In 1965, the authors published a description of the bacterium Achromobacter nematophilus Poinar and Thomas, which was found in association with strain DD-136 of the nematode Neoaplectana carpocapsae Weiser. Further studies demonstrated the importance of A. nematophilus in the reproduction and development of this entomogenous nematode. Subsequently it was shown that other geographic populations of N. carpocapsae also formed associations with identical or related bacteria. More recent studies (G. 0. Poinar, Jr., G. M. Thomas, K. A. Nelson, and M. Haygood, Soil Biol. Biochem) have shown similar associations between bacteria and entomogenous nematodes in the genus Heterorhabditis. The proposed rejection of the genus Achromobacter by Heindrie et al.  in 1974 was accepted in Bergey's Manual of Determinative Bacteriology 8W ed,. Species described as belonging to the genus Achromobacter were transferred either to the genus Acaligenes or to other genera. Achromobacter nematophilus, however, could not be accommodated as a species either in the genus Alcaligenes or in any other recognized genus. Then in December 1975, the authors received specimens of the entomogenous nematode Heterorhabditis bacteriophora Poinar collected from Hetiothis punctigera Wall. in Brecon, Australia, by Dudley E. Pinnock. The nematodes were found to carry a specific bacterium (tentatively labeled strain Hb) in the lumen of the intestine. Studies  showed the association between the Hb bacterium and the nematode to be similar to that between A. nematophilus and the nematode N. carpocapsae Weiser. These studies also showed the bacterium to be bioluminescent. The luminescent properties were further studied (Poinar et al.), Although the cultural and biological characteristics of the bacterium were studied in detail, the organism remained unclassified. Both A. nematophilus and strain Hb were subsequently studied in order to determine their taxonomic positions. The results of this study are reported here.

ABSTRACT: A new genus, Xenorhabdus, is created to accommodate large, gram-negative, rod-shaped, facultatively anaerobic, entomopathogenic bacteria which are intimately associated with entomogenous nematodes. The normal habitat of these bacteria is the intestinal lumen of nematodes or the body cavity of host insects into which they have been introduced by the nematodes. The genus is placed in the family Enterobacteriaceae since the bacteria possess most of the important characteristics of. this family. Xenorhabdus differs from other genera of Enterobacteriaceae in large cell size, failure to reduce nitrates to nitrites, intimate association with entomogenous nematodes, entomopathogenesis, and immunological characteristics. The type species is Xenorhabdus nematophilus (Poinar and Thomas) comb. nov. (synonym: Achromobacter nematophilus Poinar and Thomas). Xenorhabdus luminescens sp. nov., a bioluminescent, entomopathogenic bacterium isolated from the intestinal lumen of an entomogenous nematode, Heterorhabditis bacteriophora, is also described. In addition to their immunological differences, the two species are dissimilar in that X. luminescens is positive for bioluminescence and catalase activity, whereas X. nematophilus is not. The type strain of X. nematophilus is ATCC 19061, and that of X. luminescens is strain Hb (= ATCC 29999).

Three genera of insect pathogenic nematodes are known to have specific symbiotic associations with bacteria: Neoaplectana spp. and Heterorhabditis spp. are associated with Xenorhabdus spp. (Thomas & Poinar, 1979) and Steinernema kraussei with a Flavobacterium sp. (Mrdcek, 1977). Each species of nematode is associated with a single bacterial species; all Heterorhabditis spp. examined have Xenorhabdus luminescens as the symbiont but each species of Neoaplectana is associated with a different species of bacterium (R. J. Akhurst, unpublished results). The bacterial symbiont is carried monoxenically in the intestine of the non-feeding infective stage of the nematode. The nematode penetrates an insect host and moves to the haemocoel where it voids the bacteria. The bacteria proliferate, kill the host and establish suitable conditions for reproduction of the nematodes by providing nutrients and inhibiting the growth of other bacteria (Poinar & Thomas, 1966). The symbiotic bacteria are also capable of rendering a wide variety of artificial media suitable for nematode reproduction, thus allowing the economical mass production of the nematodes (Bedding, 1976) necessary for the control of insect pests in the field During a study of the bacterial symbionts of many isolates of several species of Neoaplectana and Heterorhabditis from Australia, Europe, New Zealand and North America, I found that all may produce two forms of colony on agar media. The results of an investigation into the significance of and the relationships between the two forms of X. nematophilus, the symbiont of N. feltiae, are presented in this paper.

