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The Journal of Biological Chemistry Apr 2017Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family...
Members of the gammaproteobacterial genus share mutualistic relationships with nematodes, and the pairs infect a wide swath of insect larvae. species produce a family of stilbenes, with two major components being 3,5-dihydroxy-4-isopropyl--stilbene (compound 1) and its stilbene epoxide (compound 2). This family of molecules harbors antimicrobial and immunosuppressive activities, and its pathway is responsible for producing a nematode "food signal" involved in nematode development. However, stilbene epoxidation biosynthesis and its biological roles remain unknown. Here, we identified an orphan protein (Plu2236) from that catalyzes stilbene epoxidation. Structural, mutational, and biochemical analyses confirmed the enzyme adopts a fold common to FAD-dependent monooxygenases, contains a tightly bound FAD prosthetic group, and is required for the stereoselective epoxidation of compounds 1 and 2. The epoxidase gene was dispensable in a nematode-infective juvenile recovery assay, indicating the oxidized compound is not required for the food signal. The epoxide exhibited reduced cytotoxicity toward its producer, suggesting this may be a natural route for intracellular detoxification. In an insect infection model, we also observed two stilbene-derived metabolites that were dependent on the epoxidase. NMR, computational, and chemical degradation studies established their structures as new stilbene-l-proline conjugates, prolbenes A (compound 3) and B (compound 4). The prolbenes lacked immunosuppressive and antimicrobial activities compared with their stilbene substrates, suggesting a metabolite attenuation mechanism in the animal model. Collectively, our studies provide a structural view for stereoselective stilbene epoxidation and functionalization in an invertebrate animal infection model and provide new insights into stilbene cellular detoxification.
Topics: Animals; Anti-Infective Agents; Biological Products; Catalysis; Chromatography, High Pressure Liquid; Crystallography, X-Ray; DNA Mutational Analysis; Epoxy Compounds; Gene Deletion; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Immunosuppressive Agents; Magnetic Resonance Spectroscopy; Molecular Conformation; Mutation; Photorhabdus; Protein Folding; Rhabditoidea; Stereoisomerism; Stilbenes; Symbiosis
PubMed: 28246174
DOI: 10.1074/jbc.M116.762542 -
Microbiology Spectrum Feb 2022Entomopathogenic bacteria (Enterobacteriaceae: Gamma-proteobacteria), the natural symbionts of nematodes, are a rich source for the discovery of biologically active...
Selective Toxicity of Secondary Metabolites from the Entomopathogenic Bacterium Photorhabdus luminescens against Selected Plant Parasitic Nematodes of the Tylenchina Suborder.
Entomopathogenic bacteria (Enterobacteriaceae: Gamma-proteobacteria), the natural symbionts of nematodes, are a rich source for the discovery of biologically active secondary metabolites (SMs). This study describes the isolation of three nematicidal SMs from culture supernatants of the Arizona-native Photorhabdus luminescens strain Caborca by bioactivity-guided fractionation. Nuclear magnetic resonance spectroscopy and comparison to authentic synthetic standards identified these bioactive metabolites as -cinnamic acid (-CA), (4)-5-phenylpent-4-enoic acid (PPA), and indole. PPA and CA displayed potent, concentration-dependent nematicidal activities against the root-knot nematode (Meloidogyne incognita) and the citrus nematode (), two economically and globally important plant parasitic nematodes (PPNs) that are ubiquitous in the United States. Southwest. Indole showed potent, concentration-dependent nematistatic activity by inducing the temporary rigid paralysis of the same targeted nematodes. While paralysis was persistent in the presence of indole, the nematodes recovered upon removal of the compound. All three SMs were found to be selective against the tested PPNs, exerting little effects on non-target species such as the bacteria-feeding nematode Caenorhabditis elegans or the entomopathogenic nematodes Steinernema carpocapsae, Heterorhabditis bacteriophora, and . Moreover, none of these SMs showed cytotoxicity against normal or neoplastic human cells. The combination of CA + PPA + indole had a synergistic nematicidal effect on both targeted PPNs. Two-component mixtures prepared from these SMs revealed complex, compound-, and nematode species-dependent interactions. These results justify further investigations into the chemical ecology of SMs, and recommend CA, PPA and indole, alone or in combinations, as lead compounds for the development of selective and environmentally benign nematicides against the tested PPNs. Two phenylpropanoid and one alkaloid secondary metabolites were isolated and identified from culture filtrates of strain Caborca. The three identified metabolites showed selective nematicidal and/or nematistatic activities against two important plant parasitic nematodes, the root-knot nematode (Meloidogyne incognita) and the citrus nematode (). The mixture of all three metabolites had a synergistic nematicidal effect on both targeted nematodes, while other combinations showed compound- and nematode-dependent interactions.
