Publications by authors named "Helge B Bode"

194 Publications

Towards the sustainable discovery and development of new antibiotics.

Nat Rev Chem 2021 Aug 19:1-24. Epub 2021 Aug 19.

Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI), and Department of Pharmacy, Saarland University Campus E8.1, Saarbrücken, Germany.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
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http://dx.doi.org/10.1038/s41570-021-00313-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8374425PMC
August 2021

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Appl Microbiol Biotechnol 2021 Jul 12;105(13):5517-5528. Epub 2021 Jul 12.

Department of Biology, Faculty of Arts and Science, Aydin Adnan Menderes University, Aydin, Turkey.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.
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http://dx.doi.org/10.1007/s00253-021-11435-3DOI Listing
July 2021

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus.

Environ Microbiol 2021 Aug 8;23(8):4260-4275. Epub 2021 Jun 8.

Senckenberg Biodiversity and Climate Research Centre (SBiK-F), Frankfurt, 60325, Germany.

Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.
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http://dx.doi.org/10.1111/1462-2920.15605DOI Listing
August 2021

Xenocoumacin 2 reduces protein biosynthesis and inhibits inflammatory and angiogenesis-related processes in endothelial cells.

Biomed Pharmacother 2021 Aug 28;140:111765. Epub 2021 May 28.

Institute of Pharmaceutical Biology, Faculty of Biochemistry, Chemistry and Pharmacy, Goethe University Frankfurt, Frankfurt, Germany; LOEWE Center for Translational Biodiversity Genomics (LOEWE-TBG), Frankfurt, Germany. Electronic address:

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E (PGE), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.
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http://dx.doi.org/10.1016/j.biopha.2021.111765DOI Listing
August 2021

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery.

Nat Commun 2021 05 28;12(1):3225. Epub 2021 May 28.

Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA.

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.
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http://dx.doi.org/10.1038/s41467-021-23502-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163882PMC
May 2021

Relative potency of a novel acaricidal compound from Xenorhabdus, a bacterial genus mutualistically associated with entomopathogenic nematodes.

Sci Rep 2021 05 27;11(1):11253. Epub 2021 May 27.

Department of Plant Protection, Faculty of Agriculture, Aydin Adnan Menderes University, Aydin, Turkey.

Our study aimed to identify the novel acaricidal compound in Xenorhabdus szentirmaii and X. nematophila using the easyPACId approach (easy Promoter Activated Compound Identification). We determined the (1) effects of cell-free supernatant (CFS) obtained from mutant strains against T. urticae females, (2) CFS of the acaricidal bioactive strain of X. nematophila (pCEP_kan_XNC1_1711) against different biological stages of T. urticae, and females of predatory mites, Phytoseiulus persimilis and Neoseiulus californicus, (3) effects of the extracted acaricidal compound on different biological stages of T. urticae, and (4) cytotoxicity of the active substance. The results showed that xenocoumacin produced by X. nematophila was the bioactive acaricidal compound, whereas the acaricidal compound in X. szentirmaii was not determined. The CFS of X. nematophila (pCEP_kan_XNC1_1711) caused 100, 100, 97.3, and 98.1% mortality on larvae, protonymph, deutonymph and adult female of T. urticae at 7 dpa in petri dish experiments; and significantly reduced T. urticae population in pot experiments. However, the same CFS caused less than 36% mortality on the predatory mites at 7dpa. The mortality rates of extracted acaricidal compound (xenocoumacin) on the larva, protonymph, deutonymph and adult female of T. urticae were 100, 100, 97, 96% at 7 dpa. Cytotoxicity assay showed that IC value of xenocoumacin extract was 17.71 μg/ml after 48 h. The data of this study showed that xenocoumacin could potentially be used as bio-acaricide in the control of T. urticae; however, its efficacy in field experiments and its phytotoxicity need to be assessed in future.
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http://dx.doi.org/10.1038/s41598-021-90726-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159955PMC
May 2021

Synthetic Zippers as an Enabling Tool for Engineering of Non-Ribosomal Peptide Synthetases*.

Angew Chem Int Ed Engl 2021 08 27;60(32):17531-17538. Epub 2021 Jun 27.

Molecular Biotechnology, Institute of Molecular Biosciences, Goethe University Frankfurt, 60438, Frankfurt am Main, Germany.

