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Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov.

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342T, isolated from Steinernema bicornutam; Xenorhabdus ehlersii sp. nov., type strain DSM 16337T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338T, isolated from Steinernema rarum.

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A novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus indica sp. nov., symbiotically associated with entomopathogenic nematode Steinernema thermophilum Ganguly and Singh, 2000.

In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28(T) = DSM 17382(T), strain 42 = DSM 17383 and strain 43 = DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28(T) and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28(T) were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28(T), DSM 17382(T), CIP 108830(T).

Animals↗

Lysogeny and bacteriocinogeny in Xenorhabdus nematophilus and other Xenorhabdus spp.

Induction by mitomycin or high-temperature treatment resulted in the production of bacteriocins and phages in both phases of Xenorhabdus nematophilus A24, indicating lysogeny. Phage DNA purified from X. nematophilus A24 hybridized to several fragments of DraI-digested A24 chromosomal DNA, confirming that the phage genome was incorporated into the bacterial chromosome. Bacteriocins and phages were detected in cultures of most other Xenorhabdus spp. after mitomycin or high-temperature treatment. Xenorhabdus luminescens K80 was not lysed by these treatments, and no phages were seen associated with this strain. However, bacteriocins were detected in limited quantities in all Xenorhabdus cultures, including X. luminescens K80, without any induction. X. nematophilus A24 bacteriocins were antagonistic for other Xenorhabdus species but not for A24 or other strains of X. nematophilus.

Bacteriocins↗

Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae.

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Umeå), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Umeå (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.

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New insight into diversity in the genus Xenorhabdus, including the description of ten novel species.

We investigated the diversity of a collection of 76 Xenorhabdus strains, isolated from at least 27 species of Steinernema nematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of the Xenorhabdus strains were highly conserved (similarity coefficient >95 %), suggesting that the common ancestor of the genus probably emerged between 250 and 500 million years ago. Based on comparisons of the 16S rRNA gene sequences, we identified 13 groups and seven unique sequences. This classification was confirmed by analysis of molecular typing profiles of the strains, leading to the classification of new isolates into the Xenorhabdus species described previously and the description of ten novel Xenorhabdus species: Xenorhabdus cabanillasii sp. nov. (type strain USTX62(T)=CIP 109066(T)=DSM 17905(T)), Xenorhabdus doucetiae sp. nov. (type strain FRM16(T)=CIP 109074(T)=DSM 17909(T)), Xenorhabdus griffiniae sp. nov. (type strain ID10(T)=CIP 109073(T)=DSM 17911(T)), Xenorhabdus hominickii sp. nov. (type strain KE01(T)=CIP 109072(T)=DSM 17903(T)), Xenorhabdus koppenhoeferi sp. nov. (type strain USNJ01(T)=CIP 109199(T)=DSM 18168(T)), Xenorhabdus kozodoii sp. nov. (type strain SaV(T)=CIP 109068(T)=DSM 17907(T)), Xenorhabdus mauleonii sp. nov. (type strain VC01(T)=CIP 109075(T)=DSM 17908(T)), Xenorhabdus miraniensis sp. nov. (type strain Q1(T)=CIP 109069(T)=DSM 17902(T)), Xenorhabdus romanii sp. nov. (type strain PR06-A(T)=CIP 109070(T)=DSM 17910(T)) and Xenorhabdus stockiae sp. nov. (type strain TH01(T)=CIP 109067(T)=DSM 17904(T)). The Xenorhabdus strains studied here had very similar phenotypic patterns, but phenotypic features nonetheless differentiated the following species: X. bovienii, X. cabanillasii, X. hominickii, X. kozodoii, X. nematophila, X. poinarii and X. szentirmaii. Based on phenotypic analysis, we identified two major groups of strains. Phenotypic group G(A) comprised strains able to grow at temperatures of 35-42 degrees C, whereas phenotypic group G(B) comprised strains that grew at temperatures below 35 degrees C, suggesting that some Xenorhabdus species may be adapted to tropical or temperate regions and/or influenced by the growth and development temperature of their nematode host.

