Xenorhabdus lircayensis sp. nov., the symbiotic bacterium associated with the entomopathogenic nematode Steinernema unicornum.

Xenorhabdus is a symbiotic group of bacteria associated with entomopathogenic nematodes of the family Steinernematidae. Although the described Steirnernema species list is extensive, not all their symbiotic bacteria have been identified. One single motile, Gram-negative and non-spore-forming rod-shaped symbiotic bacterium, strain VLST, was isolated from the entomopathogenic nematode Steinernema unicornum. Analyses of the 16S rRNA gene determined that the VLST isolate belongs to the genus Xenorhabdus, and its closest related species is Xenorhabdus szentirmaii DSM 16338T (98.2 %). Deeper analyses using the whole genome for phylogenetic reconstruction indicate that VLST exhibits a unique clade in the genus. Genomic comparisons considering digital DNA-DNA hybridization (dDDH) values confirms this result, showing that the VLST values are distant enough from the 70 % threshold suggested for new species, sharing 30.7, 30.5 and 30.3 % dDDH with Xenorhabdus khoisanae MCB, Xenorhabdus koppenhoeferi DSM 18168T and Xenorhabdus miraniensis DSM 18168T, respectively, as the closest species. Detailed physiological, biochemical and chemotaxonomic tests of the VLST isolate reveal consistent differences from previously described Xenorhabdus species. Phylogenetic, physiological, biochemical and chemotaxonomic approaches show that VLST represents a new species of the genus Xenorhabdus, for which the name Xenorhabdus lircayensis sp. nov. (type strain VLST=CCCT 20.04T=DSM 111583T) is proposed.

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 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.

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

BACKGROUND: Xenorhabdus 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. RESULTS: All 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. CONCLUSION: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

Antibacterial activity of Xenorhabdus and Photorhabdus isolated from entomopathogenic nematodes against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus, symbiotically associated with entomopathogenic nematodes (EPNs), produce a range of antimicrobial compounds. The objective of this study is to identify Xenorhabdus and Photorhabdus and their EPNs hosts, which were isolated from soil samples from Saraburi province, and study their antibacterial activity against 15 strains of drug-resistant bacteria. Fourteen isolates (6.1%), consisting of six Xenorhabdus isolates and eight Photorhabdus isolates, were obtained from 230 soil samples. Based on the BLASTN search incorporating the phylogenetic analysis of a partial recA gene, all six isolates of Xenorhabdus were found to be identical and closely related to X. stockiae. Five isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. akhurstii. Two isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. hainanensis. The remaining isolate of Photorhabdus was found to be identical to P. asymbiotica subsp. australis. The bacterial extracts from P. luminescens subsp. akhurstii showed strong inhibition the growth of S. aureus strain PB36 (MSRA) by disk diffusion, minimal inhibitory concentration, and minimal bactericidal concentration assay. The combination between each extract from Xenorhabdus/Photorhabdus and oxacillin or vancomycin against S. aureus strain PB36 (MRSA) exhibited no interaction on checkerboard assay. Moreover, killing curve assay of P. luminescens subsp. akhurstii extracts against S. aureus strain PB36 exhibited a steady reduction of 105 CFU/ml to 103 CFU/ml within 30 min. This study demonstrates that Xenorhabdus and Photorhabdus, showed antibacterial activity. This finding may be useful for further research on antibiotic production.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii.

Three strains of symbiotic bacteria were isolated from an entomopathogenic nematode Steinernema poinari retrieved from soil in eastern Poland. Using 16S rDNA, recA, gltX, gyrB, and dnaN gene sequences for phylogenetic analysis, these strains were shown to belong to the species Xenorhabdus bovienii. The nucleotide identity between the studied S. poinari microsymbionts and other X. bovienii strains calculated for 16S rDNA and concatenated sequences of four protein-coding genes was 98.7-100% and 97.9-99.5%, respectively. The phenotypic properties of the isolates also supported their close phylogenetic relationship with X. bovienii. All three tested X. bovienii strains of different Steinernema clade origin supported the recovery of infective juveniles and subsequent development of the nematode population. However, the colonization degree of new infective juvenile generations was significantly affected by the bacterial host donor/recipient. The colonization degree of infective juveniles reared on bacterial symbionts deriving from a non-cognate clade of nematodes was extremely low, but proved the possible host-switching between non-related Steinernema species.

Identification of entomopathogenic nematodes and symbiotic bacteria from Nam Nao National Park in Thailand and larvicidal activity of symbiotic bacteria against Aedes aegypti and Aedes albopictus.

Entomopathogenic nematodes (EPNs) that are symbiotically associated with Xenorhabdus and Photorhabdus bacteria can kill target insects via direct infection and toxin action. There are limited reports identifying such organisms in the National Park of Thailand. Therefore, the objectives of this study were to identify EPNs and symbiotic bacteria from Nam Nao National Park, Phetchabun Province, Thailand and to evaluate the larvicidal activity of bacteria against Aedes aegypti and Ae. albopictus. A total of 12 EPN isolates belonging to Steinernema and Heterorhabditis were obtained form 940 soil samples between February 2014 and July 2016. EPNs were molecularly identified as S. websteri (10 isolates) and H. baujardi (2 isolates). Symbiotic bacteria were isolated from EPNs and molecularly identified as P. luminescens subsp. akhurstii (13 isolates), X. stockiae (11 isolates), X. vietnamensis (2 isolates) and X. japonica (1 isolate). For the bioassay, bacterial suspensions were evaluated for toxicity against third to early fourth instar larvae of Aedes spp. The larvae of both Aedes species were orally susceptible to symbiotic bacteria. The highest larval mortality of Ae. aegypti was 99% after exposure to X. stockiae (bNN112.3_TH) at 96 h, and the highest mortality of Ae. albopictus was 98% after exposure to P. luminescens subsp. akhurstii (bNN121.4_TH) at 96 h. In contrast to the control groups (Escherichia coli and distilled water), the mortality rate of both mosquito larvae ranged between 0 and 7% at 72 h. Here, we report the first observation of X. vietnamensis in Thailand. Additionally, we report the first observation of P. luminescens subsp. akhurstii associated with H. baujardi in Thailand. X. stockiae has potential to be a biocontrol agent for mosquitoes. This investigation provides a survey of the basic diversity of EPNs and symbiotic bacteria in the National Park of Thailand, and it is a bacterial resource for further studies of bioactive compounds.

First report of a symbiotic relationship between Xenorhabdus griffiniae and an unknown Steinernema from South Africa.

Strain WS9, a mutualistic-associated bacterium, was isolated from an unknown entomopathogenic Steinernema nematode, collected from a litchi orchard in Friedenheim, Mpumalanga, South Africa. Based on phenotypic and phylogenetic data of the 16S rRNA, gltX, recA, dnaN, gyrB and infB gene sequences, strain WS9 is identified as X. griffiniae. Strain WS9 has antibacterial activity against Gram-positive and Gram-negative bacteria. This is the first report of an association between X. griffiniae and an unknown Steinernema species from South Africa.

Isolation and identification of Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes and their larvicidal activity against Aedes aegypti.

BACKGROUND: Aedes aegypti is a potential vector of West Nile, Japanese encephalitis, chikungunya, dengue and Zika viruses. Alternative control measurements of the vector are needed to overcome the problems of environmental contamination and chemical resistance. Xenorhabdus and Photorhabdus are symbionts in the intestine of entomopathogenic nematodes (EPNs) Steinernema spp. and Heterorhabditis spp. These bacteria are able to produce a broad range of bioactive compounds including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds. The objectives of this study were to identify Xenorhabdus and Photorhabdus isolated from EPNs in upper northern Thailand and to study their larvicidal activity against Ae. aegypti larvae. RESULTS: A total of 60 isolates of symbiotic bacteria isolated from EPNs consisted of Xenorhabdus (32 isolates) and Photorhabdus (28 isolates). Based on recA gene sequencing, BLASTN and phylogenetic analysis, 27 isolates of Xenorhabdus were identical and closely related to X. stockiae, 4 isolates were identical to X. miraniensis, and one isolate was identical to X. ehlersii. Twenty-seven isolates of Photorhabdus were closely related to P. luminescens akhurstii and P. luminescens hainanensis, and only one isolate was identical and closely related to P. luminescens laumondii. Xenorhabdus and Photorhabdus were lethal to Ae aegypti larvae. Xenorhabdus ehlersii bMH9.2_TH showed 100% efficiency for killing larvae of both fed and unfed conditions, the highest for control of Ae. aegypti larvae and X. stockiae (bLPA18.4_TH) was likely to be effective in killing Ae. aegypti larvae given the mortality rates above 60% at 72 h and 96 h. CONCLUSIONS: The common species in the study area are X. stockiae, P. luminescens akhurstii, and P. luminescens hainanensis. Three symbiotic associations identified included P. luminescens akhurstii-H. gerrardi, P. luminescens hainanensis-H. gerrardi and X. ehlersii-S. Scarabaei which are new observations of importance to our knowledge of the biodiversity of, and relationships between, EPNs and their symbiotic bacteria. Based on the biological assay, X. ehlersii bMH9.2_TH begins to kill Ae. aegypti larvae within 48 h and has the most potential as a pathogen to the larvae. These data indicate that X. ehlersii may be an alternative biological control agent for Ae. aegypti and other mosquitoes.

Three Novel Xenorhabdus-Steinernema Associations and Evidence of Strains of X. khoisanae Switching Between Different Clades.

Xenorhabdus species are normally closely associated with entomopathogenic nematodes of the family Steinernematidae. Strain F2, isolated from Steinernema nguyeni, was identified as Xenorhabdus bovienii and strains J194 and SB10, isolated from Steinernema jeffreyense and Steinernema sacchari as Xenorhabdus khoisanae, based on phenotypic characteristics and sequencing of 16S rRNA and housekeeping genes dnaN, gltX, gyrB, infB and recA. All three strains produced antimicrobial compounds that inhibited the growth of Gram-positive and Gram-negative bacteria. This is the first report of associations between strains of the symbiotic bacteria X. bovienii with S. nguyeni, and X. khoisanae with S. jeffreyense and S. sacchari. This provides evidence that strains of Xenorhabdus spp. may switch between nematode species within the same clade and between different clades.

Activated entomopathogenic nematode infective juveniles release lethal venom proteins.

Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

Xenorhabdus thuongxuanensis sp. nov. and Xenorhabdus eapokensis sp. nov., isolated from Steinernema species.

Two slightly yellowish-pigmented, oxidase-negative, rod-shaped and Gram-stain-negative bacterial strains (30TX1T and DL20T), isolated from Steinernema sangi and Steinernema eapokense, respectively, during soil sampling in Vietnam were studied using a polyphasic taxonomic approach. Strain 30TX1T showed highest 16S rRNA gene sequence similarity to the type strain of Xenorhabdus ehlersii (98.9 %) and strain DL20T to that of Xenorhabdus ishibashii (98.7 %). Sequence similarities to all other Xenorhabdus species were lower (<98.4 %). The two strains shared 98 % 16S rRNA gene sequence similarity. Multilocus sequence analysis (MLSA) based on concatenated partial recA, dnaN, gltX, gyrB and infB gene sequences showed a clear distinction of strains 30TX1T and DL20T among each other and to the closest related type strains. DNA-DNA hybridizations between strain DL20T and the type strain of X. ishibashii resulted in a relatedness value of 53 %. Genome-to-genome-based comparisons gave average nucleotide identities of 93.6 % (reciprocal 93.5 %) for strain 30TX1T and X. ehlersii DSM 16337T, of 92.8 % (reciprocal 93 %) for strain DL20T and X. ishibashiiDSM 22670Tand of 93.0 % (reciprocal 93.2 %) for the two novel strains. The fatty acid profile of the strains consisted of the major fatty acids C14 : 0, C16 : 0, C17 : 0 cyclo, C16 : 1ω7c and/or iso-C15 : 0 2-OH, and C18 : 1ω7c. Genome-to-genome comparison and MLSA results together with the differential biochemical and chemotaxonomic properties showed that strains 30TX1T and DL20T represent novel Xenorhabdus species, for which the names Xenorhabdus thuongxuanensis sp. nov. (type strain 30TX1T=CCM 8727T=LMG 29916T) and Xenorhabdus eapokensis sp. nov. (type strain DL20T=CCM 8728T=LMG 29917T) are proposed, respectively.

Morphological and molecular characterisation of an isolate of Steinernema diaprepesi Nguyen & Duncan, 2002 (Rhabditida: Steinernematidae) from Argentina and identification of its bacterial symbiont.

Entomopathogenic nematodes of the families Heterorhabditidae Poinar, 1976 and Steinernematidae Chitwood & Chitwood, 1937 are used for biological control of insect pests. An isolate of Steinernema diaprepesi Nguyen & Duncan, 2002 was recovered from a carrot field in the locality of Santa Rosa de Calchines (Santa Fe Province, Argentina). These nematodes were characterised based on morphological, morphometric and molecular studies. Their symbiotic bacterium was identified as Xenorhabdus doucetiae Tailliez, Pagès, Ginibre & Boemare, 2006 by sequencing the 16S rRNA gene. The isolate of S. diaprepesi studied exhibits some morphometric differences with the original description, especially in the first generation adults. This is the first description of the species in Argentina.

First report of the symbiotic bacterium Xenorhabdus indica associated with the entomopathogenic nematode Steinernema yirgalemense.

The entomopathogenic nematode Steinernema yirgalemense is considered a promising agent in the biocontrol of insects. However, little is known about the bacteria living in symbiosis with the nematode. In this study, we have identified the only available bacterial strain (157-C) isolated from S. yirgalemense, as a member of the species Xenorhabdus indica. Identification was based on 16S rDNA, recA, dnaN, gltX, gyrB and infB gene sequence analyses. The relatedness of strain 157-C to the type strain of X. indica (DSM 17 382) was confirmed with DNA-DNA hybridization. The phenotypic characteristics of strain 157-C are similar to those described for the type strain of X. indica. This is the first report associating X. indica with S. yirgalemense.

Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic association with the nematode Steinernema carpocapsae. The nematode invades and traverses the gut of susceptible insects. X. nematophila is released in the insect blood (hemolymph), where it suppresses host immune responses and functions as a pathogen. X. nematophila produces diverse antimicrobials in laboratory cultures. The natural competitors that X. nematophila encounters in the hemolymph and the role of antimicrobials in interspecies competition in the host are poorly understood. We show that gut microbes translocate into the hemolymph when the nematode penetrates the insect intestine. During natural infection, Staphylococcus saprophyticus was initially present and subsequently disappeared from the hemolymph, while Enterococcus faecalis proliferated. S. saprophyticus was sensitive to X. nematophila antibiotics and was eliminated from the hemolymph when coinjected with X. nematophila. In contrast, E. faecalis was relatively resistant to X. nematophila antibiotics. When injected by itself, E. faecalis persisted (~10(3) CFU/ml), but when coinjected with X. nematophila, it proliferated to ~10(9) CFU/ml. Injection of E. faecalis into the insect caused the upregulation of an insect antimicrobial peptide, while the transcript levels were suppressed when E. faecalis was coinjected with X. nematophila. Its relative antibiotic resistance together with suppression of the host immune system by X. nematophila may account for the growth of E. faecalis. At higher injected levels (10(6) CFU/insect), E. faecalis could kill insects, suggesting that it may contribute to virulence in an X. nematophila infection. These findings provide new insights into the competitive events that occur early in infection after S. carpocapsae invades the host hemocoel.

Extracellular novel metalloprotease from Xenorhabdus indica and its potential as an insecticidal agent.

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

Previously unrecognized stages of species-specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes.

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species-specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species-specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal-intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species-specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.

Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae.

Bacterial strain SF87(T), and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87(T) is shown to be closely related (98% similarity) to Xenorhabdus hominickii DSM 17903(T). Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96-97% similarity with Xenorhabdus miraniensis DSM 17902(T). However, strain SF87 shares only 52.7% DNA-DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87(T), SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87(T) ( =DSM 25463(T) =ATCC BAA-2406(T)).

Xenorhabdus ishibashii sp. nov., isolated from the entomopathogenic nematode Steinernema aciari.

Gram-negative bacteria of the genus Xenorhabdus exhibit a mutualistic association with steinernematid entomopathogenic nematodes and a pathogenic relationship with insects. Here we describe two isolates of the entomopathogenic nematode Steinernema aciari collected from China and Japan. 16S rRNA gene sequence similarity and phylogenetic analysis indicated that the isolates obtained from S. aciari belonged to the genus Xenorhabdus. Multilocus sequence analysis based on five universal protein-coding gene sequences revealed that the isolates were closely related to Xenorhabdus ehlersii DSM 16337(T) and Xenorhabdus griffiniae ID10(T) but that they exhibited <97 % sequence similarity with these reference strains, which indicated that the isolates were distinct from previously described species. Based on these genetic differences and several differential phenotypic traits, we propose that the isolates represent a novel species of the genus Xenorhabdus, for which we propose the name Xenorhabdus ishibashii sp. nov. The type strain is GDh7(T) ( = DSM 22670(T)  = CGMCC 1.9166(T)).