Xenorhabdus Species Research Articles

Antagonistic Efficacy of Symbiotic Bacterium Xenorhabdus sp. SCG against Meloidogyne spp.

The inhabitation and parasitism of root-knot nematodes (RKNs) can be difficult to control, as its symptoms can be easily confused with other plant diseases; hence, identifying and controlling the occurrence of RKNs in plants remains an ongoing challenge. Moreover, there are only a few biological agents for controlling these harmful nematodes. In this study, Xenorhabdus sp. SCG isolated from entomopathogenic nematodes of genus Steinernema was evaluated for nematicidal effects under in vitro and greenhouse conditions. The cell-free filtrates of strain SCG showed nematicidal activity against Meloidogyne species J2s, with mortalities of > 88% at a final concentration of 10%, as well as significant nematicidal activity against the three other genera of plant-parasitic nematodes in a dose-dependent manner. Thymine was isolated as active compounds by assay-guided fractionation and showed high nematicidal activity against M. incognita. Greenhouse experiments suggested that cell-free filtrates of strain SCG efficiently controlled the nematode population in M. incognita-infested tomatoes (Solanum lycopersicum L., cv. Rutgers). In addition, a significant increase in host plant growth was observed after 45 days of treatment. To our knowledge, this is the first to demonstrate the nematicidal activity spectrum of isolated Xenorhabdus species and their application to S. lycopersicum L., cv. Rutgers under greenhouse conditions. Xenorhabdus sp. SCG could be a promising biological nematicidal agent with plant growth-enhancing properties.

Genome sequences of key bacterial symbionts of entomopathogenic nematodes: Xenorhabdus cabanillasii DSM17905, Xenorhabdus ehlersii DSM16337, Xenorhabdus japonica DSM16522, Xenorhabdus koppenhoeferii DSM18168, and Xenorhabdus mauleonii DSM17908.

Xenorhabdus species are bacterial symbionts of entomopathogenic Steinernema nematodes, in which they produce diverse secondary metabolites implicated in pathogenesis. To expand resources for natural product prospecting and exploration of host-symbiont-pathogen relationships, the genomes of Xenorhabdus cabanillasi, Xenorhabdus ehlersii, Xenorhabdus japonica, Xenorhabdus koppenhoeferii, and Xenorhabdus mauleonii were analyzed.

Metabolites of Xenorhabdus bacteria are potent candidates for mitigating amphibian chytridiomycosis

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has caused extreme losses in amphibian biodiversity. Finding bacteria that produce metabolites with antifungal properties may turn out to be invaluable in the fight against this devastating disease. The entomopathogenic bacteria, Xenorhabdus szentirmaii and X. budapestensis produce secondary metabolites that are effective against a wide range of fungal plant pathogens. To assess whether they may also be effective against Bd, we extracted cell-free culture media (CFCM) from liquid cultures of X. szentirmaii and X. budapestensis and tested their ability to inhibit Bd growth in vitro. As a second step, using juvenile common toads (Bufo bufo) experimentally infected with Bd we also tested the in vivo antifungal efficacy of X. szentirmaii CFCM diluted to 2 and 10% (v/v), while also assessing possible malign side effects on amphibians. Results of the in vitro experiment documented highly effective growth inhibition by CFCMs of both Xenorhabdus species. The in vivo experiment showed that treatment with CFCM of X. szentirmaii applied at a dilution of 10% resulted in infection intensities reduced by ca. 73% compared to controls and to juvenile toads treated with CFCM applied at a dilution of 2%. At the same time, we detected no negative side effects of treatment with CFCM on toad survival and development. Our results clearly support the idea that metabolites of X. szentirmaii, and perhaps of several other Xenorhabdus species as well, may prove highly useful for the treatment of Bd infected amphibians.