Topics: Animals; Anthelmintics; Cinnamates; Indoles; Molecular Structure; Photorhabdus; Plant Diseases; Secondary Metabolism; Tylenchoidea
PubMed: 35138171
DOI: 10.1128/spectrum.02577-21 -
International Journal of Systematic and... Aug 2010We used the information from a set of concatenated sequences from four genes (recA, gyrB, dnaN and gltX) to investigate the phylogeny of the genera Photorhabdus and...
Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. Proposal of new taxa: X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov.,...
We used the information from a set of concatenated sequences from four genes (recA, gyrB, dnaN and gltX) to investigate the phylogeny of the genera Photorhabdus and Xenorhabdus (entomopathogenic bacteria associated with nematodes of the genera Heterorhabditis and Steinernema, respectively). The robustness of the phylogenetic tree obtained by this multigene approach was significantly better than that of the tree obtained by a single gene approach. The comparison of the topologies of single gene phylogenetic trees highlighted discrepancies which have implications for the classification of strains and new isolates; in particular, we propose the transfer of Photorhabdus luminescens subsp. thracensis to Photorhabdus temperata subsp. thracensis comb. nov. (type strain CIP 108426T =DSM 15199T). We found that, within the genus Xenorhabdus, strains or isolates that shared less than 97 % nucleotide identity (NI), calculated on the concatenated sequences of the four gene fragments (recA, gyrB, dnaN and gltX) encompassing 3395 nucleotides, did not belong to the same species. Thus, at the 97% NI cutoff, we confirm the current 20 species of the genus Xenorhabdus and propose the description of a novel species, Xenorhabdus vietnamensis sp. nov. (type strain VN01T =CIP 109945T =DSM 22392T). Within each of the three current species of the genus Photorhabdus, P. asymbiotica, P. luminescens and P. temperata, strains or isolates which shared less than 97% NI did not belong to the same subspecies. Comparisons of the four gene fragments plus the rplB gene fragment analysed separately led us to propose four novel subspecies: Photorhabdus luminescens subsp. caribbeanensis subsp. nov. (type strain HG29T =CIP 109949T =DSM 22391T), P. luminescens subsp. hainanensis subsp. nov. (type strain C8404T = CIP 109946T =DSM 22397T), P. temperata subsp. khanii subsp. nov. (type strain C1T =NC19(T) =CIP 109947T =DSM 3369T), and P. temperata subsp. tasmaniensis subsp. nov. (type strain T327T =CIP 109948T =DSM 22387T).
Topics: Animals; Bacterial Proteins; DNA, Bacterial; DNA, Ribosomal; Molecular Sequence Data; Nematoda; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; Xenorhabdus
PubMed: 19783607
DOI: 10.1099/ijs.0.014308-0 -
Inorganic Chemistry May 2022Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable...
Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active AA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu -tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of -tensors, giving confidence in our evaluation of AA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of AA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in AA10 LPMO as a foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.
Topics: Copper; Electron Spin Resonance Spectroscopy; Mixed Function Oxygenases; Photorhabdus; Polysaccharides
PubMed: 35549254
DOI: 10.1021/acs.inorgchem.2c00766 -
Biochemistry Feb 2019Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the...
Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.
Topics: Amino Acids; Biosynthetic Pathways; Carbohydrates; Glycopeptides; Multigene Family; Peptide Fragments; Peptide Synthases; Photorhabdus
PubMed: 30694662
DOI: 10.1021/acs.biochem.8b01120 -
BMC Microbiology Feb 2021The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet...
BACKGROUND
The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria's life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling.
RESULTS
Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1.
CONCLUSION
Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.
Topics: Animals; Bacterial Proteins; Gene Expression Regulation; Insecta; Nematoda; Phenotype; Photorhabdus; Symbiosis; Transcription Factors
PubMed: 33627070
DOI: 10.1186/s12866-021-02116-2 -
Systematic and Applied Microbiology Feb 2004Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to...
Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov.
Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.