ABSTRACT: Xenorhabdus spp., entornopathogenic bacteria symbiotically associated with the nematodes Neoaplectana and Heterorhabditis, occur in two forms. In general, only one form, designated the primary form, is transmitted into new hosts by the infective stage of the nematode. The significance of the relationship between the two forms has been examined with X. nematophilus, the symbiont of N. feltiae. The forms of X. nematophilus can be differentiated by their colony characteristics but by only two biochemical tests. The two forms of X. nematophilus are equally pathogenic when injected into the haemocoel of Galleria larvae. However, the primary form when injected into Galleria larvae with axenic nematodes provides better conditions for reproduction of the nematodes than the secondary form, for which a role has not been determined. Although the primary form readily converts to the secondary form in vitro and occasionally in vivo, the secondary form is usually stable. Possible causes of the instability have been investigated.

Insect pathogenic nematodes of the Heterorhabditidae and Steinernematidae are symbiotically associated with bacteria of the genus Xenorhabdus; each species of nematode is associated with a single bacterial species (Thomas & Poinar, 1979 ~ Akhurst, 1982). The bacterial symbiont is carried monoxenically within the intestine of the non-feeding infective stage nematode. After entering the insect host, the infective nematode invades the haemocoel where it releases the bacteria. The bacteria proliferate, kill the insect and enhance conditions for reproduction of the nematodes by providing nutrients and inhibiting the growth of other bacteria (Poinar & Thomas, 1966). Xenorhabdus species produce two forms of colony on agar media (Akhurst, 1980). The two forms of the symbiont of Neoaplectana feltiae can be distinguished by some biochemical tests and differ in their ability to enhance nematode reproduction in vivo and in vitro. The form isolated from the infective nematode (the primary form) was unstable, producing the secondary form which could revert to the primary. No evidence for the presence of bacteriophage or plasmid causing the differences between the forms could be detected. Dutky (1964) suggested that the bacterial symbiont of the DD 136 strain of N. feltiae (=carpocapsae) produced a wide spectrum antibiotic. Poinar et at. (1980) found that both Xenorhabdus nematophilus and X. luminescens inhibited Bacillus cereus subsp. mycoides and B. subtilis. They also reported the presence of defective bacteriophage in X. nematophilus and X. luminescens cultures and suggested that the defective phage was the bacterial agent. This paper presents the results of a study of the range activity and of some properties of the antibiotic produced by Xenorhabdus spp.

ABSTRACT: 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.
 

ABSTRACT: The taxonomy of the bacteria symbiotically associated with the insect-pathogenic nematodes Neoaplectana and Heterorhabditis was examined. The bacteria studied were isolated from 33 populations obtained from Australasia, Europe, and the United States. The symbionts of all species of Neoaplectana and Heterorhabditis examined were members of the genus Xenorhabdus, but they differed in several respects from the description of the genus Xenorhabdits, including the guanine-plus-cytosine content of the deox yri bone u cleic acid and the production of acid from carbohydrates. All bacteria isolated from Heterorhabditis spp. were identified as members of Xenorhabdus luminescens. The bacteria isolated from one Neoaplectana species were similar and were distinguishable from the bacteria isolated from other Neoaplectana species. The following three subspecies of Xenorhabdus nematophilus are proposed: Xenorhabdus nematophilus subsp. nematophilus (bacteria symbiotic with Neoaplectana feltiae (= Neoaplectana carpocapsae); type strain ATCC 19061), Xenorhabdus nematophilus subsp. bovienii (bacteria symbiotic with Neoaplectana bibionis; type strain, UQN1 2210T), and Xenorhabdus nematophilus subsp. poinarii (bacteria symbiotic with Neoaplectana glaseri; type strain, UQM 2216). These subspecies vary in host nematode, pigmentation, maximum temperature for growth, responses to tests for phenylalanine deaminase by secondary-form isolates and for lipase (Tween 80 test) and lecithinase by primary-form isolates, and coloration of primary-form isolates on MacConkey agar and media containing brornthymol blue.

