Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling.

BACKGROUND: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. RESULTS: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. CONCLUSIONS: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

Enhancement of an entomopathogenic fungal virulence against the seedcorn maggot, Delia platura, by suppressing immune responses with a bacterial culture broth of Photorhabdus temperata subsp. temperata.

In Korea, there are two maggot species in the Delia genus that commonly infest the roots and stems of the Welsh onion, thus causing serious economic damage on the crop at the seedling stage. In this study, the seedcorn maggot (Delia platura) was detected in onion fields in two different localities in Korea. After overwintering, maggot infestations occurred throughout the entire growing seasons from transplantation to harvest, but their specific patterns of occurrence varied in the two localities examined. Entomopathogenic fungi induced significant virulence against the maggot larvae, in which a strain of Beauveria bassiana was effective, though it exhibited limited mortality in its insecticidal activity. To enhance this insecticidal activity, a culture broth from an entomopathogenic bacterium, Photorhabdus temperata temperata (Ptt), was added to B. bassiana treatment. The addition of Ptt broth significantly increased the insecticidal activity of B. bassiana in a dose-dependent manner. To elucidate this enhancement in insecticidal activity, the immunosuppressive activity of Ptt broth was assessed by identifying the immune responses of the seedcorn maggots. The seedcorn maggots possessed at least three different hemocytes with plasmatocytes, crystal cells, and lamellocytes. These hemocytes exhibited nodule formation in response to the fungal infection. In addition to the cellular immunity, the maggots exhibited inducible expressions of antimicrobial peptide (AMP) genes such as cecropin and defensin. The addition of Ptt broth suppressed the nodule formation and the AMP expressions in response to the fungal infection. Altogether, this study demonstrated the innate immune responses in a non-model insect, D. platura, along with the application of immunosuppression to develop a highly efficient biological control by enhancing the virulence of B. bassiana.

Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach.

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Taxonomic and molecular characterization of a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and whole genome sequencing of its associated bacterial symbiont.

BACKGROUND: Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis. METHODS: Six Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria. RESULTS: The newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77-100 vs. 121-130 µm), V% [(anterior end of vulva/body length) × 100] (46-57 vs. 41-47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13-2.0 vs. 0.32-0.39 mm), body diameter (73-150 vs. 160-220 µm), anterior end to the excretory pore distance (135-157 vs. 174-214 µm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with Photorhabdus luminescence subsp. clarkei symbiotic bacteria. CONCLUSIONS: The discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India.

Crystal structures of PirA and PirB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

PirAB binary toxin from Photorhabdus is toxic to the larvae of dipteran and lepidopteran insect pests. However, the 3-D structures and their toxicity mechanism are not yet fully understood. Here we report the crystal structures of PirA and PirB proteins from Photorhabdus akhurstii subsp. akhurstii K-1 at 1.6 and 2.1 Å, respectively. PirA comprises of eight ß-strands depicting jelly-roll topology while PirB folds into two distinct domains, an N-terminal domain (PirB-N) made up of seven α-helices and a C-terminal domain (PirB-C) consists of ten ß-strands. Despite the low sequence identity, PirA adopts similar architecture as domain III and PirB shared similar architecture as domain I/II of the Cry δ-endotoxin of Bacillus thuringiensis, respectively. However, PirA shows significant structural variations as compared to domain III of lepidopteran and dipteran specific Cry toxins (Cry1Aa and Cry11Ba) suggesting its role in virulence among range of insect pests and hence, in receptor binding. High structural resemblance between PirB-N and domain I of Cry toxin raises the possibility that the putative PirAB binary toxin may mimic the toxicity mechanism of the Cry protein, particularly its ability to perform pore formation. The mixture of independently purified PirA and PirB proteins are not toxic to insects. However, PirA-PirB protein complex purified from expression of pir operon with non-coding Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences found toxic to Galleria mellonella larvae with LD50 value of 1.62 µg/larva. This suggests that toxic conformation of PirA and PirB are achieved in-vivo with the help of ERIC sequences.

Production and Characterization of Photorin, a Novel Proteinaceous Protease Inhibitor from the Entomopathogenic Bacteria Photorhabdus laumondii.

