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.     

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.     

Biochemical Characterization and Agglutinating Properties of Xenorhabdus nematophilus F1 Fimbriae

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus.     

Transformation of Xenorhabdus nematophilus

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Transformation of Xenorhabdus nematophilus.

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Purification and characterization of xenorhabdicin, a phage tail-like bacteriocin, from the lysogenic strain F1 of Xenorhabdus nematophilus.

Xenorhabdicin, the phage tail-like bacteriocins of Xenorhabdus nematophilus, and phage head particles, elements produced together after mitomycin induction in X. nematophilus lysogenic strain F1 cultures, were separated by DEAE chromatography, examined by transmission electron microscopy, and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis of xenorhabdicin showed two major subunits of 43 and 20 kDa corresponding to the sheath and the inner core, respectively. At least five other minor subunits of 67, 54, 35, 28, and 16 kDa were also characterized. Electrophoresis of the phage head capsids showed a major 40-kDa subunit and two minor 50- and 34-kDa subunits. Bactericidal activity recorded against closely related bacterial species and spontaneously produced by X. nematophilus resides in the xenorhabdicin particles and is another antimicrobial barrier to save the symbiotic association.     

A homoserine lactone autoinducer regulates virulence of an insect-pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae).

N-beta-Hydroxybutanoyl homoserine lactone (HBHL), the autoinducer of the luminescent system of Vibrio harveyi, has been identified as the first small compound to restore virulence to avirulent mutants of Xenorhabdus nematophilus. HBHL stimulated the level of lipase activity excreted by avirulent X. nematophilus and lowered the phenoloxidase activity in the hemolymph of insects infected with X. nematophilus, parameters that are both associated with insect pathogenesis. Moreover, mortality of the insects infected with avirulent X. nematophilus was restored upon injection with HBHL. Chloroform extraction of medium conditioned with wild-type but not avirulent X. nematophilus led to the isolation of a compound with the same chromatographic mobility as HBHL as well as the ability to stimulate the luminescence of a dim autoinducer-dependent mutant of V. harveyi. Transfer of the V. harveyi lux operon into avirulent and wild-type X. nematophilus generated dim and bright luminescent strains, respectively, which responded to HBHL and an agonist and antagonist in a manner analogous to their effects on the luminescence of dim autoinducer-deficient and bright wild-type strains of V. harveyi, indicating that similar HBHL-dependent regulatory systems exist in these two bacterial species.     

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

The bacterium Xenorhabdus nematophilus depresses nodulation reactions to infection by inhibiting eicosanoid biosynthesis in tobacco hornworms,Manduca sexta

The bacterium, Xenorhabdus nematophilus, is a virulent insect pathogen. We tested the hypothesis that this bacterium impairs insect cellular immune defense reactions by inhibiting biosynthesis of eicosanoids involved in mediating cellular defense reactions. Fifth instar tobacco hornworms, Manduca sexta, produced melanized nodules in reaction to challenge with living and heat-killed X. nematophilus. However, the nodulation reactions were much attenuated in insects challenged with living bacteria (approximately 20 nodules/larva for living bacteria vs. approximately 80 nodules/larva in insects challenged with heat-killed bacteria). The nodule-inhibiting action of living X. nematophilus was due to a factor that was present in the organic, but not aqueous, fraction of the bacterial cultural medium. The nodule-inhibiting factor in the organic fraction was labile to heat treatments. The immunodepressive influence of the factor in the organic fraction was reversed by treating challenged hornworms with arachidonic acid. The factor also depressed nodulation reactions to challenge with the plant pathogenic bacteria, Pseudomonas putida and Ralstonia solanacearum. These findings indicate that one or more factors from X. nematophilus depress nodulation reactions in tobacco hornworms by inhibiting eicosanoid biosynthesis.     

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 (Ume?), 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 Ume? (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.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.     

Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.