Xenorhabdus spp. are bacteria mutualistically associated with entomopathogenic nematodes of the families Steinernematidae and Heterorhabditidae (Thomas & Poinar, 1979; Akhurst, 1983). The bacteria are pathogenic for the insect host when released into the haemolymph by the nematodes. They support nematode reproduction by producing nutrients and antimicrobial agents that inhibit the growth of a wide range of organisms (Poinar & Thomas, 1966; Akhurst, 1982). All Xenorhabdus isolates have been shown to produce two colony forms when cultured in vitro. These colony forms are indicative of two phases that differ in some biochemical characteristics (Akhurst 1980, 1986; Boemare & Akhurst, 1988). Field-collected infective-stage nematodes carry only primary-phase Xenorhabdus in their intestine. This phase is unstable in vitro, and occasionally in vivo, producing the secondary phase; this may also be unstable, reverting to the primary phase. There has been some difficulty in describing the taxonomic characteristics of Xenorhabdus, with various workers presenting conflicting data (Poinar et al., 1971 ; Thomas & Poinar, 1979, 1983; Akhurst, 1982, 1983; Boemare, 1983; Farmer, 1984; Grimont et al., 1984). Boemare & Akhurst (1988) suggested that one of the reasons for the disparity in results may have been the phase variation. They tested both phases of Xenorhabdus isolated from 21 strains (13 species) of Steinernematidae and Heterorhabditidae for 240 characters to examine the consequence of phase variation on the taxonomy of Xenorhabdus. This paper reports numerical analyses of the data of Boemare & Akhurst (1988). The analyses were undertaken to examine more closely the significance of phase variation for the taxonomy of Xenorhabdus and to evaluate taxonomic relationships within the genus.

ABSTRACT: Data from a study of both hases of 21 strains of Xenorhabdus examined for 240 characters were subjected to numerical analysis. Only 60 characters were used for the analyses, since 169 characters were common to all isolates, and the acidification data essentially duplicated the assimilation tests. The data were arranged in seven ways to determine the significance of characters affected by phase change and of weak responses. Most of the analyses involved calculation of similarities by the Jaccard coefficient and clustering by single linkage, complete linkage and centroid sorting algorithms. The resultant dendrograms emphasized the importance of recognizing phase-related characteristics in examining the taxonomy of Xenorhabdus. They also demonstrated a close correspondence between the taxonomic groupings of Xenorhabdus and those of their nematode associates. It is proposed that the subspecies of X. nematophilus be elevated to species, X. netnatophilus, X. bovienii, X. poinard and X. beddingii.

Xenorhabdus spp. (members of the family Enterobacteriaceae) are symbiotically associated with entornopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. The nematodes act as vectors, transporting their bacterial symbionts; into the hemocoel of the insect host. Xenorhabdus spp. contribute to the symbiotic relationship by providing nutritional requirements for their nematode partners. Xenorhabdus strains spontaneously produce two colony form variants  which have been called phase variants. Phase 1 variants adsorb dyes on agar plates, produce lecithinase, emit light (in bioluminescent strains only  and have cytoplasmic paracrystalline inclusions, while these properties are either absent or greatly reduced in phase 2 . Phase 1 variants also produce nonprotein agardiffusible antibiotics that minimize the secondary invasion of the insect cadaver by other microorganisms, while phase 2 variants produce no such compounds. Some of these antibiotics have been identified as indole derivatives, trans-stilbenes, xenorhabdins, and xenocoumacins. Antibiotic activity has also been attributed to elements like the phage tails that were detected in Xenorhabdus luminescens. These elements, interpreted as virulent for Bacillus cereus, were designated defective bacteriophages. Subsequently, a bacteriophage that was lytic for phase 1 but not for phase 2 of X luminescens was reported. It has been hypothesized that this phage was the cause of phase variation in Xenorhabdus spp.. However, the lytic activity of this phage occurred without any induction, indicating that the phage may have originated from another host. To test the hypothesis that lysogenic phages; were present in Xenorhabdus spp., we attempted to induce lysis by several treatments and determine the nature of the elements causing lysis.

ABSTRACT: 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 fragmeats 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 lurninescens 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. nernatophilus.

