Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens.

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 PlAA10 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 g-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 g-tensors, giving confidence in our evaluation of PlAA10 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 PlAA10. 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 PlAA10 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.

A Whole-Cell Bacterial Biosensor for Blood Markers Detection in Urine.

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases (e.g., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter (luxCDABE) from Photorhabdus luminescens. To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts (luxCDE and luxAB). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 104 to 5 × 105 RBC per mL of human urine.

Identification and Characterization of an Efficient Phenylalanine Ammonia-Lyase from Photorhabdus luminescens.

A putative aromatic amino acid ammonia-lyase gene (named Pl-pal) was discovered in Photorhabdus luminescens DSM 3368. BLAST and phylogenetic analyses predicted that this enzyme is a histidine ammonia-lyase, whereas sequence alignment suggested that it is more likely a phenylalanine ammonia-lyase (PAL). This gene was amplified from P. luminescens and expressed in Escherichia coli BL21(DE3). The function of Pl-PAL (58 kDa) was characterized by in vitro enzymatic reactions with L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), L-histidine (L-His), and L-tryptophan (L-Trp). Pl-PAL can convert L-Phe and L-Tyr to trans-cinnamic acid and p-coumaric acid, respectively, but had no function on L-His and L-Trp. The optimum temperature and pH were determined to be 40 °C and 11.0, respectively. Under the optimal conditions, Pl-PAL had a kcat/Km value of 0.52 s-1 mM-1 with L-Phe as the substrate, while only 0.013 s-1 mM-1 for L-Tyr. Therefore, the primary function of Pl-PAL was determined to be PAL. The Pl-pal-harboring E. coli strain was used as a whole-cell biocatalyst to produce trans-cinnamic acid from L-Phe. The overall molar conversion rate and productivity were 65.98% and 228.10 mg L-1 h-1, respectively, after the cells were repeatedly utilized 7 times. This work thus provides a promising strain for industrial production of trans-cinnamic acid.

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

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.

Synthesis of acridone derivatives via heterologous expression of a plant type III polyketide synthase in Escherichia coli.

BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.

Characterization of a bacterial copper-dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes involved in the degradation of recalcitrant polysaccharides such as cellulose or chitin. LPMOs act in synergy with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving a number of glycosidic bonds at the surface of their crystalline substrate(s). Besides their role in biomass degradation, some bacterial LPMOs have been found to be virulence factors in some human and insect pathogens. Photorhabdus luminescens is a nematode symbiont bacterium that is pathogenic to a wide range of insects. A single gene encoding a LPMO is found in its genome. In this work, we report the characterization of this LPMO, referred to as PlAA10. Surprisingly, PlAA10 lacks the conserved alanine residue (substituted by an isoleucine) found in the second coordination sphere of the copper-active site in bacterial LPMOs. PlAA10 was found to be catalytically active on both α- and ß-chitin, and exhibits a C1-oxidation regiospecificity, similarly to other chitin-active LPMOs. The 1.6 Å X-ray crystal structure confirmed that PlAA10 adopts the canonical immunoglobulin-like fold typical for LPMOs. The geometry of the copper-active site is not affected by the nearby isoleucine, as also supported by electron paramagnetic resonance. Nevertheless, the bulkier side chain of isoleucine protrudes from the substrate-binding surface. A bioinformatic study on putative bacterial LPMOs unveiled that they exhibit some variability at the conserved active-site alanine position with a substitution in about 15% of all sequences analyzed. DATABASE: Structural data (atomic coordinates and structure factors) reported for PlAA10 are available in the Protein Data Bank under accession number 6T5Z. ENZYMES: PlAA10, EC1.14.99.53.

Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts.

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex.

Production of a photohexapeptide library from entomopathogenic Photorhabdus asymbiotica PB68.1.

A new natural product compound library, photohexapeptide library, was identified from entomopathogenic Photorhabdus asymbiotica PB68.1 after the NRPS-encoding gene phpS was activated via promoter exchange. Peptide structures, including the absolute configurations of amino acids, were determined by using a combination of bioinformatics analysis and isotopic labelling experiments followed by detailed HPLC-MS analysis. Additionally, their structures were confirmed by chemical synthesis and NMR after preparative isolation. The chemical diversity of the photohexapeptides results from promiscuous adenylation domain specificity being an excellent example of how to create libraries in nature.

Synthesis of Three Bioactive Aromatic Compounds by Introducing Polyketide Synthase Genes into Engineered Escherichia coli.

