Entomopathogenic Bacterium Xenorhabdus Research Articles

OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

The evolution of entomopathogeny in nematodes.

Understanding how parasites evolved is crucial to understand the host and parasite interaction. The evolution of entomopathogenesis in rhabditid nematodes has traditionally been thought to have occurred twice within the phylum Nematoda: in Steinernematidae and Heterorhabditidae families, which are associated with the entomopathogenic bacteria Xenorhabdus and Photorhabdus, respectively. However, nematodes from other families that are associated with entomopathogenic bacteria have not been considered to meet the criteria for "entomopathogenic nematodes." The evolution of parasitism in nematodes suggests that ecological and evolutionary properties shared by families in the order Rhabditida favor the convergent evolution of the entomopathogenic trait in lineages with diverse lifestyles, such as saprotrophs, phoretic, and necromenic nematodes. For this reason, this paper proposes expanding the term "entomopathogenic nematode" considering the diverse modes of this attribute within Rhabditida. Despite studies are required to test the authenticity of the entomopathogenic trait in the reported species, they are valuable links that represent the early stages of specialized lineages to entomopathogenic lifestyle. An ecological and evolutionary exploration of these nematodes has the potential to deepen our comprehension of the evolution of entomopathogenesis as a convergent trait spanning across the Nematoda.

Computational exploration of the genomic assignments, molecular structure, and dynamics of the ccdABXn 2 toxin-antitoxin homolog with its bacterial target, the DNA gyrase, in the entomopathogen Xenorhabdus nematophila

Toxin-antitoxin (TA) modules, initially discovered on bacterial plasmids and subsequently identified within chromosomal contexts, hold a pivotal role in the realm of bacterial physiology. Among these, the pioneering TA system, ccd (Control of Cell Death), primarily localized on the F-plasmid, is known for its orchestration of plasmid replication with cellular division. Nonetheless, the precise functions of such systems within bacterial chromosomal settings remain a compelling subject that demands deeper investigation. To bridge this knowledge gap, our study focuses on exploring ccdABXn2 , a chromosomally encoded TA module originating from the entomopathogenic bacterium Xenorhabdus nematophila. We meticulously delved into the system’s genomic assignments, structural attributes, and functional interplay. Our findings uncovered intriguing patterns—CcdB toxin homologs exhibited higher conservation levels compared to their CcdA antitoxin counterparts. Moreover, we constructed secondary as well as tertiary models for both the CcdB toxin and CcdA antitoxin using threading techniques and subsequently validated their structural integrity. Our exploration extended to the identification of key interactions, including the peptide interaction with gyrase for the CcdB homolog and CcdB toxin interactions for the CcdA homolog, highlighting the intricate TA interaction network. Through docking and simulation analyses, we unequivocally demonstrated the inhibition of replication via binding the CcdB toxin to its target, DNA gyrase. These insights provide valuable knowledge about the metabolic and physiological roles of the chromosomally encoded ccdABXn 2 TA module within the context of X. nematophila, significantly enhancing our comprehension of its functional significance within the intricate ecosystem of the bacterial host. Communicated by Ramaswamy H. Sarma

Discovery of a new highly pathogenic toxin involved in insect sepsis.

