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 Ume? 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 Ume? 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 Ume?, 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.     

Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes

Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.     

Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.     

Comparative analysis of P2-type remnant prophage loci in Xenorhabdus bovienii and Xenorhabdus nematophila required for xenorhabdicin production

The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor. Both xnp1 and xbp1 lack typical P2-type lysis genes but contain a predicted endolysin gene (enp) that may be involved in cell lysis. The main tail fibers of xnp1 and xbp1 are mosaic structures with divergent C-terminal regions suggesting they differ in host specificity. Several genes encoding C-terminal tail fiber fragments are present in the same position in xnp1 and xbp1. Recombination between the main fiber genes and the C-terminal fragments could potentially expand the host range specificity of xenorhabdicin in the respective strains.     

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).     

Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Xenorhabdus japonicus sp. nov. associated with the nematode Steinernema kushidai

A new species, Xenorhabdus japonicus, is proposed as the bacterial symbiont of Steinernema kushidai isolated from field soil in Shizuoka Prefecture, Japan. Xenorhabdus japonicus could be distinguished phenotypically and genetically from other Xenorhabdus spp. The type strain of the species, SK-1, a Gram-negative, facultative anaerobe and peritrichously flagellated rod, has colonies with primary and secondary forms. The strain can be differentiated from the type strain of Xenorhabdus nematophilus by several characters, including the formation of arginine dehydrolase, phenylalanine deaminase and lysine decarboxylase, the assimilation of inosine and L-proline and acid production from inositol. The major cellular fatty acids are 16:0, cyclo 17:0 and 18:1. The ubiquinone system is Q-8. The G+C content of DNA is 45.9 mol%. The DNA of strain SK-1 has 20 to 58% homology with that of the type strains of other Xenorhabdus spp.Y. Nishimura, A. Hagiwara and T. Suzuki are with the Department of Applied Biological Science, Science University of Tokyo, Noda 278, Japan, and SDS Biotech K.K., Tsukuba Technology Centre, Tsukuba 300-26, Japan     

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.     

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.     

A numerical taxonomic study of the genus Xenorhabdus (Enterobacteriaceae) and proposed elevation of the subspecies of X. nematophilus to species

Data from a study of both phases 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. nematophilus, X. bovienii, X. poinarii and X. beddingii.     

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.     

Whole cell fatty acid patterns of Xenorhabdus species

Thirty-three strains of the nematode-associated bacterium Xenorhabdus were characterized by traditional biochemical tests and whole cell fatty acid analysis. In traditional tests 26 strains were found to belong to X. luminescens and 7 to X. nematophilus (sensu latu). No further subdivision could be made. In fatty acid analysis, however, X. luminescens strains could be divided into three subgroups. The amount of distinction in fatty acids is similar to that at subspecies or species level found in other bacteria. Xenorhabdus nematophilus could be clearly differentiated from X. luminescens , key acids are 12: 0, 15: 0 iso, 16: 0, 17: 0 iso, 17: 0 cyclo, 18: 1 cis 11 and 19: 0 cyclo. Separation is almost at genus level. The presence of branched and hydroxy acids in Xenorhabdus and its aberrant morphology make the placement of this genus in the Enterobacteriaceae questionable. This is the first report on fatty acid profiles of Xenorhabdus species.     

Tn5-induced Xenorhabdus bovienii lecithinase mutants demonstrate reduced virulence for Galleria mellonella larvae

A derivative of Tn5 was used to construct a variety of stable insertion mutations in the entomopathogenic bacterium, Xenorhabdus bovienii T228/1. Mutants which had altered expression of Congo Red binding ability, ampicillin resistance, haemolytic activity and lecithinase were isolated. Isolates with altered lecithinase activity had either lost ability to produce this enzyme or showed reduced expression. The role of lecithinase in pathogenesis of X. bovienii T228/1 for Galleria mellonella larvae was examined by LD₅₀ analysis. Maximum killing of G. mellonella was observed at 72 h post infection for both the wild-type parent strain and a lecithinase mutant 34(45). However, the LD₅₀ value for the wild-type parent strain (8·7 cells per larva) was significantly less than that calculated for the lecithinase mutant (35·5 cells per larva). The data suggested that although lecithinase is a virulence factor produced by X. bovienii , lecithinase activity alone is not sufficient for killing of G. mellonella larvae.     

Analysis of the PixA inclusion body protein of Xenorhabdus nematophila

The symbiotic pathogenic bacterium Xenorhabdus nematophila produces two distinct intracellular inclusion bodies. The pixA gene, which encodes the 185-residue methionine-rich PixA inclusion body protein, was analyzed in the present study. The pixA gene was optimally expressed under stationary-phase conditions but its expression did not require RpoS. Analysis of a pixA mutant strain showed that PixA was not required for virulence towards the insect host or for colonization of or survival within the nematode host, and was not essential for nematode reproduction. The pixA gene was not present in the genome of Xenorhabdus bovienii, which also produces proteinaceous inclusions, indicating that PixA is specifically produced in X. nematophila.     