We show that inactivation of envZ, the gene encoding the histidine kinase sensor protein, EnvZ, of Xenorhabdus nematophilus, affected the production of several outer membrane proteins (Opns). X. nematophilus produced five major Opns during exponential growth. Insertional inactivation of envZ led to a decrease in the production of OpnP, the OmpF-like pore-forming protein which constitutes approximately 50% of the total outer membrane protein in X. nematophilus. OpnA production was also reduced, while the remaining Opns were produced normally. During the transition to stationary phase, three new outer membrane proteins, OpnB, OpnS, and OpnX, were induced in the wild-type strain. The envZ-minus strain, ANT1, did not produce OpnB and OpnX, while OpnS was induced at markedly reduced levels. These results suggest that EnvZ was required for the high-level production of OpnP during exponential growth and may be involved in the production of OpnB, OpnS, and OpnX during stationary-phase growth. We also show that ANT1 was more pathogenic than the wild-type strain when as few as five cells were injected into the hemolymph of the larval stage of the tobacco hornworm (Manduca sexta). The larvae died before significant numbers of bacteria were detectable in the hemolymph. These results are discussed in relation to the role of EnvZ in the life cycle of X. nematophilus.     

Haemolymph proteins of larvae of Galleria mellonella detoxify endotoxins of the insect pathogenic bacteria Xenorhabdus nematophilus (Enterobacteriaceae)

Haemolymph of non-vaccinated Galleria mellonella larvae contains two proteins, LBP-1 (17.2kDa) and LBP-2 (26.0kDa) that:bond to the surfaces of the insect pathogenic bacteria, Xenorhabdus nematophilus;prevented lipid A-binding dye attaching to the lipid A of X. nematophilus endotoxin; andreduced endotoxin activity on the haemocytes.Protein LBP-1 also blocked the inhibition of prophenoloxidase activation by the endotoxins. It is proposed that proteins LBP-1 and LBP-2 are part of the containment responses of the insects to bacteria.     

Inactivation of a novel gene produces a phenotypic variant cell and affects the symbiotic behavior of Xenorhabdus nematophilus

Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.     

Swarming and Swimming Changes Concomitant with Phase Variation in Xenorhabdus nematophilus

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Phase I, the variant naturally isolated from the infective-stage nematode, provides better conditions than the phase II variant for nematode reproduction. This study has shown that Xenorhabdus phase I variants displayed a swarming motility when they were grown on a suitable solid medium (0.6 to 1.2% agar). Whereas most of the phase I variants from different Xenorhabdus spp. were able to undergo cycle of rapid and coordinately population migration over the surface, phase II variants were unable to swarm and even to swim in semisolid agar, particularly in X. nematophilus. Optical and electron microscopic observations showed nonmotile cells with phase II variants of X. nematophilus F1 which lost their flagella. Flagellar filaments from strain F1 phase I variants were purified, and the molecular mass of the flagellar structural subunit was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 36.5 kDa. Flagellin from cellular extracts or culture medium of phase II was undetectable with antiserum against the denatured flagellin by immunoblotting analysis. This suggests that the lack of flagella in phase II cells is due to a defect during flagellin synthesis. The importance of such a difference of motility between both phases is discussed in regard to adaptation of these bacteria to the insect prey and the nematode host.     

Pathogenicity of Bacteria Symbiotically Associated with Insect Pathogenic Nematodes against the Greater Wax Moth,Galleria Mellonella (L.)

The bacterial symbionts isolated from the entomopathogenic nematodes were compared for their pathogenicity to last instar larvae of G. mellonella at both Phases I and II. Most bacterial symbionts at Phase I cause 100% mortality within 2-3 days post-injection with 1 times; 10 3 cells/larva. The pathogenicity of Phase I decreased in the following order: Xenorhabdus nematopbilus, Flavimonas oryzihabitans, Photorhabdus luminescens and Xenorhabdus bovienii with LD 50 values of 40, 55, 70 and 170 cells/larva. The injection of Phase II of the bacterial symbionts did not give 100% mortality even after 4 days post-injection. The time mortality response of G. mellonella larvae to both phases of the bacterial symbiont was significantly different usually at the two highest concentrations tested. The significancy in case of Phase I was in the following order from lowest to highest, F . oryzihabitans , X . nematophilus , P. luminescens and X. bovienii . It was 20.57, 23.96, 23.99 and 53.76 h, respectively. Also, F. oryzihabitans gave the lowest LT 50 value for its Phase II form. It was 36.85 h, and this is followed by X. bovienii , X. nematophilus , and P. luminescens , the LT 50 values of which were 69.29, 74.08 and 74.49 h, respectively. The results suggest that there is a direct correlation between toxin concentration and rate of killing the larvae. On the other hand, there is an inverse correlation between the LT 50 values and the injected concentration.     

Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae)

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96-160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.     

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.