Non-ribosomal peptide synthetases (NRPSs) are the origin of a wide range of natural products, including many clinically used drugs. Efficient engineering of these often giant biosynthetic machineries to produce novel non-ribosomal peptides (NRPs) is an ongoing challenge. Here we describe a cloning and co-expression strategy to functionally combine NRPS fragments of Gram-negative and -positive origin, synthesising novel peptides at titres up to 220 mg L . Extending from the recently introduced definition of eXchange Units (XUs), we inserted synthetic zippers (SZs) to split single protein NRPSs into independently expressed and translated polypeptide chains. These synthetic type of NRPS (type S) enables easier access to engineering, overcomes cloning limitations, and provides a simple and rapid approach to building peptide libraries via the combination of different NRPS subunits.
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http://dx.doi.org/10.1002/anie.202102859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362031PMC
August 2021

Cooperation between a T Domain and a Minimal C-Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS.

Angew Chem Int Ed Engl 2021 06 14;60(25):14171-14178. Epub 2021 May 14.

Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, 60438, Frankfurt am Main, Germany.

Non-ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N- and C-terminal docking domains ( DDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of "designer" NRPS from proteins that normally do not interact. The multiprotein "peptide-antimicrobial-Xenorhabdus" (PAX) peptide-producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small DD of PaxA cooperates with its preceding thiolation (T ) domain to bind the DD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.
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http://dx.doi.org/10.1002/anie.202103498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8251938PMC
June 2021

Structure and biosynthesis of deoxy-polyamine in Xenorhabdus bovienii.

J Ind Microbiol Biotechnol 2021 Jun;48(3-4)

Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, 60438 Frankfurt, Germany.

Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans.
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http://dx.doi.org/10.1093/jimb/kuab006DOI Listing
June 2021

NMR resonance assignments for a docking domain pair with an attached thiolation domain from the PAX peptide-producing NRPS from Xenorhabdus cabanillasii.

Biomol NMR Assign 2021 04 5;15(1):229-234. Epub 2021 Mar 5.

Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, 60438, Frankfurt am Main, Germany.

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 H, C and  N NMR resonance assignments for a C-terminal di-domain construct containing a thiolation (T) domain followed by a C-terminal docking domain (DD) from PaxA and for its binding partner - the N-terminal docking domain (DD) 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.
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http://dx.doi.org/10.1007/s12104-021-10010-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973640PMC
April 2021

Microbial Cationic Peptides as a Natural Defense Mechanism against Insect Antimicrobial Peptides.

ACS Chem Biol 2021 03 17;16(3):447-451. Epub 2021 Feb 17.

Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe-Universität Frankfurt, Frankfurt am Main 60438, Germany.

Bacteria produce a plethora of specialized metabolites (SM), with the ecological function of most of them not known. A major group of SM are peptides derived from nonribosomal peptide synthetases (NRPS). In entomopathogenic bacteria of the genus , PAX (peptide-antimicrobial-) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in and found the majority bound to the cells. We derivatized PAX with fluorophores and show binding to cells when added externally using super-resolution microscopy. Externally added PAX in and as well as inducible PAX production in showed a protective effect against various antimicrobial peptides (AMPs) from insects, where they are used as a defense mechanism against pathogens. Because AMPs are often positively charged, our results suggest a PAX-induced repulsive force due to positive charge at the bacterial cell wall.
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http://dx.doi.org/10.1021/acschembio.0c00794DOI Listing
March 2021

subsp. subsp. nov., subsp. subsp. nov., subsp. subsp. nov., subsp. subsp. nov., and sp. nov. isolated from entomopathogenic nematodes.

Int J Syst Evol Microbiol 2021 Jan 19;71(1). Epub 2021 Jan 19.

Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1, Q614 and PB68.1, isolated from the digestive system of entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family , and to the genus . Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1 is closely related to , that Q614 is closely related to and that PB68.1 is closely related to genomic comparisons confirm these observations: BA1 and 15138 share 68.8 % digital DNA-DNA hybridization (dDDH), Q614 and SF41 share 75.4 % dDDH, and PB68.1 and DSM 17609 share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1 as a new species within the genus , Q614 as a new subspecies within and PB68.1 as a new subspecies within . Hence, the following names are proposed for these strains: sp. nov. with the type strain BA1(=DSM 111180=CCOS 1943=LMG 31957), subsp subsp. nov. with the type strain Q614 (=DSM 111144=CCOS 1944=LMG 31959) and subsp subsp. nov. with the type strain PB68.1 (=DSM 111145=CCOS 1942). These propositions automatically create subsp subsp. nov. with SF41 as the type strain (currently classified as ) and subsp. subsp. nov. with DSM17609 as the type strain (currently classified as ).
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http://dx.doi.org/10.1099/ijsem.0.004610DOI Listing
January 2021

A Desaturase-Like Enzyme Catalyzes Oxazole Formation in Pseudomonas Indolyloxazole Alkaloids.