Plasmids and phase variation in Xenorhabdus spp.

Three strains of Xenorhabdus nematophilus (A24, F1, NC116) and strain Dan of Xenorhabdus bovienii were tested to evaluate whether the phase variation observed in these bacteria was in any way connected with plasmids. The plasmid patterns of both phases of A24 and F1 strains were the same, whereas the two NC116 phases had only one band each. No difference was observed between the undigested or digested plasmid patterns of the two phases from the three strains. No plasmid was detected in either phase of strain Dan. The plasmid probes were prepared from the six bands of A24 phase 1. By hybridization studies, three plasmids in two forms (open circular and supercoiled) were detected in the strain A24. Two were estimated at 12 kb, and the smallest was about 4 kb. Attempts to hybridize plasmid probes with either undigested or digested chromosomal DNA of the two phases of strain A24 were unsuccessful. The results suggest that neither a difference in plasmid content nor a plasmid recombination with the chromosome is involved in phase variation. The hybridizations revealed homologous DNA sequences among the three plasmids of strain A24 and among the plasmids of strains such as A24 and NC116, which were isolated from geographically distant countries, suggesting that plasmids may encode similar proteins.     

Plasmids and phase variation in Xenorhabdus spp.

Three strains of Xenorhabdus nematophilus (A24, F1, NC116) and strain Dan of Xenorhabdus bovienii were tested to evaluate whether the phase variation observed in these bacteria was in any way connected with plasmids. The plasmid patterns of both phases of A24 and F1 strains were the same, whereas the two NC116 phases had only one band each. No difference was observed between the undigested or digested plasmid patterns of the two phases from the three strains. No plasmid was detected in either phase of strain Dan. The plasmid probes were prepared from the six bands of A24 phase 1. By hybridization studies, three plasmids in two forms (open circular and supercoiled) were detected in the strain A24. Two were estimated at 12 kb, and the smallest was about 4 kb. Attempts to hybridize plasmid probes with either undigested or digested chromosomal DNA of the two phases of strain A24 were unsuccessful. The results suggest that neither a difference in plasmid content nor a plasmid recombination with the chromosome is involved in phase variation. The hybridizations revealed homologous DNA sequences among the three plasmids of strain A24 and among the plasmids of strains such as A24 and NC116, which were isolated from geographically distant countries, suggesting that plasmids may encode similar proteins.     

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.     

The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes

Members of the Steinernema genus of nematodes are colonized mutualistically by members of the Xenorhabdus genus of bacteria. In nature, Steinernema carpocapsae nematodes are always found in association with Xenorhabdus nematophila bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific. X. nematophila requires the nilA, nilB, and nilC genes to colonize S. carpocapsae. In this work, we showed that of all the Xenorhabdus species examined, only X. nematophila has the nilA, nilB, and nilC genes. By exposing S. carpocapsae to other Xenorhabdus spp., we established that only X. nematophila is able to colonize S. carpocapsae; therefore, the S. carpocapsae-X. nematophila interaction is species specific. Further, we showed that introduction of the nilA, nilB, and nilC genes into other Xenorhabdus species enables them to colonize the same S. carpocapsae host tissue that is normally colonized by X. nematophila. Finally, sequence analysis supported the idea that the nil genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.     

Statistical optimization of process variables for antibiotic activity of Xenorhabdus bovienii

The production of secondary metabolites with antibiotic properties is a common characteristic to entomopathogenic bacteria Xenorhabdus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities of medicinal and agricultural interests. Culture variables are critical to the production of secondary metabolites of microorganisms. Manipulating culture process variables can promote secondary metabolite biosynthesis and thus facilitate the discovery of novel natural products. This work was conducted to evaluate the effects of five process variables (initial pH, medium volume, rotary speed, temperature, and inoculation volume) on the antibiotic production of Xenorhabdus bovienii YL002 using response surface methodology. A 2(5-1) factorial central composite design was chosen to determine the combined effects of the five variables, and to design a minimum number of experiments. The experimental and predicted antibiotic activity of X. bovienii YL002 was in close agreement. Statistical analysis of the results showed that initial pH, medium volume, rotary speed and temperature had a significant effect (P<0.05) on the antibiotic production of X. bovienii YL002 at their individual level; medium volume and rotary speed showed a significant effect at a combined level and was most significant at an individual level. The maximum antibiotic activity (287.5 U/mL) was achieved at the initial pH of 8.24, medium volume of 54 mL in 250 mL flask, rotary speed of 208 rpm, temperature of 32.0°C and inoculation volume of 13.8%. After optimization, the antibiotic activity was improved by 23.02% as compared with that of unoptimized conditions.     

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 16342^T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337^T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336^T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338^T, isolated from Steinernema rarum.