Insect sepsis is a severe consequence that arises from the invasion of the hemocoel by symbionts of entomopathogenic nematodes and bacteria. In the present study, we unveiled the heightened virulence of the entomopathogenic nematode Steinernema feltiae and the entomopathogenic bacteria Xenorhabdus bovienii, which operate symbiotically, against the wax moth Galleria mellonella. Maximum mortality was observed at 25°C while the optimal infestation efficiency was 20 nematodes per host. After infestation, G. mellonella displayed rapid darkening and softening, accompanied by an escalated esterase activity at 9 h. The X. bovienii, released by S. feltiae, underwent substantial proliferation and discharged toxins that attacked hemocytes, thus triggering extensive hemolysis and sepsis. The host G. mellonella was usually killed within 24 h due to disseminated septicemia. Additionally, X. bovienii infestation led to the upregulation of metabolites like 3-hydroxyanthranilic acid. Strikingly, we identified the perilous actinomycin D, generated through kynurenine metabolites, representing a novel biomarker of insect sepsis. Furthermore, a comprehensive transcriptomic analysis unveiled a noteworthy upregulation of gene expression associated with actinomycin D. Overall, X. bovienii induced apoptosis and sepsis through actinomycin D production, indicating its pivotal role in infestation activity. These findings open up new avenues for studying the mechanism of sepsis and developing innovative biotic pesticides. IMPORTANCE As a current biocontrol resource, entomopathogenic nematodes and their symbiotic bacterium can produce many toxin factors to trigger insect sepsis, having the potential to promote sustainable pest management. In this study, we found Steinernema feltiae and Xenorhabdus bovienii were highly virulent against the insects. After infective juvenile injection, Galleria mellonella quickly turned black and softened with increasing esterase activity. Simultaneously, X. bovienii attacked hemocytes and released toxic components, resulting in extensive hemolysis and sepsis. Then, we applied high-resolution mass spectrometry-based metabolomics and found multiple substances were upregulated in the host hemolymph. We found extremely hazardous actinomycin D produced via 3-hydroxyanthranilic acid metabolites. Moreover, a combined transcriptomic analysis revealed that gene expression of proteins associated with actinomycin D was upregulated. Our research revealed actinomycin D might be responsible for the infestation activity of X. bovienii, indicating a new direction for exploring the sepsis mechanism and developing novel biotic pesticides.

The aptness of entompathogenic bacteria against sawtoothed grain beetle (Oryzaephilus surinamensis [L.]) (Coleoptera: Silvanidae) in dates under laboratory conditions

ObjectiveThe sawtoothed grain beetle, Oryzaephilus surinamensis (L). is known as one of the most devastating insect pests in stored date fruits, infrequently found in newly harvested date fruits and raisins but developed a high plenty in dry fruits particularly after long storage. Indiscriminate use of pesticides and fumigants to control insects have resulted in insecticide resistance, environmental hazards, residual toxicity, and pest resurgence so the control trends have been changed and particularly biological control agents like entomopathogenic bacteria Photorhabdus temperata and Xenorhabdus nematophila being used to manage sawtoothed grain beetle. MethodsDuring present study, the pathogenicity of P. temperata and X. nematophila was evaluated against this crucial pest of stored grains. The bacterial concentrations, 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 cells/ml of both bacteria were prepared and used for the experiments. The prepared solutions were applied directly to the date fruits infested with sawtoothed grain beetles and data were recorded. ResultsSignificant results were observed for both bacterial treatments as the highest concentration of 108 cells/ml showed to be very effective against the beetle in terms of fecundity, adult emergence, and mortality. In terms of fecundity, 8.66 to 4.33 mean number of eggs were produced at bacterial P. temperata concentrations of 1 × 104 and 1 × 108 cells/ml, respectively. Similarly, the F1 adult emergence for P. temperata was recorded 15.33 and 9 adults at concentrations of 1 × 104 and 1 × 108 cells/ml, respectively. Results showed a similar trend for the X. nematophila species. ConclusionsThese results indicate that utilizing bacteria as biological control agents can be advantageous for reducing insect populations in storage facilities and preventing the loss of grains and date fruits that have been stored in storage.

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.

Diverse Roles for a Conserved DNA-Methyltransferase in the Entomopathogenic Bacterium Xenorhabdus.

In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria.

In-silico analysis of genomic distribution and functional association of hipBA toxin-antitoxin (TA) homologs in entomopathogen Xenorhabdus nematophila

• Presence of three hipBA TA paralogs in the genome of X. nematophila. • HipA homologs represents a class of serine/threonine-protein kinase. • HipB homologs represents a class of transcriptional regulator. • hipBA TA paralogs in the genome of X. nematophila are functionally associated. Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli . Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila . This bacterium is a symbiotic model with the nematode Steinernema carpocapsae . We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila .

Genomic assortment and interactive insights of the chromosomal encoded control of cell death (ccd) toxin-antitoxin (TA) module in Xenorhabdus nematophila

In the present circumstances, toxin-antitoxin (TA) modules have a great consideration due to their elusive role in bacterial physiology. TA modules consist of a toxic part and a counteracting antitoxin part and these are abundant genetic loci harbored on bacterial plasmids and chromosomes. The control of cell death (ccd) TA locus was the first identified TA module and its unitary function (such as plasmid maintenance) has been described, however, the function of its chromosomal counterparts is still ambiguous. Here, we are exploring the genomic assortment, structural and functional association of chromosomally encoded ccdAB TA homolog (ccdABXn 1) in the genome of an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae that infects and kills the host insect. By genomic assortment analysis, our observations suggested that CcdA antitoxin homologs are not more closely related than CcdB toxin homologs. Further results suggest that the ccdABXn 1 TA homolog has sulphonamide (such as 4C6, for CcdA homolog) and peptide (such as gyrase, for CcdB homolog) ligand partners with a typical TA interaction network that may affect essential cellular metabolism of the X. nematophila. Collectively, our results improve the knowledge and conception of the metabolic interactive role of ccdAB TA homologs in X. nematophila physiology. Communicated by Ramaswamy H. Sarma

The Effects of Various Organic and Inorganic Fertilizers on the Density of Entomopathogenic Nematodes Symbiont

Entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora and their bacterial symbionts Xenorhabdus nematophila and Photorhabdus luminescens (P. luminescens), represent a specific agricultural niche. The successful integration of entomopathogenic nematodes (EPN) as regular use biological control agents require specific knowledge and understanding of the adaptation and establishment of applied biological control agents in agricultural ecosystems. For many years, the pest management capabilities of these pathogenic partnerships have been implemented in biological control settings. In this study, ten fertilizers were examined to explore the effects of nitrogen, phosphorus, and potassium (NPK) on the production of entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus luminescens. Laboratory exposure to fertilizer concentrations (0.5-2.5%) was used to determine the production of each bacterial species. Results conclude that P. luminescens are generally more sensitive to fertilizer than X. nematophila. Moreover, fertilizers containing high nitrogen suppressed bacterial densities more readily than those with lesser amounts. This paper summarizes the effects of the three important nutrients found in various concentrations of organic and inorganic fertilizers on entomopathogenic bacteria production.

A Conserved Nonribosomal Peptide Synthetase in Xenorhabdus bovienii Produces Citrulline-Functionalized Lipopeptides.

The entomopathogenic bacterium Xenorhabdus bovienii exists in a mutualistic relationship with nematodes of the genus Steinernema. Free-living infective juveniles of Steinernema prey on insect larvae and regurgitate X. bovienii within the hemocoel of a host larva. X. bovienii subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While Xenorhabdus species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in Xenorhabdus species. Heterologous expression of this NRPS gene from X. bovienii in E. coli led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type X. bovienii cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.

Biocontrol potential and mode of action of entomopathogenic bacteria Xenorhabdus budapestensis C72 against Bipolaris maydis

Southern corn leaf blight (SCLB) caused by Bipolaris maydis is an important foliar disease of maize. In this study, a nematode-symbiotic bacterium Xenorhabdus budapestensis strain C72 was identified with remarkable inhibiting effect on mycelial growth and spore germination of B. maydis. The in vitro assay revealed that C72 cell-free culture media (CFCM) with thermostability exhibited broad-spectrum antifungal activities against other several important plant pathogenic fungi. The early colonization of B. maydis were significantly impaired by CFCM treatment under phytotron condition. This antagonism is likely to be the main contributor to the highly efficient plant protection of 40% (v/v) CFCM treatment against B. maydis, and the relative control effect reached to 59.15% and 77.96% in greenhouse and field experiments, which was comparable to the effect of fungicides. Moreover, we found that extracellular enzymes secreted by symbiotic bacterium may be one of the reasons for the antifungal potential of C72. Beside direct antagonistic effects provided by the bacterium, defense related genes were induced in maize after CFCM treatment. In summary, this study reported the first systematic evaluation of the effect of X. budapestensis C72 in controlling SCLB and exploration of its mode of action, then indicating that entomopathogenic bacteria have the potential to become a new and efficient biological control resource for plant fungal disease management.

