dynamic studies of this protein complex.
A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B–D (2–4), were identified by detailed HPLC–MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 μm. The biosynthesis of phototemtides A–D (1–4) was also proposed.
Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.
Nonribosomal peptides produced by minimal and engineered synthetases with terminal reductase domains
(2020)
Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2‐electron reduction of the enzyme‐bound thioester releasing the generated peptides as C‐terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde‐generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.
Several members of the genus Legionella cause Legionnaires’ disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella.
Background: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.
Results: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.
Conclusions: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila
(2019)
The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.
Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.
Biosynthetic gene content of the "Perfume Lichens" Evernia prunastri and Pseudevernia furfuracea
(2019)
Lichen-forming fungi produce a vast number of unique natural products with a wide variety of biological activities and human uses. Although lichens have remarkable potential in natural product research and industry, the molecular mechanisms underlying the biosynthesis of lichen metabolites are poorly understood. Here we use genome mining and comparative genomics to assess biosynthetic gene clusters and their putative regulators in the genomes of two lichen-forming fungi, which have substantial commercial value in the perfume industry, Evernia prunastri and Pseudevernia furfuracea. We report a total of 80 biosynthetic gene clusters (polyketide synthases (PKS), non-ribosomal peptide synthetases and terpene synthases) in E. prunastri and 51 in P. furfuracea. We present an in-depth comparison of 11 clusters, which show high homology between the two species. A ketosynthase (KS) phylogeny shows that biosynthetic gene clusters from E. prunastri and P. furfuracea are widespread across the Fungi. The phylogeny includes 15 genomes of lichenized fungi and all fungal PKSs with known functions from the MIBiG database. Phylogenetically closely related KS domains predict not only similar PKS architecture but also similar cluster architecture. Our study highlights the untapped biosynthetic richness of lichen-forming fungi, provides new insights into lichen biosynthetic pathways and facilitates heterologous expression of lichen biosynthetic gene clusters.
The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. Bfunction is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.
The pyrrolobenzodiazepine tilivalline (1) was originally identified in the human gut pathobiont Klebsiella oxytoca, the causative agent of antibiotic-associated hemorrhagic colitis. Here we show the identification of tilivalline and analogs thereof in the entomopathogenic bacterium Xenorhabdus eapokensis as well as the identification of its biosynthesis gene cluster encoding a bimodular non-ribosomal peptide synthetase. Heterologous expression of both genes in E. coli resulted in the production of 1 and from mutasynthesis and precursor directed biosynthesis 11 new tilivalline analogs were identified in X. eapokensis. These results allowed the prediction of the tilivalline biosynthesis being similar to that in K. oxytoca.
The Gram-negative bacteria Photorhabdus and Xenorhabdus are known to produce a variety of different natural products (NP). These compounds play different roles since the bacteria live in symbiosis with nematodes and are pathogenic to insect larvae in the soil. Thus, a fine tuned regulatory system controlling NP biosynthesis is indispensable. Global regulators such as Hfq, Lrp, LeuO and HexA have been shown to influence NP production of Photorhabdus and Xenorhabdus. Additionally, photopyrones as quorum sensing (QS) signals were demonstrated to be involved in the regulation of NP production in Photorhabdus. In this study, we investigated the role of another possible QS signal, autoinducer-2 (AI-2), in regulation of NP production. The AI-2 synthase (LuxS) is widely distributed within the bacterial kingdom and has a dual role as a part of the activated methyl cycle pathway, as well as being responsible for AI-2 precursor production. We deleted luxS in three different entomopathogenic bacteria and compared NP levels in the mutant strains to the wild type (WT) but observed no difference to the WT strains. Furthermore, the absence of the small regulatory RNA micA, which is encoded directly upstream of luxS, did not influence NP levels. Phenotypic differences between the P. luminescens luxS deletion mutant and an earlier described luxS deficient strain of P. luminescens suggested that two phenotypically different strains have evolved in different laboratories.
Janthinobacterium and Duganella are well-known for their antifungal effects. Surprisingly, almost nothing is known on molecular aspects involved in the close bacterium-fungus interaction. To better understand this interaction, we established the genomes of 11 Janthinobacterium and Duganella isolates in combination with phylogenetic and functional analyses of all publicly available genomes. Thereby, we identified a core and pan genome of 1058 and 23,628 genes. All strains encoded secondary metabolite gene clusters and chitinases, both possibly involved in fungal growth suppression. All but one strain carried a single gene cluster involved in the biosynthesis of alpha-hydroxyketone-like autoinducer molecules, designated JAI-1. Genome-wide RNA-seq studies employing the background of two isolates and the corresponding JAI-1 deficient strains identified a set of 45 QS-regulated genes in both isolates. Most regulated genes are characterized by a conserved sequence motif within the promoter region. Among the most strongly regulated genes were secondary metabolite and type VI secretion system gene clusters. Most intriguing, co-incubation studies of J. sp. HH102 or its corresponding JAI-1 synthase deletion mutant with the plant pathogen Fusarium graminearum provided first evidence of a QS-dependent interaction with this pathogen.
Heterogeneous regulation of bacterial natural product biosynthesis via a novel transcription factor
(2016)
Biological diversity arises among genetically equal subpopulations in the same environment, a phenomenon called phenotypic heterogeneity. The life cycle of the enteric bacterium Photorhabdus luminescens involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic forms designated as primary (1°) and secondary (2°). In contrast to 1° cells, 2° cells are non-pigmented due to the absence of natural compounds, especially anthraquinones (AQs). We identified a novel type of transcriptional regulator, AntJ, which activates expression of the antA-I operon responsible for AQ production. AntJ heterogeneously activates the AQ production in single P. luminescens 1° cells, and blocks AQ production in 2° cells. AntJ contains a proposed ligand-binding WYL-domain, which is widespread among bacteria. AntJ is one of the rare examples of regulators that mediate heterogeneous gene expression by altering activity rather than copy number in single cells.
In search for new natural products, which may lead to the development of new drugs for all kind of applications, novel methods are needed. Here we describe the identification of electrophilic natural products in crude extracts via their reactivity against azide as a nucleophile followed by their subsequent enrichment using a cleavable azide-reactive resin (CARR). Using this approach, natural products carrying epoxides and α,β-unsaturated enones as well as several unknown compounds were identified in crude extracts from entomopathogenic Photorhabdus bacteria.
The red yeast Xanthophyllomyces dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a β-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4’-diketo-nostoxanthin (2,3,2’,3’-tetrahydroxy-4,4’-diketo-β-carotene) and 4-keto-nostoxanthin (2,3,2’,3’-tetrahydroxy-4-monoketo-β-carotene). Both keto carotenoids and the resulting 4,4’-dihydroxy-nostoxanthin (2,3,4,2’,3’,4’-hexahydroxy-β-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2’3’-pentahydroxy-β-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.
In China and other countries of East Asia, so-called Ling-zhi or Reishi mushrooms are used in traditional medicine since several centuries. Although the common practice to apply the originally European name ‘Ganoderma lucidum’ to these fungi has been questioned by several taxonomists, this is still generally done in recent publications and with commercially cultivated strains. In the present study, two commercially sold strains of ‘G. lucidum’, M9720 and M9724 from the company Mycelia bvba (Belgium), are compared for their fruiting body (basidiocarp) morphology combined with molecular phylogenetic analyses, and for their secondary metabolite profile employing an ultra-performance liquid chromatography–electrospray ionization mass spectrometry (UPLC–ESIMS) in combination with a high resolution electrospray ionization mass spectrometry (HR-ESI-MS). According to basidiocarp morphology, the strain M9720 was identified as G. lucidum s.str. whereas M9724 was determined as Ganoderma lingzhi. In molecular phylogenetic analyses, the M9720 ITS and beta-tubulin sequences grouped with sequences of G. lucidum s.str. from Europe whereas those from M9724 clustered with sequences of G. lingzhi from East Asia. We show that an ethanol extract of ground basidiocarps from G. lucidum (M9720) contains much less triterpenic acids than found in the extract of G. lingzhi (M9724). The high amount of triterpenic acids accounts for the bitter taste of the basidiocarps of G. lingzhi (M9724) and of its ethanol extract. Apparently, triterpenic acids of G. lucidum s.str. are analyzed here for the first time. These results demonstrate the importance of taxonomy for commercial use of fungi.
Background: Bacteria within the genus Photorhabdus maintain mutualistic symbioses with nematodes in complicated lifecycles that also involves insect pathogenic phases. Intriguingly, these bacteria are rich in biosynthetic gene clusters that produce compounds with diverse biological activities. As a basis to better understand the life cycles of Photorhabdus we sequenced the genomes of two recently discovered representative species and performed detailed genomic comparisons with five publically available genomes.
Results: Here we report the genomic details of two new reference Photorhabdus species. By then conducting genomic comparisons across the genus, we show that there are several highly conserved biosynthetic gene clusters. These clusters produce a range of bioactive small molecules that support the pathogenic phase of the integral relationship that Photorhabdus maintain with nematodes.
Conclusions: Photorhabdus contain several genetic loci that allow them to become specialist insect pathogens by efficiently evading insect immune responses and killing the insect host.