Xenorhabdus aichiensis sp. nov., Xenorhabdus anantnagensis sp. nov., and Xenorhabdus yunnanensis sp. nov., Isolated from Steinernema Entomopathogenic Nematodes

Three bacterial strains, XENO-2T, XENO-7T, and XENO-10T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel Xenorhabdus species. In this study, we describe these new species by whole-genome and whole-proteome phylogenomic reconstructions, by calculating sequence identity scores using core genome sequences, and by phenotypic characterization. Phylogenomic reconstructions using ribosomal and house-keeping genes, and whole-genome and whole-proteome sequences show that XENO-2T and XENO-10T are closely related to Xenorhabdus japonica DSM 16522T and that XENO-7T is closely related to Xenorhabdus bovienii subsp. africana XENO-1T and to X. bovienii subsp. bovienii T228T. The dDDH values between XENO-2T and XENO-10T and between XENO-2T and X. japonica DSM 16522T are 56.4 and 51.8%, respectively. The dDDH value between XENO-10T and X. japonica DSM 16522T is 53.4%. The dDDH values between XENO-7T and X. bovienii subsp. africana XENO-1T and between XENO-7T and X. bovienii subsp. bovienii T228T are 63.6 and 69.4%, respectively. These dDDH values are below the 70% divergence threshold for prokaryotic species delineation. The newly described species are highly pathogenic to G. mellonella larvae, grow at pH between 5 and 9 (optimum 5–7), at salt concentrations of 1–3% (optimum 1–2%), and temperatures between 20 and 37 °C (optimum 28–30 °C). Biochemical tests such as lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, citrate utilization, indole and acetoin production, and cytochrome oxidase tests allow to differentiate the novel species from their more closely related species. Considering these genetic and phenotypic divergencies, we propose the following new species: Xenorhabdus aichiensis sp. nov. with XENO-7T (= CCM 9233T = CCOS 2024T) as the type strain, Xenorhabdus anantnagensis sp. nov., with XENO-2T (= CCM 9237T = CCOS 2023T) as the type strain, and Xenorhabdus yunnanensis sp. nov., with XENO-10T (= CCM 9322T = CCOS 2071T) as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Draft genomes, phylogenomic reconstruction and comparative genome analysis of three Xenorhabdus strains isolated from soil-dwelling nematodes in Kenya.

As a proven source of potent and selective antimicrobials, Xenorhabdus bacteria are important to an age plagued with difficult-to-treat microbial infections. Yet, only 27 species have been described to date. In this study, a novel Xenorhabdus species was discovered through genomic studies on three isolates from Kenyan soils. Soils in Western Kenya were surveyed for steinernematids and Steinernema isolates VH1 and BG5 were recovered from red volcanic loam soils from cultivated land in Vihiga and clay soils from riverine land in Bungoma respectively. From the two nematode isolates, Xenorhabdus sp. BG5 and Xenorhabdus sp. VH1 were isolated. The genomes of these two, plus that of X. griffiniae XN45 - this was previously isolated from Steinernema sp. scarpo that also originated from Kenyan soils - were sequenced and assembled. Nascent genome assemblies of the three isolates were of good quality with over 70 % of their proteome having known functions. These three isolates formed the X. griffiniae clade in a phylogenomic reconstruction of the genus. Their species were delineated using three overall genome relatedness indices: an unnamed species of the genus, Xenorhabdus sp. BG5, X. griffiniae VH1 and X. griffiniae XN45. A pangenome analysis of this clade revealed that over 70 % of species-specific genes encoded unknown functions. Transposases were linked to genomic islands in Xenorhabdus sp. BG5. Thus, overall genome-related indices sufficiently delineated species of two new Xenorhabdus isolates from Kenya, both of which were closely related to X. griffiniae . The functions encoded by most species-specific genes in the X. griffiniae clade remain unknown.

Rattus norvegicus (Berkenhout, 1769) (Rodentia: Muridae)’un entomopatojen bakteriler ile enfekte böcek kadavralarına tepkisinin belirlenmesi

Xenorhabdus Thomas & Poinar (Enterobacterales: Morganellaceae) and Photorhabdus Thomas & Poinar (Enterobacterales: Morganellaceae) bacteria are mutualistically associated with Steinernema Travassos, 1927 (Rhabditida: Steinernematidae) and Heterorhabditis Poinar, 1976 (Rhabditida: Heterorhabditidae) nematodes, respectively, and are known to produce several secondary metabolites that protect nematode-killed insects from different competitors. One of these compounds called “the scavenger deterrent factor” (SDF) is known to deter different arthropod, bird, and fish species from feeding on insects killed by Xenorhabdus or Photorhabdus bacteria. The effects of SDF from five different Xenorhabdus and one Photorhabdus species against the Norway rat, Rattus norvegicus (Berkenhout, 1769) (Rodentia: Muridae) were investigated using either a one-choice or two-choice experimental design during 2019-2020 in Aydın Adnan Menderes University. Rats were given four-day-old bacteria-killed Galleria mellonella (L., 1758) (Lepidoptera: Pyralidae) larvae and feeding behavior was observed and recorded. The results demonstrate that the Norway rat is deterred from feeding on insects killed by certain Xenorhabdus and Photorhabdus species and it is likely due to the distastefulness of these cadavers. Ecologically, the data suggest that insects killed by the entomopathogenic nematode/bacterium complex in nature may be protected from attack from insectivorous mammals, especially those that feed on soil-dwelling insects.

