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1.
Biomol NMR Assign ; 15(1): 229-234, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33675014

RESUMO

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.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33693901

RESUMO

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.

3.
ACS Chem Biol ; 16(3): 447-451, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33596038

RESUMO

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 Xenorhabdus, PAX (peptide-antimicrobial-Xenorhabdus) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in Xenorhabdus doucetiae 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 X. doucetiae and E. coli as well as inducible PAX production in X. doucetiae 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.

5.
Artigo em Inglês | MEDLINE | ID: mdl-33464198

RESUMO

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis 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 Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).


Assuntos
Nematoides/microbiologia , Photorhabdus/classificação , Filogenia , Animais , Austrália , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Sistema Digestório/microbiologia , Egito , Hibridização de Ácido Nucleico , Photorhabdus/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Tailândia
6.
Chembiochem ; 2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33452852

RESUMO

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.

7.
Angew Chem Int Ed Engl ; 60(16): 8781-8785, 2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33460275

RESUMO

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.

8.
Artigo em Inglês | MEDLINE | ID: mdl-33218994

RESUMO

Ustilago maydis 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 U. maydis 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 (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B synthase (vbs1). Metabolic profiles of deletion mutants in this gene cluster suggested that Pks3 and Pks4 act in concert as heterodimer 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 U. maydis 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 an insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCE The fungus Ustilago maydis represents one of the major threats for 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 U. maydis to protect itself against harsh environments, 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 multimodular enzymes and their evolution in the fungal kingdom.

10.
Nat Microbiol ; 5(12): 1481-1489, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33139881

RESUMO

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, 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.

11.
Microb Ecol ; 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32827089

RESUMO

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.

12.
ACS Synth Biol ; 9(9): 2418-2426, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32818377

RESUMO

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 vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro 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.

13.
Nat Chem ; 12(8): 755-763, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32632186

RESUMO

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/ß 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.

14.
Nat Microbiol ; 5(8): 987-994, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514073

RESUMO

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.


Assuntos
Archaea/classificação , Bactérias/classificação , Archaea/genética , Bactérias/genética , DNA Bacteriano , Metagenoma , Filogenia , Células Procarióticas/classificação , Análise de Sequência de DNA , Terminologia como Assunto
16.
Chembiochem ; 21(19): 2750-2754, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32378773

RESUMO

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.

17.
Beilstein J Org Chem ; 16: 956-965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32461774

RESUMO

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 Xenorhabdus or Photorhabdus. 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 Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine (fcl) 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-MS2 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.

18.
Angew Chem Int Ed Engl ; 59(32): 13463-13467, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32329545

RESUMO

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.

19.
ACS Chem Biol ; 15(4): 982-989, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32167274

RESUMO

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 Xenorhabdus bovienii 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 (C/NDDs). Here, we investigate the structural basis for the specific interaction between the CDD from the protein PaxB and the NDD 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 CDD/PaxC NDD complex represents an architecturally new type of DD interaction motif.

20.
Nat Biotechnol ; 38(5): 600-608, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32066956

RESUMO

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.


Assuntos
Aquaporinas/genética , Benzoxazinas/farmacologia , Farmacorresistência Bacteriana , Nematoides/microbiologia , Photorhabdus/fisiologia , Zea mays/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Evolução Molecular , Engenharia Genética , Mutação , Nematoides/patogenicidade , Controle Biológico de Vetores , Photorhabdus/efeitos dos fármacos , Photorhabdus/genética , Doenças das Plantas/prevenção & controle , Zea mays/parasitologia
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