Topics: Animals; Base Sequence; Cluster Analysis; DNA, Bacterial; Molecular Sequence Data; Nematoda; Photorhabdus; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Ribotyping; Sequence Alignment; Sequence Analysis, DNA; Soil Microbiology; Turkey
PubMed: 15053319
DOI: 10.1078/0723-2020-00255 -
Developmental and Comparative Immunology May 2018Photorhabdus bacteria enter into a mutualistic symbiosis with Heterorhabditis nematodes to infect insect larvae. However, they rapidly kill the model nematode...
Photorhabdus bacteria enter into a mutualistic symbiosis with Heterorhabditis nematodes to infect insect larvae. However, they rapidly kill the model nematode Caenorhabditis elegans. One hypothesis for these divergent outcomes is that the nematode defense responses differ. To begin testing this hypothesis, we have systematically analyzed available data on the transcriptional response of C. elegans to P. luminescens strain Hb. From a starting pool of over 7000 differentially expressed genes, we carefully chose 21 Heterorhabditis-conserved genes to develop as comparative markers. Using newly designed and validated qRT-PCR primers, we measured expression of these genes in C. elegans exposed to the sequenced TT01 strain of P. luminescens, on two different media types. Almost all (18/21) of the genes showed a significant response to P. luminescens strain TT01. One response is dependent on media type, and a subset of genes may respond differentially to distinct strains. Overall, we have established useful resources and generated new hypotheses regarding how C. elegans responds to P. luminescens infection.
Topics: Animals; Biodiversity; Caenorhabditis elegans; Enterobacteriaceae; Gene Expression Regulation, Developmental; Infections; Insecta; Larva; Photorhabdus; Species Specificity; Symbiosis; Transcriptome
PubMed: 29203330
DOI: 10.1016/j.dci.2017.09.005 -
Toxicon : Official Journal of the... Feb 2018The entomopathogenic Photorhabdus luminescens TT01 is a promoting bacterium that controls effectively many insect pests. Indeed, it exhibited a mortality rate of 32.36%...
The entomopathogenic Photorhabdus luminescens TT01 is a promoting bacterium that controls effectively many insect pests. Indeed, it exhibited a mortality rate of 32.36% against the first instar larvae of the turnip moth Agrotis segetum, when it was used at a concentration of 5 × 10 cells/ml but no toxicity against the second instar larvae in the same condition. P. luminescens TT01 oral toxicity is associated to septicaemia since cells fraction exhibited the highest mortality rate of 34%. In order to enhance P. luminescens TT01 insecticidal potential, combination with Bacillus thuringiensis Vip3Aa16 toxin was tested. An improvement of insecticidal activity was shown. Indeed, 100% mortality of A. segetum first instar larvae was obtained after 2 days of treatment, when using TT01 cells and Vip3Aa16 toxin at a concentration of 5 × 10 cells/ml and 9.025 ng/cm, respectively. Moreover, growth inhibition rate of 45% of the second instar larvae was observed, when using the same combination. A. segetum mortality could be the result of several alterations in the midgut epithelium caused by Vip3Aa16 toxin, allowing a rapid invasion of the hemocoel by TT01 cells as demonstrated by histopathological study. Clear symptoms of intoxication were observed for all combinations tested, including swelling, vesicle formation, cytoplasm vacuolization and brush border membrane lysis. Taken together, these results promote the use of P. luminescens TT01 as a potent bioinsecticide to control effectively A. segetum by oral treatment in a mixture with Vip3Aa16 toxin.
Topics: Animals; Bacterial Proteins; Insecticides; Larva; Moths; Photorhabdus
PubMed: 29305079
DOI: 10.1016/j.toxicon.2017.12.054 -
Indian Journal of Microbiology Mar 2017is an insect-pathogenic Gram negative enterobacterium found in the gut of nematodes. is highly virulent to insects, and can kill insects rapidly upon injection at...
is an insect-pathogenic Gram negative enterobacterium found in the gut of nematodes. is highly virulent to insects, and can kill insects rapidly upon injection at very low concentrations of one to few cells. We characterized the virulence of symbionts isolated from the nematodes collected from various parts of India by injecting different concentrations of bacterial cells into fourth instar larval stage of insect . ssp strain IARI-SGMG3 from Meghalaya was identified as the most virulent of all the tested strains on the basis of LT and LC values. This study forms a basis for further investigations on the genetic basis of virulence in bacteria.
PubMed: 28148990
DOI: 10.1007/s12088-016-0628-y