Assessment of biocontrol potential of different Steinernema species and their bacterial symbionts, Xenorhabdus species against larvae of almond moth, Ephestia cautella (Walker)

The almond moth, Ephestia cautella is a serious pest of stored food commodities including dried figs, dates, raisins, cocoa beans. The control of E. cautella infestations in storage that mostly relies on toxic insecticides or fumigants has caused emergence of resistant pest populations as well as residues on the commodities. Entomopathogenic nematodes (EPNs) and their endosymbionts are one of the emerging approaches in eco-friendly pest management and pose no health risk to humans and the environment. Therefore, here we evaluated the toxicity of different EPN species (Steinernema feltiae, S. carpocapsae, and S. bicornotum) and their endosymbiotic bacteria (Xenorhabdus nematophila, X. bovienii, and X. budapestensis) on the 3rd and 4th instars larvae of E. cautella under controlled conditions. The IJs of EPNs were applied to concrete arenas in Petri dishes at three concentrations (50, 100, and 200 IJs/cm2). Oral and contact toxicity of the cell-free supernatants and cell suspension of symbiotic bacteria (4x107 cells/mL) were tested against both larval instars of E. cautella. Steinernema carpocapsae and S. feltiae induced mortalities over 80% at their lowest concentrations against both larval instars after 24 h of application. Oral and contact treatments of cell-free supernatants and cell suspensions were lethal for both larval instars; however, higher mortalities were generally obtained from direct exposure of larvae to cell-free supernatants and cell suspensions. The contact treatment of X. nematophila exhibited the highest virulence, causing 70 and 82.5% mortalities in 3rd and 4th instar larvae after 72 h of application, respectively. The anti-feedant activity of cell-free supernatants and cell suspensions was also observed in both instars in oral treatments. The findings of this study suggest that EPNs and their symbiotic bacteria have enormous potential for the eco-friendly control of E. cautella.

Potato waste as an effective source of electron generation and bioremediation of pollutant through benthic microbial fuel cell

The goal of this research was to improve electron generation in BMFC by utilizing potatoes waste as an electron donor source for bacterial species. Pollutant remediation is a secondary applications of BMFC, hence the current research investigated heavy metal remediation from wastewater and energized the system using potato waste. Within 20 days of operation, the present study provided 112 mV, with a maximum current density of 36.84 mA/m2. The cell measured an internal resistance of 1557 Ω, which is bit higher than the external resistance. Several electrochemical studies were also carried out to validate the BMFC findings. On day 30, the specific capacitance was measured with a cyclic voltameter at 0.00057F/g to investigate the biofilm. It demonstrates that the biofilm remains stable throughout the BMFC process. Overall, the electrochemical studies demonstrated that potato waste was a good source of bacterial activity. The Enterobacter, Proteus, and Xenorhabdus species are detected on the surface of anode. Lastly, within 40 days, the maximum efficiency of metal remediation was found to be 94.20 % (Cd2+), 97.34 % (Pb2+), and 84.12 % (Cr3+).

Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

Identification of novel prophage regions in Xenorhabdus nematophila genome and gene expression analysis during phage-like particle induction.

BackgroundEntomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species.MethodsIn the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response (recA and lexA) associated with mitomycin C and UV exposure.ResultsWe described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions.

Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

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.

A study on Xenorhabdus and Photorhabdus isolates from Northeastern Thailand: Identification, antibacterial activity, and association with entomopathogenic nematode hosts.

Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5_TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates.

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.

Insecticidal Effect of Entomopathogenic Nematodes and the Cell-Free Supernatant from Their Symbiotic Bacteria against Philaenus spumarius (Hemiptera: Aphrophoridae) Nymphs.

Simple SummaryThe disease caused by Xylella fastidiosa affects economically relevant crops such as olives, almonds, and grapevine. Since curative means are not available, its current management principally consists of broad-spectrum pesticide applications to control vectors like the meadow spittlebug Philaenus spumarius, the most important one in Europe. Exploring environmentally sound alternatives is a primary challenge for sustainable agriculture. Entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling arthropods. Recent technological advances for field applications, including improvements in obtaining cell-free supernatants from EPN symbiotic bacteria, allow their successful implementation against aerial pests. Here, we investigated the impact of four EPN species and their cell-free supernatants on nymphs of the meadow spittlebug. First, we observed that the exposure to the foam produced by this insect does not affect the nematode virulence. Indeed, direct applications of certain EPN species reached up to 90–78% nymphal mortality rates after five days of exposure, while specific cell-free supernatants produced 64% mortality rates. Overall, we demonstrated the great potential of EPN and cell-free supernatant of their symbiont bacteria applications against this vector, opening new venues to develop novel biopesticides for integrated management practices and organic productions.The meadow spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the primary vector of Xylella fastidiosa (Proteobacteria: Xanthomonadaceae) in Europe, a pest–disease complex of economically relevant crops such as olives, almonds, and grapevine, managed mainly through the use of broad-spectrum pesticides. Providing environmentally sound alternatives to reduce the reliance on chemical control is a primary challenge in the control of P. spumarius and, hence, in the protection of crops against the expansion of its associated bacterial pathogen. Entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling arthropods. Recent technological advances in field applications, including improvements in obtaining cell-free supernatant from their symbiotic bacteria, allow their successful implementation against aerial pests. Thus, this study aimed to evaluate, for the first time, the efficacy of EPN applications against nymphal instars of P. spumarius. We tested four EPN species and the cell-free supernatant of their corresponding symbiotic bacteria: Steinernema feltiae–Xenorhabdus bovienii, S. carpocapsae–X. nematophila, S. riojaense–X. kozodoii, and Heterorhabditis bacteriophora–Photorhabdus laumondii subsp. laumondii. First, we showed that 24 and 72 h exposure to the foam produced by P. spumarius nymphs did not affect S. feltiae virulence. The direct application of steinernematid EPNs provided promising results, reaching 90, 78, and 53% nymphal mortality rates after five days of exposure for S. carpocapsae, S. feltiae, and S. riojaense, respectively. Conversely, the application of the cell-free supernatant from P. laumondii resulted in nymphal mortalities of 64%, significantly higher than observed for Xenorhabdus species after five days of exposure. Overall, we demonstrated the great potential of the application of specific EPNs and cell-free supernatant of their symbiont bacteria against P. spumarius nymphs, introducing new opportunities to develop them as biopesticides for integrated management practices or organic vineyard production.

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria.

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of "pro-pyrazinones" featuring a hetero-tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes.

Mode of entry of secondary metabolites of the bacteria Xenorhabdus szentirmaii and X. nematophila into Tetranychus urticae, and their toxicity to the predatory mites Phytoseiulus persimilis and Neoseiulus californicus

The bacterial metabolites in supernatants of Xenorhabdus species have acaricidal activity, but this mode of entry into mites has not yet been elucidated. Herein, we report on the possible mode of entry of Xenorhabdus szentirmaii and Xenorhabdus nematophila supernatants into Tetranychus urticae (Acari: Tetranychidae) adult females. We also assessed the toxicity of the supernatants against the developmental stages of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae). Experiments were conducted at 25 ± 1 °C, 70 ± 5% relative humidity, and 16:8h light:dark conditions. Our data showed that the bioactive acaricidal compound is most effective (86.5 to 89% mortality) when the entire integument of T. urticae comes in contact with it compared to contact of the ventral side only (26.5–34%). Against P. persimilis and N. californicus at 6 days post-application (dpa), the eggs were not affected by the X. szentirmaii or X. nematophila supernatant, whereas mortality of the mobile stages (larva, protonymph, deutonymph, adult) was 18.5% to 39.2%. Overall, the predatory mites were less affected by the bacterial metabolites than T. urticae. We hypothesize that the differences in morphology such as longer legs and thicker cuticle, as well as the diet of the predatory mites, reduce the contact of the body parts to the supernatant-treated surfaces. We need to isolate, identify, and characterize the X. szentirmaii and X. nematophila metabolite(s) and demonstrate efficacy to pestiferous mites and safety to plants, non-target organisms and the environment before it can be used as an acaricide.