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Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae.

A wide range of micro-organisms, including yeasts, was found to be inhibited by the primary form of Xenorhabdus spp., but not by the secondary form. Only one Xenorhabdus strain, the symbiont of Neoaplectana glaseri, did not inhibit any of the micro-organisms tested; it is suggested that this strain may not have been isolated in the primary form. Gram-positive bacteria were sensitive to all active isolates of Xenorhabdus; each of the yeasts and almost all of the Gram-negative bacteria were sensitive to some but not all Xenorhabdus isolates. Each Xenorhabdus isolate was sensitive to some other Xenorhabdus isolates. The antibiotic activity of X. nematophilus was unaffected by autoclaving but was lost after dialysis. Anaerobically incubated Xenorhabdus spp. did not exhibit antibiotic activity.

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Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus).

BACKGROUND: Symbioses between invertebrates and prokaryotes are biological systems of particular interest in order to study the evolution of mutualism. The symbioses between the entomopathogenic nematodes Steinernema and their bacterial symbiont Xenorhabdus are very tractable model systems. Previous studies demonstrated (i) a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and (ii) that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. The goal of the present study was to address the question of the impact of Xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different Steinernema species. For this, we monitored experimental interspecific competition between (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an experimental insect-host (Galleria mellonella). Three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs (i.e. naturally associated with their symbionts) of both species. RESULTS: We found that both the progression and the outcome of interspecific competition between entomopathogenic nematodes were influenced by their bacterial symbionts. Thus, the results obtained with aposymbiotic nematodes were totally opposite to those obtained with symbiotic nematodes. Moreover, the experimental introduction of different ratios of Xenorhabdus symbionts in the insect-host during competition between Steinernema modified the proportion of each species in the adults and in the global offspring. CONCLUSION: We showed that Xenorhabdus symbionts modified the competition between their Steinernema associates. This suggests that Xenorhabdus not only provides Steinernema with access to food sources but also furnishes new abilities to deal with biotic parameters such as competitors.

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Specialization of the entomopathogenic nematode Steinernema scapterisci with its mutualistic Xenorhabdus symbiont.

The level of specialization of the entomopathogenic nematode Steinernema scapterisci with its native Xenorhabdus symbiont was investigated by testing (1) the influence of non-native bacterial strains on nematode fitness within an insect-host (Galleria mellonella) and (2) specificity of the association between the nematode infective juveniles and non-native bacteria. All non-native Xenorhabdus spp. or Photorhabdus spp. strains tested were mutualistically associated with other entomopathogenic nematodes in nature. We showed that most of the Xenorhabdus spp. strains tested led to an insignificant difference of the nematode's fitness compared to the one obtained with the native bacterium. Conversely, Photorhabdus spp. strains almost entirely abolished nematode reproduction. The phylogenetic analysis of bacterial strains tested, showed that there was a negative correlation between S. scapterisci's reproduction rate with a bacterial strain and the genetic distance of this bacterial strain from the native one. We also showed that the native bacterium was the only one which was transmitted by S. scapterisci's infective juveniles. All these results, suggested a specialization between S. scapterisci and its native Xenorhabdus. As the same phenomenon was already demonstrated in the association between S. carpocapsae and X. nematophila, specialization between partners would not be an exception in entomopathogenic nematode-bacteria interactions. Nevertheless, S. scapterisci showed a dramatically higher compatibility with non-native Xenorhabdus spp. strains than did S. carpocapsae, suggesting differences in the co-evolutionary processes between nematodes and bacteria in these two model systems.

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Isolation and entomotoxic properties of the Xenorhabdus nematophilus F1 lecithinase.

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C18 reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.

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Effect of bacterial symbionts Xenorhabdus on mortality of infective juveniles of two Steinernema species.

Steinernema species are entomopathogenic nematodes associated with Xenorhabdus bacteria. The life cycle of these associations is composed of two stages: (1) a free stage in the soil, where infective juveniles (IJs), which carry bacteria in their guts, search for new insect hosts; and (2) a parasitic stage, where the IJs infect insects, release their Xenorhabdus symbionts and reproduce in order to produce new IJs. Previous studies clearly showed benefits to the association for several Steinernema species during the parasitic stage. Nevertheless, no study has so far explored, during the free stage, the existence of costs or benefits to the association for different Steinernema. Here, we compared the survival of both symbiotic and aposymbiotic IJs in two nematode species: (1) Steinernema carpocapsae-exhibiting IJs that carry a high number of Xenorhabdus cells in their guts; and (2) its closely relative species, S. scapterisci-exhibiting IJs, that carry very few Xenorhabdus cells in their guts. We showed that the bacterial symbionts were costly for S. carpocapsae by increasing IJs' mortality but not for S. scapterisci. This difference in cost induced by bacteria to IJs during the free stage could be correlated with the difference in the numbers of bacteria carried by IJs of each nematode species.

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Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode, Steinernema riobrave (Rhabditida: Steinernematidae).

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.

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Growth-mediated variations in fatty acids of Xenorhabdus sp.

The bacterium Xenorhabdus sp. is symbiotically associated with the entomopathogenic nematode Steinernema riobravis. This nematode is produced in monoxenic culture with Xenorhabdus sp. and is sold as a biological insecticide. Acceptable yields in fermentors can only be achieved in the presence of vigorous growth of the bacterium. We investigated the fatty acid composition of Xenorhabdus species when grown at 15, 20, 25 or 30 degrees C on media containing one of two primary carbon sources: glucose or lipids from the insect host, Galleria mellonella. Both temperature and primary carbon source significantly affected lipid quantity and quality in Xenorhabdus sp. Bacteria grown with insect lipids as a primary carbon source accumulated more lipids with greater proportion of longer chain fatty acids than bacteria grown with glucose as a primary carbon source. Cells grown with insect lipids at 15 degrees C had a lower lipid content than cells grown on the same media at 20, 25 or 30 degrees C. Increasing growth temperature increased saturated fatty acids and decreased unsaturated fatty acids, irrespective of carbon source. We recommend addition of complex fatty acid sources that resemble natural host lipids to growth medium for mass producing entomopathogenic nematodes. This could provide nematode quality similar to in vivo-produced nematodes.

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Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts.

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

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The effect of temperature on the fatty acids and isozymes of a psychrotrophic and two mesophilic species of Xenorhabdus, a bacterial symbiont of entomopathogenic nematodes.

In the first part of this study, generation times relative to temperature, together with cardinal and conceptual temperatures, were determined for four strains of Xenorhabdus bacteria that represented three geographically distinct species. The data showed that the NF strain of Xenorhabdus bovienii, like the Umeå strain of the same species, is psychrotrophic, while Xenorhabdus sp. TX strain resembles Xenorhabdus nematophila All strain in being mesophilic. In the second part, the capacity of these bacteria to adapt to changes in temperature, shown by changes in fatty acid composition, was investigated. As temperature declined, the proportions of the two major unsaturated fatty acids, palmitoleic (16:1omega7) acid and oleic (18:1omega9) acid, increased significantly in all of the strains. The proportion of the prevalent saturated fatty acid, which was palmitic acid (16:0), decreased. In the All, NF, and Umeå strains, myristic acid (14:0), margaric acid (17:0), cyclopropane (17:0c), and arachidic acid (20:0) decreased with decreasing temperature. In the third part of the study, the synthesis of isozymes in response to changing temperature was investigated. For the seven enzymes studied, the numbers for which isozyme synthesis was temperature related were as follows: five for Umeå, four for All, three for NF, and two for TX. Where the study dealt with fatty acid composition and isozyme synthesis, the results show a broad capacity for physiological temperature adaptation among strains of different climatic origin.

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Phylogenetic relationships of entomopathogenic nematophilic bacteria: Xenorhabdus spp. and Photorhabdus sp.

Phylogenetic relationships of Xenorhabdus spp. and Photorhabdus sp. were investigated on the basis of 16S rRNA gene sequences. Xenorhabdus spp. and Photorhabdus sp. were grouped together with Proteus vulgaris and Arsenophonus nasoniae. This group was distant from other members of the family Enterobacteriaceae. Xenorhabdus japonicus, previously proposed as a new species, was nearly located to Xenorhabdus nematophilus. Signature nucleotides of X. japonicus were identified that distinguish it other members of the family Enterobacteriaceae.

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Neoaplectana species: specificity of association with bacteria of the genus Xenorhabdus.

Each of five Neoaplectana (Nematoda: Steinernematidae) species was cultured monoxenically with various Xenorhabdus (Eubacteriales: Enterobacteriaceae) isolates. The nematodes were usually able to reproduce when cultured with the bacterial symbiont of any one of the five Neoaplectana spp. but never with Xenorhabdus luminescens, symbiotic with Heterorhabditis spp., or with the Xenorhabdus sp. isolated from an undescribed steinernematid species. Only Neoaplectana bibionis could be cultured with the Xenorhabdus symbiont of Steinernema kraussei. A high proportion of infectives were able to carry within their intestine X. nematophilus isolated from other strains of the same nematode species; a small proportion of infectives were able to carry X. nematophilus isolated from other nematode species.

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Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus: phase variation leads to differential transcription of two flagellar genes (fliCD).

The insect-pathogenic bacterium Xenorhabdus undergoes spontaneous phase variation involving a large number of phenotypes. Our previous study indicated that phase I variants were motile, whereas phase II variants of X. nematophilus F1 were nonflagellated cells which did not synthesize flagellin [Givaudan A., Baghdiguian, S., Lanois, A. and Boemare, N. (1995) Appl. Environ. Microbiol. 61, 1408-1413]. In order to approach the study of the flagellar switching, a locus containing two ORFs from X. nematophilus F1 (phase I) was identified by using functional complementation of flagellin-negative E. coli. The sequence analysis revealed that the first ORF corresponds to the fliC gene coding for flagellin, and showed a high degree of homology between the N-terminal and C-terminal of Xenorhabdus FliC and flagellins from other bacteria. The second identified ORF in the opposite orientation encodes a homologue of the enterobacterial hook-associated protein 2, FliD. Both Xenorhabdus fliCD genes were required for the entire restoration of E. coli motility. A sequence highly homologous to the sigma 28 consensus promoter was identified upstream from the coding sequences from both genes. The structure of the fliC gene and its surrounding region was shown to be the same in both phase variants, but Northern blot analysis revealed that fliC and fliD were, respectively, not and weakly transcribed in phase II variants. In addition, complementation experiments showed that motility and flagellin synthesis of phase II cannot be recovered by placing in trans fliCD genes from phase I. These latter results suggest that a gene(s) higher in the transcriptional hierarchy of the flagellar regulon is switched off in Xenorhabdus phase II variants.

Amino Acid Sequence↗

Development and application of oligonucleotide probes for molecular identification of Xenorhabdus species.

Synthetic deoxyoligonucleotide probes that hybridized against the region at positions 455 through 480 of 16S rRNA were developed for the identification of all five Xenorhabdus species. Sequence variation in the respective rRNA region between two strains of Xenorhabdus luminescens in addition allowed the construction of two strain-specific probes. Of 27 isolates determined to be Xenorhabdus strains by phenotypic characterization, 24 could be assigned to four of the five species. Two strains (HL-1 and HL-2) isolated from a Heterorhabditis sp. and a single strain (D-1.1) isolated from Steinernema affinis showed no hybridization signal with any of the five species-specific probes. With regard to the available species descriptions of nematodes, the results presented here confirm that, except for Steinernema affinis, the individual nematode hosts harbor only a single Xenorhabdus species.

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