Entomopathogenic bacteria of the genus Photorhabdus secrete protease S (PrtS), which is considered a virulence factor. We found that in the Photorhabdus genomes, immediately after the prtS genes, there are genes that encode small hypothetical proteins homologous to emfourin, a recently discovered protein inhibitor of metalloproteases. The gene of emfourin-like inhibitor from Photorhabdus laumondii subsp. laumondii TT01 was cloned and expressed in Escherichia coli cells. The recombinant protein, named photorin (Phin), was purified by metal-chelate affinity and gel permeation chromatography and characterized. It has been established that Phin is a monomer and inhibits activity of protealysin and thermolysin, which, similar to PrtS, belong to the M4 peptidase family. Inhibition constants were 1.0 ± 0.3 and 10 ± 2 µM, respectively. It was also demonstrated that Phin is able to suppress proteolytic activity of P. laumondii culture fluid (half-maximal inhibition concentration 3.9 ± 0.3 nM). Polyclonal antibodies to Phin were obtained, and it was shown by immunoblotting that P. laumondii cells produce Phin. Thus, the prtS genes in entomopathogenic bacteria of the genus Photorhabdus are colocalized with the genes of emfourin-like inhibitors, which probably regulate activity of the enzyme during infection. Strict regulation of the activity of proteolytic enzymes is essential for functioning of all living systems. At the same time, the principles of regulation of protease activity by protein inhibitors remain poorly understood. Bacterial protease-inhibitor pairs, such as the PrtS and Phin pair, are promising models for in vivo studies of these principles. Bacteria of the genus Photorhabdus have a complex life cycle with multiple hosts, being both nematode symbionts and powerful insect pathogens. This provides a unique opportunity to use the PrtS and Phin pair as a model for studying the principles of protease activity regulation by proteinaceous inhibitors in the context of bacterial interactions with different types of hosts.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Photorhabdus-Derived Secondary Metabolites Reduce Root Infection by Meloidogyne incognita in Cowpea.

Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from Photorhabdus bacteria, trans-cinnamic acid (t-CA), and (4E)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN Meloidogyne incognita in a cowpea. Factorial combinations of five t-CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 µg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the t-CA + PPA mixture significantly reduced the penetration of M. incognita infective juveniles (J2s) into the cowpea roots. The potential toxicity of t-CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of t-CA + PPA × nematode inoculation interactions and the t-CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll b content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of t-CA and PPA reduces M. incognita J2's ability to infect the roots without impairing plant growth or chlorophyll content.

A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

Photorhabdus aballayi sp. nov. and Photorhabdus luminescens subsp. venezuelensis subsp. nov., isolated from Heterorhabditis amazonensis entomopathogenic nematodes.

Six Gram-negative, rod-shaped bacterial strains isolated from Heterorhabditis amazonensis entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gyrB gene sequences indicate that they belong to the class Gammaproteobacteria, family Morganellaceae and genus Photorhabdus, and that some of them are conspecifics. Two of them, APURET and JART, were selected for further molecular characterization using whole genome- and whole-proteome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions using whole genome and whole proteome sequences show that strains APURET and JART are closely related to Photorhabdus luminescens subsp. luminescens ATCC 29999T and to P. luminescens subsp. mexicana MEX47-22T. Moreover, digital DNA-DNA hybridization (dDDH) values between APURET and P. luminescens subsp. luminescens ATCC 29999T, APURET and P. luminescens subsp. mexicana MEX47-22T, and APURET and JART are 61.6, 61.2 and 64.1 %, respectively. These values are below the 70 % divergence threshold that delimits prokaryotic species. dDDH scores between JART and P. luminescens subsp. luminescens ATCC 29999T and between JART and P. luminescens subsp. mexicana MEX47-22T are 71.9 and 74.8 %, respectively. These values are within the 70 and 79 % divergence thresholds that delimit prokaryotic subspecies. Based on these genomic divergence values, APURET and JART represent two different taxa, for which we propose the names: Photorhabdus aballayi sp. nov. with APURET (=CCM 9236T =CCOS 2019T) as type strain and Photorhabdus luminescens subsp. venezuelensis subsp. nov. with JART (=CCM 9235T =CCOS 2021T) as type strain. Our study contributes to a better understanding of the biodiversity of an important bacterial group with enormous biotechnological and agricultural potential.

Biochemical compositions and histopathology of the young and aged nymphs of the desert locust Schistocerca gregaria Forsskål (Orthoptera: Acrididae) affected by Photorhabdus luminescens (Enterobacterales: Morganellaceae).

Locusts occasionally represent a danger in Africa despite intensive management measures, leading to severe yield loss and a commensurate loss of food and money. The laboratory assessment of the toxicity of the Photorhabdus luminescens bacteria and its cell-free filtrate used Schistocerca gregaria nymphs in the second and fifth nymphs as test insects. Greater mortality was seen in locust nymphs of the second and fifth instars due to the high levels of toxicity produced by the bacterial suspension and its cell-free filtrate. The amounts of protein, fat, and carbohydrates in the treated locusts were drastically reduced. For the treated second and fifth instar nymphs of the desert locust, adverse effects on the muscular layers of the midgut and the muscles in the jumping legs were investigated.

The deterrent ability of Xenorhabdus nematophila and Photorhabdus laumondii compounds as a potential novel tool for Lobesia botrana (Lepidoptera: Tortricidae) management.

The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had âˆ¼ 55% and âˆ¼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in âˆ¼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.

A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments.

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.

Programmable protein delivery with a bacterial contractile injection system.

Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells1-3, raising the possibility that these systems could be harnessed for therapeutic protein delivery. However, whether eCISs can function in human cells remains unknown, and the mechanism by which these systems recognize target cells is poorly understood. Here we show that target selection by the Photorhabdus virulence cassette (PVC)-an eCIS from the entomopathogenic bacterium Photorhabdus asymbiotica-is mediated by specific recognition of a target receptor by a distal binding element of the PVC tail fibre. Furthermore, using in silico structure-guided engineering of the tail fibre, we show that PVCs can be reprogrammed to target organisms not natively targeted by these systems-including human cells and mice-with efficiencies approaching 100%. Finally, we show that PVCs can load diverse protein payloads, including Cas9, base editors and toxins, and can functionally deliver them into human cells. Our results demonstrate that PVCs are programmable protein delivery devices with possible applications in gene therapy, cancer therapy and biocontrol.

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

The role of Photorhabdus-induced bioluminescence and red cadaver coloration on the deterrence of insect scavengers from entomopathogenic nematode-infected cadavers.

Photorhabdus spp. and Xenorhabdus spp. bacteria produce a variety of molecules that inhibit bacterial and fungal contamination as well as deter scavenging invertebrates and some vertebrates in soil. Certain Heterorhabditis/Photorhabdus-infected insect cadavers can be bioluminescent in the dark and/or turn red from the production of anthraquinone pigments. The role of these traits remains unresolved. The aim of the present study was to evaluate the role of red color (anthraquinone) and bioluminescence on the deterrence of insect scavengers. Our data shows that scavenger deterrent factor (SDF) is not related to red cadaver coloration or bioluminescence activity as crickets and ants did not consume Galleria mellonella cadavers infected by P. laumondii strain 48-02 and X. bovienii. Both bacteria exhibit SDF activity but do not produce anthraquinone. Also, the insects were not affected by anthraquinone in agar plugs prepared with supernatant from induced P. laumondii Δpptase Pcep-KM-antA (SVS-275) mutant strain, which overproduces anthraquinone. Since bioluminescence and anthraquinone are not responsible for SDF activity against insect scavengers, more studies are needed to elucidate the SDF compound from Xenorhabdus and Photorhabdus bacteria.

RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria.

BACKGROUND: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria. RESULTS: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified. CONCLUSIONS: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

Photorhabdus antumapuensis sp. nov., a novel symbiotic bacterial species associated with Heterorhabditis atacamensis entomopathogenic nematodes.

One motile, Gram-negative, non-spore-forming and rod-shaped symbiotic bacterium, strain UCH-936T, was isolated from Heterorhabditis atacamensis nematodes. Results of biochemical, physiological, molecular and genomic analyses suggest that it represents a new species, which we propose to name Photorhabdus antumapuensis sp. nov. Digital DNA-DNA hybridization shows that strain UCH-936T is more closely related to Photorhabdus kleinii DSM 23513T, but shares solely 50.5 % similarity, which is below the 70% cut-off value that delimits species boundaries in bacteria. Phylogenetic reconstructions using whole-genome sequences show that strain UCH-936T forms a unique clade, suggesting its novel and distinct taxonomic status again. Similarly, comparative genomic analyses shows that the virulence factor flagella-related gene fleR, the type IV pili-related gene pilL and the vibriobactin-related gene vibE are present in the genome of strain UCH-936T but absent in the genomes of its closest relatives. Biochemically and physiologically, UCH-936T differs also from all closely related Photorhabdus species. Therefore, Photorhabdus antumapuensis sp. nov. is proposed as a new species with the type strain UCH-936T (CCCT 21.06T=CCM 9188T=CCOS 1991T).

Purification, characterization and toxicity assessment of PirAB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

Photorhabdus insect related proteins A & B (PirA, PirB) from Photorhabdus and Xenorhabdus bacteria exhibit both oral and injectable toxicity against lepidopteran and dipteran insect pest. The pirA, pirAt (encoding 6 N-terminal truncated PirA), pirB genes, pirA-pirB (with ERIC sequences), pirA-pirB-mERIC (modified pirA-pirB with mutated ERIC sequences) and polycistronic-pirAB were cloned and expressed in Escherichia coli. However, PirA protein was expressed in insoluble form and therefore the pirA gene was modified to produce PirAt. Moreover, pirA-pirB-mERIC, polycistronic-pirAB and co-transformed pirA/pirB genes were not expressed in the studied prokaryotic expression systems. None of the single purified proteins or mixtures of the individually expressed and purified proteins were toxic to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. However, PirA-PirB protein mixtures purified from pirA-pirB operon plasmid were toxic to A. aegypti and C. quinquefasciatus larvae with LC50 values of 991 and 614 ng/ml, respectively. The presence of ERIC sequences between the two orfs of the pirA-pirB operon could help to obtain the proteins in biologically active form. Further, results confirm that PirA-PirB proteins of P. akhurstii subsp. akhurstii K-1 are binary insecticidal toxins and ERIC sequences could play an important role in expression of Pir proteins. Reports of biophysical characterization of individually purified PirAt, PirB and expressed PirA-PirB toxin mixture could provide the structural insight into these proteins.