Angew Chem Int Ed Engl 2021 04 5;60(16):8781-8785. Epub 2021 Mar 5.

Eidgenössische Technische Hochschule (ETH) Zürich, Institute of Microbiology, Vladimir-Prelog-Weg 4, 8093, Zürich, Switzerland.

Indolyloxazole alkaloids occur in diverse micro- and macroorganisms and exhibit a wide range of pharmacological activities. Despite their ubiquitous occurrence and simple structures, the biosynthetic pathway remained unknown. Here, we used transposon mutagenesis in the labradorin producer Pseudomonas entomophila to identify a cryptic biosynthetic locus encoding an N-acyltransferase and a non-heme diiron desaturase-like enzyme. Heterologous expression in E. coli demonstrates that both enzymes are sufficient to produce indolyloxazoles. Probing their function in stable-isotope feeding experiments, we provide evidence for an unusual desaturase mechanism that generates the oxazole by decarboxylative cyclization.
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http://dx.doi.org/10.1002/anie.202014491DOI Listing
April 2021

Activation, Structure, Biosynthesis and Bioactivity of Glidobactin-like Proteasome Inhibitors from Photorhabdus laumondii.

Chembiochem 2021 May 3;22(9):1582-1588. Epub 2021 Mar 3.

Molecular Biotechnology, Department of Biosciences, Goethe University Frankfurt, 60438, Frankfurt am Main, Germany.

The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
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http://dx.doi.org/10.1002/cbic.202100014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248439PMC
May 2021

An Unconventional Melanin Biosynthesis Pathway in Ustilago maydis.

Appl Environ Microbiol 2021 01 15;87(3). Epub 2021 Jan 15.

Department of Biology, Philipps University Marburg, Marburg, Germany

is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well-established model organism for the study of plant-microbe interactions, its potential to produce specialized metabolites, which might contribute to this interaction, has not been studied in detail. By analyzing the genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (, , and ), a cytochrome P450 monooxygenase (), and a protein with similarity to versicolorin B synthase (). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimers to generate orsellinic acid (OA), which is reduced to the corresponding aldehyde by Pks5. The OA-aldehyde can then react with triacetic acid lactone (TAL), also derived from Pks3/Pks4 heterodimers to form larger molecules, including novel coumarin derivatives. Our findings suggest that synthesizes a novel type of melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or l-3,4-dihydroxyphenylalanine (l-DOPA). Along with these observations, this work also provides insight into the mechanisms of polyketide synthases in this filamentous fungus. The fungus represents one of the major threats to maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by to protect itself against harsh environments, such as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in an unconventional way, requiring more than one polyketide synthase for producing melanin precursors, gives a different perspective on the complexity of these multidomain enzymes and their evolution in the fungal kingdom.
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http://dx.doi.org/10.1128/AEM.01510-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848912PMC
January 2021

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Nat Microbiol 2020 12 2;5(12):1481-1489. Epub 2020 Nov 2.

Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe-Universität Frankfurt, Frankfurt, Germany.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence. Although regulators have been implicated in the regulation of these specialized metabolites, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.
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http://dx.doi.org/10.1038/s41564-020-00797-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610847PMC
December 2020

Competition and Co-existence of Two Photorhabdus Symbionts with a Nematode Host.

Microb Ecol 2021 Jan 21;81(1):223-239. Epub 2020 Aug 21.

Department of Biology, Maynooth University, Maynooth, County Kildare, Ireland.