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.

Modification and de novo design of non-ribosomal peptide synthetases using specific assembly points within condensation domains.

Non-ribosomal peptide synthetases (NRPSs) are giant enzyme machines that activate amino acids in an assembly line fashion. As NRPSs are not restricted to the incorporation of the 20 proteinogenic amino acids, their efficient manipulation would enable microbial production of a diverse range of peptides; however, the structural requirements for reprogramming NRPSs to facilitate the production of new peptides are not clear. Here we describe a new fusion point inside the condensation domains of NRPSs that results in the development of the exchange unit condensation domain (XUC) concept, which enables the efficient production of peptides, even containing non-natural amino acids, in yields up to 280 mg l-1. This allows the generation of more specific NRPSs, reducing the number of unwanted peptide derivatives, but also the generation of peptide libraries. The XUC might therefore be suitable for the future optimization of peptide production and the identification of bioactive peptide derivatives for pharmaceutical and other applications.

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells.

Photorhabdus luminescens Tc toxins consist of the cell-binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP-ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP-ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP-ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP-ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.

Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging.

Bioluminescence imaging of single cells is often complicated by the requirement of exogenous luciferins that can be poorly cell-permeable or produce high background signal. Bacterial bioluminescence is unique in that it uses reduced flavin mononucleotide as a luciferin, which is abundant in all cells, making this system purely genetically encodable by the lux operon. Unfortunately, the use of bacterial bioluminescence has been limited by its low brightness compared with other luciferases. Here, we report the generation of an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in Escherichia coli; ilux can be used to image single E. coli cells with enhanced spatiotemporal resolution over several days. In addition, since only metabolically active cells produce bioluminescent signal, we show that ilux can be used to observe the effect of different antibiotics on cell viability on the single-cell level.

Functional Characterization of a Condensation Domain That Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of biologically important small molecules. NRPSs can interface with other enzymes to form hybrid biosynthetic systems that expand the structural and functional diversity of their products. The pepteridines are metabolites encoded by an unprecedented pteridine-NRPS-type hybrid biosynthetic gene cluster in Photorhabdus luminescens, but how the distinct enzymatic systems interface to produce these molecules has not been examined at the biochemical level. By an unknown mechanism, the genetic locus can also affect the regulation of other enzymes involved in autoinducer and secondary metabolite biosynthesis. Here, through in vitro protein biochemical assays, we demonstrate that an atypical NRPS condensation (C) domain present in the pathway condenses acyl units derived from α-keto acids onto a free 5,6,7,8-tetrahydropterin core, producing the tertiary cis-amide-containing pepteridines. Solution studies of the chemically synthesized molecules led to the same amide regiochemistries that were observed in the natural products. The biochemical transformations mediated by the C domain destroy the radical scavenging activity of its redox active tetrahydropterin substrate. Secondary metabolite analyses revealed that the pepteridine locus affects select metabolic pathways associated with quorum sensing, antibiosis, and symbiosis. Taken together, the results suggest that the pathway likely regulates cellular redox and specialized metabolic pathways through engagement with the citric acid cycle.

Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo.

Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed ccdB counterselection combined with oligonucleotide-mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from Photorabdus luminescens directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene plu3263 in E. coli led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site-directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi-copy plasmids.

The actin and Rho-modifying toxins PTC3 and PTC5 of Photorhabdus luminescens: enzyme characterization and induction of MAL/SRF-dependent transcription.

PTC3 and PTC5 are tripartite Tc (toxin complex) toxins from Photorhabdus luminescens, which consist of the binding component TcdA1, the linker component TcdB2 and the enzyme components TccC3 and TccC5 respectively. While PTC5 adenosine diphosphate (ADP)-ribosylates Rho proteins at Gln61/63 resulting in constitutive activation of the GTPases, PTC3 ADP-ribosylates actin at Thr148 thereby inducing actin polymerization. Here, we identified amino acids involved in ADP-ribosyltransferase activity of TccC3 and TccC5 and analysed the substrate specificity of Rho-activating TccC5. We compared the time dependency of Rho protein activation by PTC5 in HeLa cells with the effects of Escherichia coli cytotoxic necrotizing factor 1, which activates Rho GTPases by deamidation of Gln61/63. Using a luciferase reporter assay, we show that PTC5 and PTC3 stimulated gene transcription via myocardin-related transcription factor A (also called MAL) and AP1. MAL activation by PTC5 involved Rho kinase and formins. Activation of AP1 by PTC5 occurred via two MAP kinase pathways involving extracellular signal-regulated kinase and Jun kinase respectively.