The genus Xenorhabdus (Enterobacteriaceae) consists almost entirely of the bacterial symbionts of entomopatho ... genic nematodes belonging to the families Steinernematidae and Heterorhabditidae . The bacterial symbionts ... are carried monoxenically in a special vesicle in the infective stage (L3 juveniles) of members of the Steinernematidae and throughout the whole intestine of infective juveniles Heterorhabditidae, which provide protection and trans port for their bacterial symbionts. The nematodes also provide protection for their symbionts against the host's immune response; during the early stage of the infection, L4 and adult steinernernatids produce an inhibitor of the insect inducible immune protein. Both nematodes and Xenorhabdus spp. are pathogenic for most insects when they  are released into the hemolymph. The bacterial symbionts also contribute to the symbiotic relationship by establishing and maintaining suitable conditions for nematode reproduction, providing nutrients and antimicrobial substances that inhibit the growth of a wide range of micro organisms . Phase variants of 21 strains of Xenorhabdus spp. from 14 nematode species isolated from various parts of the world were characterized biochemically and physiologically, and the data were analyzed by numerical taxonomy. On the basis of this evidence, the elevation of four subspecies of Xenorhabdus nematophilus to species status as X. nematophilus, Xenorhabdus bovienii, Xenorhabdus poinarii, and Xenorhabdus beddingii was proposed, and an emended description of the species Xenorhabdus luminescens was given.

The first genotypic study of Xenorhabdus spp. was an investigation of DNA relatedness . This demonstrated that there were substantial differences among X. nematophilus, X. luminescens, and a third group that clustered about what is now known as X. bovienii. Furthermore, the X. luminescens group was shown to contain four DNA relatedness groups. Farmer et al. subsequently identified a fifth DNA group in X luminescens when they examined some isolates taken from clinical specimens. None of the DNA relatedness groups was described as a separate species because too few strains had been There were some apparent anomalies in the distribution of studied X. luminescens into DNA relatedness groups. The Type Culture Collection type strain of X. luminescens was American assigned to a different DNA relatedness group than clones apparently isolated from the same nematode species. More over, two clones obtained from strain 18 were also assigned to different groups. Grimont et al.  suggested that dimorphism detected in Xenorhabdus spp.  might be explained by some strains being "mixed cultures containing two different luminous Xenorhabdus species." We undertook a study of DNA-DNA relatedness in
Xenorhabdus spp. to test the hypothesis of Grimont et al. (18)concerning dimorphism by using defined phase one and phase two cultures. We also sought to test and possibly extend the taxonomic scheme for Xenorhabdus spp. described by Akhurst and Boemare .

ABSTRACT: The levels of DNA relatedness for a broad sample of Xenorhabdus strains isolated from different species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) and from different geographical sources were estimated by the hydroxyapatite method. The level of DNA-DNA relatedness for the two phases of each isolate tested was not significantly different from 100%, demonstrating unequivocally that the phase variation demonstrated by all Xenorhabdus spp. is not due to contamination. The isolates of the described Xenorhabdus species coalesced into different DNA relatedness groups, confirming that Xenorhabdus nematophilus, Xenorhabdus bovienii, Xenorhabdus poinarii, and Xenorhabdus beddingii, defined on the basis of phenotypic differences, are valid species. The symbiont of Steinernema intermedia also coalesced with the X. bovienii isolates. This was the only symbiont of seven recently described and unamed Steinernema spp. (including Steinernema ritteri, Steinernema rara, and Steinernema anomali) that formed a group with any of the previously described Xenorhabdus species; new species descriptions are required to accommodate the other taxa, but too few isolates were available to allow satisfactory descriptions of them. The DNA relatedness data also showed that the bacteria currently classified as Xenorhabdus luminescens are significantly different from all other Xenorhabdus strains. These data strongly support indications from previous studies of phenotypic characteristics, cellular fatty acids, and DNA relatedness that X. luminescens should be classified as a separate genus. A new genus, Photorhabdus, with an amended description of the type species, Photorhabdus luminescens, is proposed.