Photorhabdus spp. (Enterobacteriales: Morganellaceae) occur exclusively as symbionts of Heterorhabditis nematodes for which they provide numerous services, including killing insects and providing nutrition and defence within the cadavers. Unusually, two species (Photorhabdus cinerea and Photorhabdus temperata) associate with a single population of Heterorhabditis downesi at a dune grassland site. Building on previous work, we investigated competition between these two Photorhabdus species both at the regional (between insects) and local (within insect) level by trait comparison and co-culture experiments. There was no difference between the species with respect to supporting nematode reproduction and protection of cadavers against invertebrate scavengers, but P. cinerea was superior to P. temperata in several traits: faster growth rate, greater antibacterial and antifungal activity and colonisation of a higher proportion of nematodes in co-culture. Moreover, where both bacterial symbionts colonised single nematode infective juveniles, P. cinerea tended to dominate in numbers. Differences between Photorhabdus species were detected in the suite of secondary metabolites produced: P. temperata produced several compounds not produced by P. cinerea including anthraquinone pigments. Bioluminescence emitted by P. temperata also tended to be brighter than that from P. cinerea. Bioluminescence and pigmentation may protect cadavers against scavengers that rely on sight. We conclude that while P. cinerea may show greater local level (within-cadaver) competitive success, co-existence of the two Photorhabdus species in the spatially heterogeneous environment of the dunes is favoured by differing specialisations in defence of the cadaver against differing locally important threats.
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http://dx.doi.org/10.1007/s00248-020-01573-yDOI Listing
January 2021

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

ACS Synth Biol 2020 09 3;9(9):2418-2426. Epub 2020 Sep 3.

Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, Frankfurt am Main, 60438, Germany.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms and KJ12.1, respectively. These produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.
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http://dx.doi.org/10.1021/acssynbio.0c00186DOI Listing
September 2020

Structural snapshots of the minimal PKS system responsible for octaketide biosynthesis.

Nat Chem 2020 08 6;12(8):755-763. Epub 2020 Jul 6.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Garching, Germany.

Type II polyketide synthases (PKSs) are multi-enzyme complexes that produce secondary metabolites of medical relevance. Chemical backbones of such polyketides are produced by minimal PKS systems that consist of a malonyl transacylase, an acyl carrier protein and an α/β heterodimeric ketosynthase. Here, we present X-ray structures of all ternary complexes that constitute the minimal PKS system for anthraquinone biosynthesis in Photorhabdus luminescens. In addition, we characterize this invariable core using molecular simulations, mutagenesis experiments and functional assays. We show that malonylation of the acyl carrier protein is accompanied by major structural rearrangements in the transacylase. Principles of an ongoing chain elongation are derived from the ternary complex with a hexaketide covalently linking the heterodimeric ketosynthase with the acyl carrier protein. Our results for the minimal PKS system provide mechanistic understanding of PKSs and a fundamental basis for engineering PKS pathways for future applications.
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http://dx.doi.org/10.1038/s41557-020-0491-7DOI Listing
August 2020

Roadmap for naming uncultivated Archaea and Bacteria.

Nat Microbiol 2020 08 8;5(8):987-994. Epub 2020 Jun 8.

National Museum of Natural Sciences, CSIC, Madrid, Spain.

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.
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http://dx.doi.org/10.1038/s41564-020-0733-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381421PMC
August 2020

Fabclavine diversity in bacteria.

Beilstein J Org Chem 2020 7;16:956-965. Epub 2020 May 7.

Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.

The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera or . In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected and mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine () biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.
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http://dx.doi.org/10.3762/bjoc.16.84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214866PMC
May 2020

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains.

Chembiochem 2020 10 25;21(19):2750-2754. Epub 2020 Jun 25.

Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe-Universität Frankfurt, 60438, Frankfurt am Main, Germany.

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.
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http://dx.doi.org/10.1002/cbic.202000176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586950PMC
October 2020

Artificial Splitting of a Non-Ribosomal Peptide Synthetase by Inserting Natural Docking Domains.

Angew Chem Int Ed Engl 2020 08 27;59(32):13463-13467. Epub 2020 May 27.

Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt am Main, Germany.

The interaction in multisubunit non-ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit-to-subunit interaction. We introduced natural docking domains into the three-module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC-ESI-MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS-PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split-module cases compared to the full-length wild-type enzyme.
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http://dx.doi.org/10.1002/anie.201915989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496407PMC
August 2020

A New Docking Domain Type in the Peptide-Antimicrobial-Xenorhabdus Peptide Producing Nonribosomal Peptide Synthetase from .

ACS Chem Biol 2020 04 25;15(4):982-989. Epub 2020 Mar 25.

Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of different natural products from amino acid precursors. In contrast to single protein NRPS, the NRPS of the bacterium producing the peptide-antimicrobial-Xenorhabdus (PAX) peptide consists of three individual proteins (PaxA/B/C), which interact with each other noncovalently in a linear fashion. The specific interactions between the three different proteins in this NRPS system are mediated by short C- and N-terminal docking domains (DDs). Here, we investigate the structural basis for the specific interaction between the DD from the protein PaxB and the DD from PaxC. The isolated DD peptides feature transient α-helical conformations in the absence of the respective DD partner. Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) titration experiments showed that the two isolated DDs bind to each other and form a structurally well-defined complex with a dissociation constant in the micromolar range as is typical for many DD interactions. Artificial linking of this DD pair via a flexible glycine-serine (GS) linker enabled us to solve the structure of the DD complex by NMR spectroscopy. In the complex, the two DDs interact with each other by forming a three helix bundle arranged in an overall coiled-coil motif. Key interacting residues were identified in mutagenesis experiments. Overall, our structure of the PaxB DD/PaxC DD complex represents an architecturally new type of DD interaction motif.
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http://dx.doi.org/10.1021/acschembio.9b01022DOI Listing
April 2020

Engineering bacterial symbionts of nematodes improves their biocontrol potential to counter the western corn rootworm.

Nat Biotechnol 2020 05 17;38(5):600-608. Epub 2020 Feb 17.

Institute of Plant Sciences, University of Bern, Bern, Switzerland.

The western corn rootworm (WCR) decimates maize crops worldwide. One potential way to control this pest is treatment with entomopathogenic nematodes (EPNs) that harbor bacterial symbionts that are pathogenic to insects. However, WCR larvae sequester benzoxazinoid secondary metabolites that are produced by maize and use them to increase their resistance to the nematodes and their symbionts. Here we report that experimental evolution and selection for bacterial symbionts that are resistant to benzoxazinoids improve the ability of a nematode-symbiont pair to kill WCR larvae. We isolated five Photorhabdus symbionts from different nematodes and increased their benzoxazinoid resistance through experimental evolution. Benzoxazinoid resistance evolved through multiple mechanisms, including a mutation in the aquaporin-like channel gene aqpZ. We reintroduced benzoxazinoid-resistant Photorhabdus strains into their original EPN hosts and identified one nematode-symbiont pair that was able to kill benzoxazinoid-sequestering WCR larvae more efficiently. Our results suggest that modification of bacterial symbionts might provide a generalizable strategy to improve biocontrol of agricultural pests.
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http://dx.doi.org/10.1038/s41587-020-0419-1DOI Listing
May 2020

Phenylethylamides derived from bacterial secondary metabolites specifically inhibit an insect serotonin receptor.

Sci Rep 2019 12 30;9(1):20358. Epub 2019 Dec 30.

Department of Plant Medicals, College of Life Sciences, Andong National University, Andong, 36729, Korea.

Serotonin (5-hydroxytryptamine: 5-HT) is a biogenic monoamine that mediates immune responses and modulates nerve signal in insects. Se-5HTR, a specific receptor of serotonin, has been identified in the beet armyworm, Spodoptera exigua. It is classified into subtype 7 among known 5HTRs. Se-5HTR was expressed in all developmental stages of S. exigua. It was expressed in all tested tissues of larval stage. Its expression was up-regulated in hemocytes and fat body in response to immune challenge. RNA interference (RNAi) of Se-5HTR exhibited significant immunosuppression by preventing cellular immune responses such as phagocytosis and nodulation. Treatment with an inhibitor (SB-269970) specific to 5HTR subtype 7 resulted in significant immunosuppression. Furthermore, knockout mutant of Se-5HTR by CRISPR-Cas9 led to significant reduction of phagocytotic activity of S. exigua hemocytes. Such immunosuppression was also induced by bacterial secondary metabolites derived from Xenorhabdus and Photorhabdus. To determine specific bacterial metabolites inhibiting Se-5HTR, this study screened 37 bacterial secondary metabolites with respect to cellular immune responses associated with Se-5HTR and selected 10 potent inhibitors. These 10 selected compounds competitively inhibited cellular immune responses against 5-HT and shared phenylethylamide (PEA) chemical skeleton. Subsequently, 46 PEA derivatives were screened and resulting potent chemicals were used to design a compound to be highly inhibitory against Se-5HTR. The designed compound was chemically synthesized. It showed high immunosuppressive activities along with specific and competitive inhibition activity for Se-5HTR. This study reports the first 5HT receptor from S. exigua and provides its specific inhibitor designed from bacterial metabolites and their derivatives.
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http://dx.doi.org/10.1038/s41598-019-56892-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935581PMC
December 2019
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