Photorhabdus luminescens toxins TccC3 and TccC5: insecticidal ADP-ribosyltransferases that modify threonine and glutamine.

The ADP-ribosyltransferases TccC3 and TccC5 are the biologically active TcC components of the tripartite Photorhabdus luminescens Tc toxin, which consist of TcA, TcB, and TcC components. TcA is the binding and membrane translocation component. TcB is a functional linker between TcC and TcA and also involved in the translocation of the toxin. While TccC3 ADP-ribosylates actin at threonine 148, TccC5 modifies Rho proteins at glutamine 61/63. Both modifications result in major alteration of the actin cytoskeleton. Here we discuss structure and function of the Tc toxin and compare its ADP-ribosyltransferase activities with other types of actin and Rho modifying toxins.

An atypical orphan carbohydrate-NRPS genomic island encodes a novel lytic transglycosylase.

Microbial genome sequencing platforms have produced a deluge of orphan biosynthetic pathways suspected of biosynthesizing new small molecules with pharmacological relevance. Genome synteny analysis provides an assessment of genomic island content, which is enriched in natural product gene clusters. Here we identified an atypical orphan carbohydrate-nonribosomal peptide synthetase genomic island in Photorhabdus luminescens using genome synteny analysis. Heterologous expression of the pathway led to the characterization of five oligosaccharide metabolites with lysozyme inhibitory activities. The oligosaccharides harbor a 1,6-anhydro-ß-D-N-acetyl-glucosamine moiety, a rare structural feature for natural products. Gene deletion analysis and biochemical reconstruction of oligosaccharide production led to the discovery that a hypothetical protein in the pathway is a lytic transglycosylase responsible for bicyclic sugar formation. The example presented here supports the notion that targeting select genomic islands with reduced reliance on known protein homologies could enhance the discovery of new metabolic chemistry and biology.

Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange.

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.

Improved detection of antibiotic compounds by bacterial reporter strains achieved by manipulations of membrane permeability and efflux capacity.

The occurrence of pharmaceuticals, including antibacterial compounds, in the environment has been acknowledged as an emerging and troubling issue in environmental safety; their usage is constantly on the rise, and their effects on the environment are only partially understood. Such compounds can accumulate, contaminate the ecosystem, and contribute to the spreading of antibiotic resistance among bacteria, hindering human health. Bioluminescent Escherichia coli reporter strains, engineered to detect antibiotic compounds by fusing the promoter of the global regulator soxS to the Photorhabdus luminescens luxCDABE cassette, were further modified by altering their membrane permeability and efflux capabilities. This was accomplished by introducing several mutations in the efflux system (ΔemrE, ΔacrB, and ΔtolC) and by overexpressing OmpF, a porin located in the outer membrane that allows passive diffusion of molecules. Combinations of these alterations had a cumulative effect in lowering the detection threshold of several antibiotics, in some of the cases to concentrations reported from pharmaceutical-polluted environments.

The chaperone Hsp90 and PPIases of the cyclophilin and FKBP families facilitate membrane translocation of Photorhabdus luminescens†ADP-ribosyltransferases.

TccC3 and TccC5 from Photorhabdus luminescens are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively, thereby inducing polymerization and clustering of actin. The bacterial proteins are components of the Photorhabdus toxin complexes, consisting of the binding and translocation component TcdA1, a proposed linker component TcdB2 and the enzymatic component TccC3/5. While the action of the toxins on target proteins is clearly defined, uptake and translocation of the toxins into the cytosol of target cells are not well understood. Here we show by using pharmacological inhibitors that heat shock protein 90 (Hsp90) and peptidyl prolyl cis/trans isomerases (PPIases) including cyclophilins and FK506-binding proteins (FKBPs) facilitate the uptake of the ADP-ribosylating toxins into the host cell cytosol. Inhibition of Hsp90 and/or PPIases resulted in decreased intoxication of target cells by Photorhabdus toxin complexes determined by cell rounding and reduction of transepithelial electrical resistance of cell monolayers. ADP-ribosyltransferase activity of toxins and toxin-induced pore formation were notimpaired by the inhibitors of Hsp90 and PPIases. The Photorhabdus toxins interacted with Hsp90, FKBP51, Cyp40 and CypA, suggesting a role of these host cell factors in translocation and/or refolding of the ADP-ribosyltransferases.