The genus Xenorhabdus consists of specific bacterial symbionts of the entornopathogenic nematodes Steinernemat idae, and the genus Photorhabdus consists almost entirely of those of the entornopathogenic nematodes Heterorhabditidae. They are both members of the Enterobacteriaceae. These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, inducing a lethal septiceamia. Moreover, the two bacterial genera contribute to the symbiotic relationship by providing nutritional requirements for their nematode partners during their reproduction in the insect cadavers. Most of the Xenorhabdus strains are lysogenic. They produce few enterphages and a lot of bacteriocins after induction by mitomycin C or high temperature treatments . Consequently, lysogeny was concomitant with an important bacteriocinogeny (as the ability to produce bacteriocins after an induction treatment). They were bactericidal against other species. The term 'bacterio cin' was coined by Jacob et al. for antibacterial agents which were synthetized by bacteria and required specific receptors. Among several types, one group includes phage tail-like particles consisting of contractile tails also called particulate bacteriocins, lethal phages or defective phages . Bacteriocins evidenced in Xenorhabdus cul tures belong to this category. They have a high molecular mass, sediment easily and appear as phage tail-like particles in the electron microscope as do numbers of other described contractile tails. Electronic examination of the purified suspensions shows bacteriocins with extended and contracted sheaths.
A natural occurrence of a bacteriocinogeny was also revealed by examination of cultures in stationary growth of Photorhabdus strains where few bacteriocins were neighbouring many healthy bacterial cells. We failed to show a total lysis with more bacteriocins than the natural occurrence in supernatants of cultures treated by heating, UV irradiation or H2O2. Paracrystalline inclusions have been reported in all the cells after logarithmic growth of Xenorhabdus and Photorhabdus cultures in bacteriological media without any additive compounds, such as nutrient broth or nutrient agar, in rich media, such as tryptic soy agar and yolk nutritive agar, or during growth in the hemolymph of insects. They can be observed directly with an optical micro scope by examination of fresh suspensions giving an important refringence within bacilli, or by electron microscopy examination showing the paracrystalline weft responsible for a refractive effect in optical transmission They are produced during the stationary growth of the cultures in the protoplasm of the cells of both genera in many
quantities (often more than one per cell) without any de generative consequence. Their purification allowed testing of possible toxicity against insect, mammalian and insect cell cultures, an antibiotic action or a pharmaco logical effect, but to date no biochemical or physio logical role was attributed to these proteinic crystals. Moreover, in natural conditions other ultrastructural elements were described in Photorhabdus cells as 'lattice structures'. Due to their ultrastructural similarities, they were interpreted as 'photosomes', ie structures devoted to the production of light which is one of the most relevent properties of these bacteria. But later it was noticed that the scarcity of these lattice structure in a small proportion of the cells of a luminous strain of Photorhabdus sp. did not necessarily indicate that they were involved in bioluminescence.
The aim of the present study was to clarify the ultra structural features observed in the past in the protoplasm of Xenorhabdus and Photorhabdus and to distinguish which are the structures which can be related to the recently shown bacteriocinogeny. Firstly we attempted to show  how the genesis of bacteriocins (bacteriocinogenesis) occurs in the cells after a chemical induction of the Xenorhabdus cultures. In order to recognize bacteriocins in infected cells, bacteriocin purified suspensions were prepared from the same induced cultures to examine their ultrastructure and to analyze them by image processing. Secondly, the natural bacteriocinogenesis occuring in some cells of Photorhabdus cultures, without any chemical induction, was examined and compared with knowledge previously obtained from Xenorhabdus studies.

ABSTRACT: Xenorhabdus nematophilus F1 strain and Photorhabdus luminescens NC19 strain produced bacteriocins after mitomycin C treatment and under natural conditions respectively. The ultrastructure of these two strains was described and compared to the ultrastructure of untreated or normal cells. After image processing of purified bacteriocins we found morphological homology in infected cells with protoplasmic rods in longitudinal section and hexagonal aggregates in transversal section. We concluded that these particular structures, so-called 'lattice structures' and previously interpreted as 'photosomes', are in fact the early stages of in situ production of bacteriocins in these two bacterial genera. Natural occurrence of Photorhabdus spp bacteriocinogenesis was observed in other strains, while other lysogenic strains of Xenorhabdus spp are lysed after a mitomycin C treatment.

ABSTRACT:  The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes (Steinernema spp. and Heterorhabditis spp.) was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). 117 strains were studied, most of which were isolated from the Caribbean basin after exhaustive soil sampling.
The collection consisted of 77 isolates recovered from entomopathogenic  nematodes on 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. 30 distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.

                             ABSTRACT: 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 C-18 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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