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

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.

Structure and biosynthesis of deoxy-polyamine in Xenorhabdus bovienii.

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.

Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity

BackgroundXenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus.ResultsAll bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities.ConclusionVirulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

Evaluation of insecticidal activity of entomopathogenic bacteria Photorhabdus and Xenorhabdus against shoot and fruit borer Earias vittella (Lepidoptera: Noctuidae) of vegetable crops

The shoot and fruit borer, Earias vittella is one of the major polyphagous pest prefer various host plants attacking almost 38 plant species and causes yield loss of 15-45% in vegetable crops annually in India. In this study Entomopathogenic bacteria (EPB) were used for biocontrol larvae of Earias vittella under in vitro condition. Whole cell culture and cell supernatant of EPB was screened for insecticidal activity against the larvae and results has shown that the lethality of larvae started after 12-24h of treatment. The percent mortality treated with intact cell was significantly higher in larvae treated with EPB3 with 90% mortality at 72h followed by EPB1 and EPB3. The larvae treated with cell supernatant of EPB had shown lethality in the range of the mortality of larvae where in the range of 50-70% at 72h of treatment and there was no significant difference among the bacteria treated. The LD50 value represent the concentration of cells required for the 50% population to be lethal and EPB3 had expressed significantly lower concentration of 4.28x104 CFU / ml followed by EPB9 and EPB8. The LD50 value for the larvae treated with cell supernatant were in the range of 1010 to 1012 CFU / ml. The larvae treated with EPB3 cell culture has taken significantly less time for mortality of fifty percent of population with a media lethal time (LT50) of 24h followed by EPB1 (30h) and EPB8 (32h). Among all the isolates EPB3 (Xenorhabdus nematophila) and EPB1 (Photorhabdus luminescens) had more virulence against the Earias vittella and can be further tested for biocontrol efficiency under greenhouse and field conditions.

The hipBAXn operon from Xenorhabdus nematophila functions as a bonafidetoxin-antitoxin module.

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.

Two novel cyclic depsipeptides Xenematides F and G from the entomopathogenic bacterium Xenorhabdus budapestensis.

Two novel depsipeptides xenematides F and G (1, 2), were isolated from entomopathogenic Xenorhabdus budapestensis SN84 along with a known compound xenematide B. The structures of the two new molecules were elucidated using NMR, MS and Marfey's method. The xenematide G (2) contains α-aminoheptanoic acid, a non-protein amino acid that is rarely found in secondary metabolites from entomopathogenic bacteria. Xenematides F and G were tested for antibacterial activity. Xenematide G (2) exhibited moderate antibacterial activity.

Dual phenazine gene clusters enable diversification during biosynthesis.

Biosynthetic gene clusters (BGCs) bridging genotype and phenotype continuously evolve through gene mutations and recombinations to generate chemical diversity. Phenazine BGCs are widespread in bacteria, and the biosynthetic mechanisms of the formation of the phenazine structural core have been illuminated in the last decade. However, little is known about the complex phenazine core-modification machinery. Here, we report the diversity-oriented modifications of the phenazine core through two distinct BGCs in the entomopathogenic bacterium Xenorhabdus szentirmaii, which lives in symbiosis with nematodes. A previously unidentified aldehyde intermediate, which can be modified by multiple enzymatic and non-enzymatic reactions, is a common intermediate bridging the pathways encoded by these BGCs. Evaluation of the antibiotic activity of the resulting phenazine derivatives suggests a highly effective strategy to convert Gram-positive specific phenazines into broad-spectrum antibiotics, which might help the bacteria-nematode complex to maintain its special environmental niche.

Inhibition of Spodoptera frugiperda phenoloxidase activity by the products of the Xenorhabdus rhabduscin gene cluster.

We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa.