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Appl Environ Microbiol, July 1998, p. 2367-2373, Vol. 64, No. 7
Laboratoire de Pathologie Comparée,
Université Montpellier II, Institut National de la Recherche
Agronomique, Centre National de la Recherche Scientifique (URA
INRA-CNRS no. 2209), 34095 Montpellier Cedex 05, France
Received 1 December 1997/Accepted 12 April 1998
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.
Bacterial symbionts of
entomopathogenic nematodes in the families Steinernematidae and
Heterorhabditidae are members of the family
Enterobacteriaceae and belong to the genera
Xenorhabdus and Photorhabdus, respectively
(7, 29). These bacteria are carried in an intestinal vesicle
of the nonfeeding infective stage of members of the Steinernematidae
(5) and throughout the whole intestine of infective
juveniles of members of the Heterorhabditidae (14). The
nematodes release their bacterial symbionts into the hemocoels of the
insects, where growth induces a lethal septicemia and contributes to
the symbiotic relationship by providing nutrients required by nematode
partners during reproduction in insect cadavers (23).
All Xenorhabdus strains spontaneously produce two distinct
physiological states in vitro (2), phase I and II variants
(6). Phase I variants absorb dyes on agar plates, produce
several antibiotics, secrete a variety of proteins (e.g., lipases and
proteases), and produce fimbriae and flagella, while these properties
are either apparently absent or greatly reduced in phase II variants
(6, 17, 21). Both types are pathogens of insect larvae, but
phase I variants are associated only with infective nematodes that
naturally parasitize insects (2). The entomotoxicity
mechanisms of these bacteria and the benefits provided by the bacteria
to their host nematodes are not well-documented, but it has been
suggested that extracellular molecules produced by
Xenorhabdus spp. may participate in both virulence and
symbiosis with nematodes (3, 15).
Production of phosphatidylcholine-hydrolyzing phospholipases (or
lecithinase) is detected on solid media as opalescent zones surrounding
colonies grown on agar supplemented with egg yolk (the egg yolk test)
(30). A wide variety of gram-positive and gram-negative
bacteria have been found to produce lecithinases when this reaction is
used as an assay (30). Many of these lecithinases have been
purified and characterized as single secreted-polypeptide proteins.
Lecithinases are toxic determinants, as well as a means of securing
bacterial supplies of phosphates (9, 30). They may also have
an important role in the induction of pathogenicity in host organisms
(27). For example, the ability of Bacillus thuringiensis to develop is thought to be due in part to its
high-level production of phospholipases (11). Because phase
I variants of Photorhabdus and Xenorhabdus
species have been reported to give positive results in the egg yolk
test (6), it was apposite to define any toxic property of
this enzymatic activity. More recently, it was found that some
Tn5-induced Xenorhabdus bovienii lecithinase
mutants exhibited reduced virulence for Galleria mellonella (22).
Phospholipases which hydrolyze the glycerophospholipids belong to the
lipase subgroup (glycerol ester hydrolases; EC 3.1.1.3) of esterases
(carboxyl esterases; EC 3.1.1.1). Phospholipases A1,
A2, B, C, and D are characterized by the sites of cleavage of the phospholipids (18, 19). Phospholipases
A1, A2, and B are carboxyl esterases, while
phospholipases C and D are phosphoryl esterases. It was shown
previously that bacterial symbionts of entomopathogenic nematodes
produced positive reactions on both Tween agar and egg yolk agar,
suggesting that these bacteria exhibit lipase activities
(6). The variability of the lipase activities noticed during
phase variation suggested that at least two different lipases were
produced by the bacterial symbionts.
To elucidate the nature of lipase production by symbiotic bacteria of
entomopathogenic nematodes, we compared the enzymatic activities of
several symbionts on Tween and lecithin (phosphatidylcholine) agar
plates. The enzymatic activities were determined for a range of
Xenorhabdus sp. and Photorhabdus sp. strains
from our worldwide laboratory collection. After comparing the lipase
activities of members of both genera, we concentrated our efforts on
Xenorhabdus nematophilus in order to characterize the
lipases of this species biochemically and pathologically. The X. nematophilus F1 lecithinase was purified and partially
characterized in a first step, and then the entomotoxic and cytotoxic
activities of the purified molecules were studied.
Bacterial strains and growth conditions.
All of the
bacterial strains used in this study are listed in Table
1. For each subculture, the phase status
was determined by culturing on NBTA and measuring antibacterial
activity against Micrococcus luteus (8). Phase I
colonies are blue on NBTA and produce agar-diffusible antibiotics,
while phase II colonies are red and produce reduced or no antibacterial
activity. Phases I and II of strains are indicated as suffixes (/1 and
/2, respectively) attached to strain designations. Bacterial cells were
grown in Luria-Bertani broth for liquid cultures and on nutrient agar
(NA) manufactured by Pasteur Institute or on Luria-Bertani agar for solid cultures.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Isolation and Entomotoxic Properties of the
Xenorhabdus nematophilus F1 Lecithinase
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Activities of phase I variants of Xenorhabdus
and Photorhabdus species grown on NA plates containing 1%
(vol/vol) Tween and NA plates containing 0.01% (wt/vol) lecithin after
2 days of incubation at 28°C
Measurements of lipase activity. Qualitative assays of lipase activity were performed by plating solid bacterial cultures on Tween agar plates as described previously (8). Lipase-releasing colonies were identified by the formation of precipitates of water-insoluble fatty acids from hydrolyzed Tween surrounding them (26).
Glycerol ester hydrolase activity was qualitatively assayed by plating solid bacterial cultures or liquid culture supernatants on triolein solid medium plates; pure triolein (Sigma) was added to NA at 45°C to a final concentration of 1% (vol/vol) and was emulsified by sonication at 125 W for 5 min with a Branson 2210 Sonifier. Lipase-releasing colonies or supernatants were recognized by the formation of precipitates of water-insoluble fatty acids from the hydrolyzed triglyceride. A photometric assay of lipase activity was performed with p-nitrophenylpalmitate as the substrate, as previously described (28, 32).Measurements of lecithinase activity. Lecithinase activity was assayed by spotting on NA containing 0.01% (wt/vol) lecithin prepared as described previously (8). After 48 h of incubation, lecithinase-releasing colonies were recognized by the formation of precipitation zones of water-insoluble fatty acids from hydrolyzed lecitin surrounding them (30). The substrate specificity of the purified X. nematophilus F1 lecithinase was determined by replacing the crude egg yolk lecithin with different highly purified phospholipids from egg yolk (see Table 4) (99% pure; Sigma). Agarose (1%, wt/vol) was dissolved in 0.1 M NaCl-0.02 M Tris buffer (pH 9, 45°C), and each phospholipid was emulsified as described above at a final concentration of 0.01% (wt/vol) before it was spread over colonies grown on NA.
A semiquantitative lecithinase assay, adapted from the assay described by Giskow et al. (16), was used to monitor lecithinase production. It was performed as a diffusion speed assay with thin 1% agarose gels containing 0.01% lecithin, 0.1 M NaCl, and 0.02 M Tris (pH 9). Three-microliter aliquots were added to wells (diameter, 1 mm) made in the gels with a Pasteur pipette. The presence of lecithinase induced a precipitation-diffusing zone around each hole. We found that there was a linear relationship between the logarithm of a serial dilution of a concentrated sample and the radius of the precipitating zone in the gel (data not shown). After incubation at 28°C, enzymatic activity was determined by recording the radius (in millimeters) of the precipitation zone in each gel at given times. Lecithinase activity was calculated by determining the change in the precipitation radius (in millimeters) per unit of time (24 h), and specific activity was calculated by determining the change in the precipitation radius (in millimeters) per unit of time (24 h) and unit of material (microgram of protein or milligram of dried bacterial material). The protein concentration was measured by the bicinchoninic acid (Mallet SA; Pierce) method with serum albumin as the standard (34).IEF and blotting of lecithinase activity. Isoelectric focusing (IEF) was performed by using an LKB apparatus (Pharmacia, Uppsala, Sweden) and broad isoelectric point (pHi) precast gels (pH 3 to 10) according to the manufacturer's instructions. A broad-pHi calibration kit (pH 3 to 10; Pharmacia) was used to determine the lecithinase pHi. After electrophoresis, the position of lecithinase in the IEF gels was determined by washing the gels five times in 100 ml of Tris-NaCl buffer (0.1 M NaCl, 0.02 M Tris [pH 9]) (30 min each) and overlapping them with a 1% agarose layer containing 0.01% lecithin in Tris-NaCl buffer. After 2 h of incubation at 28°C, the position of lecithinase activity was observed as a white precipitation zone in the agarose-lecithin gel.
Purification of lecithinase.
X. nematophilus F1/1 was
cultivated at 28°C in 100 ml of Luria-Bertani broth for 3 days. Cells
were removed by low-speed centrifugation (6,000 × g,
10 min, 4°C), and a filter-sterilized (pore size, 0.22 µm)
supernatant was dialyzed overnight at 4°C against 10 liters of
Tris-NaCl buffer. The dialyzed supernatant (100 ml) was subjected to
cation-exchange chromatography on a SP MemSep cartridge column (void
volume, 1.4 ml; Millipore) at a flow rate of 1.4 ml · min
1 and was washed with 14 ml of Tris-NaCl
buffer. The lecithinase activity was eluted once with 0.5 M NaCl-0.02
M Tris buffer (pH 9). Active fractions were pooled and diluted with 1 volume (1.5 ml) of Tris-NaCl buffer.
1 with solvent A for 20 min. Proteins were eluted with
a 0 to 80% solvent B gradient generated with a Waters delivery system
over a period of 30 min. Peptides were detected with a photodiode array detector (model 990; Waters), and the optical density at 220 nm was
recorded. Eluted fractions (500 µl) were evaporated in a speed vacuum
apparatus, and the dried material was resuspended in 50 µl of
Tris-NaCl buffer.
During the purification procedure, lecithinase activity was routinely
monitored by spotting on NA plates containing 0.01% lecithin and was
quantitatively determined by the radial diffusion assay.
Toxicity assays. The common cutworm, Spodoptera littoralis, was reared on an artificial diet (24) at 24°C, and the wax moth, G. mellonella, was reared on pollen and wax at 28°C. A locust, Locusta migratoria, was reared on grass at 28°C. Eggs of the tobacco hornworm, Manduca sexta, were obtained from Monika Stengl (University of Regensburg, Regensburg, Germany). M. sexta larvae were reared on an artificial diet (1) at 27°C with light-dark cycles consisting of 16 h of light and 8 h of darkness. Fifth-instar larvae of each insect species were selected and surface sterilized with 70% (vol/vol) ethanol prior to intrahemocoelic injection. The larvae were divided into groups of 12, and each larva was injected with 10 µl of one of the purified HPLC fractions, corresponding to a dose of 0.1 µg per insect, or with phosphate-buffered saline (PBS). The treated larvae were incubated individually for up to 96 h, and then the number of dead insects was recorded.
A liquid hemolysis assay with sheep erythrocytes (25) was used to determine hemolytic activity in purified HPLC fractions. Cytolytic assays were performed with insect hemocytes by collecting hemolymph samples from S. littoralis larvae in an anticoagulant buffer (4). Hemocytes were centrifuged, rinsed in PBS to remove plasmatic factors, and resuspended in the same buffer (2 × 104 hemocytes · ml1). The
suspensions (10 µl) were each mixed with 10 µl of a purified HPLC
fraction, corresponding to a 0.1-µg dose, deposited on a slide, and
incubated for 20 min at 28°C. Hemocytes with PBS were used as a
control. Cell lysis was observed with a light microscope and was
recorded.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Lipase- and lecithinase-producing Xenorhabdus and Photorhabdus strains. No precipitation zones were observed surrounding the Photorhabdus luminescens K122 colonies when they were grown on NA containing 0.01% lecithin even when incubation was prolonged by 1 week. Since this strain is known to secrete a Tween 80 lipase in solid cultures (31), we deduced that the lecithin and Tween assays may be used to discriminate between the two different enzymes (a lecithinase and a lipase, respectively). Screening the lecithinase and lipase activities in the wide range of symbiotic bacteria from entomopathogenic nematodes allowed us to distinguish the following three groups of symbionts: (i) lipase-producing strains which were strongly positive on NA plates containing 1% Tween but failed to generate any precipitates on NA containing 0.01% lecithin; (ii) lecithinase-producing strains which were strongly positive on NA plates containing 0.01% lecithin but failed to generate any precipitates on NA containing 1% Tween; and (iii) strains which produced both lipase and lecithinase (Table 1). Most of the Photorhabdus strains belonged to the first group, while several X. bovienii strains belonged to the second group. X. nematophilus and Xenorhabdus beddingii strains were able to produce both lecithinase and lipase activities.
When both phase I and phase II variants of the lecithinase-producing strains were grown on NA plates containing 0.01% lecithin for 2 days, the phase I variants had large halos around the colonies, while the phase II variants did not. Nevertheless, when incubation was extended by 7 days, the X. nematophilus phase II variants also produced a weak zone of precipitation (Fig. 1). The X. bovienii phase II variants were still negative even after an additional 1 week of incubation. On the other hand, the Tween activities of all of the Tween-positive Xenorhabdus spp. were always found to be greater for the phase II variants than for the phase I variants (Table 2).
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Lecithinase activity during broth growth of X. nematophilus F1. Lecithinase production by phase I variants of X. nematophilus occurred after 16 to 24 h of incubation at 28°C in a shaking bath incubator, whereas a phase II variant culture failed to produce any lecithinase activity even after several days. Figure 2 shows that production of lecithinase by X. nematophilus F1/1 was growth phase dependent. No lecithinase activity was observed during the exponential growth phase, but when growth began to slow down and entered the stationary phase, there was a sudden burst of expression, which rapidly increased to the maximum level. Further incubation revealed that the maximum level of lecithinase activity remained constant for several weeks.
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, growth, as determined by absorbance at 600 nm; 
,
lecithinase specific activity (per milligram of dried material), as
determined by measuring the rate of enzyme diffusion (in millimeters
per 24 h) on an NA plate containing 0.01% lecithin. Values are
the means from three experiments. Error bars indicate standard
deviations.
Purification of the X. nematophilus F1 lecithinase. IEF of a 10-fold-concentrated supernatant sample from a 3-day-old X. nematophilus F1/1 culture was performed on a 5% acrylamide gel containing ampholines ranging from pH 3 to 9.3. After the preparation was blotted with a 0.01% lecithin-1% agarose layer, the lecithinase activity was observed at the cathode, indicating that the pHi was greater than pH 9.3 (data not shown). This basic feature of the lecithinase was used to perform a first chromatographic procedure with an SP-MemSep cation-exchange cartridge at pH 9. Under these basic conditions most proteins from the supernatant (theoretically all proteins with a pHi less than pH 9) were excluded, while the lecithinase was kept on the column. After a single elution with 0.5 M NaCl-0.02 M Tris (pH 9) buffer, the lecithinase was eluted, and the positive fractions were subjected to reverse-phase HPLC on a C18 column. This second chromatographic procedure allowed us to separate five peaks (peaks 5 through 9) containing lecithinase activity (Fig. 3), which eluted at about 50% acetonitrile. Each of these peaks was passed separately a second time through the same C18 column to check its purity and activity (data not shown). Quantification by the diffusion speed assay on the 0.01% lecithin-1% agarose gel showed that peaks 6 and 8 possessed most of the total activity. Nevertheless, the specific activities (in millimeters per 24 h per milligram) were found to be similar in all peaks and increased by a factor of about 350 during the purification procedure (Table 3).
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Biochemical properties and substrate specificity of the HPLC
isolates.
Each isolate was very stable, as shown by full recovery
of lecithinase activity after long-term storage at room temperature or
after 30 min of exposure to 100°C. Moreover, the activity was not
affected by preincubation of the purified lecithinase with a commercial
lipase or with several proteases. However, treatment with pronase E
from Streptomyces griseus (Sigma) completely destroyed the
lecithinase. Spectral analysis at wavelengths between 200 and 600 nm
showed that the five C18 HPLC peaks exhibited the same narrow absorption spectrum at a 
max of ca. 207 nm (data
not shown). The substrate specificity of each of the isolates was
assayed separately by spotting aliquots of a purified sample onto 1%
agarose gels containing different pure phospholipids.
Phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylinositol,
lyso-phosphatidylinositol, sphingomyelin, and phosphatidic acid were
found to produce an opaque zone with all five lecithinase isolates,
whereas phosphatidylethanolamine, diacylglycerol, and triolein did not
(Table 4). These data indicate that there
was a preferential affinity for polar lipids. Enzymatic assays in
liquid failed to reveal any enzymatic reaction with p-nitrophenylphosphorylcholine or
p-nitrophenylpalmitate substrates despite the use of several
detergents and buffers.
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Toxic properties. None of the five isolates exhibited any cytolytic activities against either sheep erythrocytes or insect hemocytes. Although preliminary experiments showed that G. mellonella, S. littoralis, M. sexta, and L. migratoria were very susceptible to intrahemocoelic injection of X. nematophilus F1/1 (16a), injection into the hemocoel of 0.1-µg portions of the purified molecule had no effect on the mortality of fifth-instar larvae of these insects.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The previously described lipase-producing organism P. luminescens K122 (31) gave a positive reaction on Tween 80 agar plates but did not produce any precipitate on phospholipid-containing medium, such as the lecithin-NA plates used in our study. The data described here demonstrate that the Tween and lecithin agar assays are enzyme specific and suitable for discriminating lipase-producing strains from lecithinase-producing strains when the bacteria symbiotically associated with the entomopathogenic nematodes are examined. Generally, we found that X. bovienii had lecithinase properties and Photorhabdus spp. had lipase properties. We report here that 13 of 15 Photorhabdus strains were lecithinase negative on lecithin agar, in contrast to previously published results (obtained by using the egg yolk agar test) reported for three Photorhabdus strains formerly considered Xenorhabdus luminescens strains (6). X. nematophilus and X. beddingii formed an intermediate group having both activities. When the bacteria produced only one of the two enzymes, this biochemical characteristic was specific to the phase I variant. The strains that released both enzymes exhibited differential production; the phase I variants were the lecithinase producers, while the phase II variants were the major lipase producers. This last observation provided another demonstration that lecithinase and lipase activities were due to two different enzymes which were conversely regulated during phase variation. P. luminescens K122 lipase was found to possess a triacyl glycerol esterase activity, as indicated by the production of an opaque zone when the organism was grown on a triolein-NA plate. However, this enzyme appeared to be specific to nonpolar lipids because K122 failed to exhibit any reactions with the phospholipids tested. Strains which produced precipitates only on lecithin-NA plates were found to be unable to hydrolyze p-nitrophenylpalmitate, suggesting that a particular lipase activity (lecithinase) was present.
X. nematophilus F1 lecithin precipitate-associated molecules were purified by cation-exchange chromatography and hydrophobic C18 HPLC. Surprisingly, HPLC generated five peaks representing compounds which were capable of inducing precipitates on lecithin-NA plates. These five purified compounds showed the same substrate specificity, forming some precipitates with the polar phospholipids, such as phosphatidylcholine. Moreover, the substrates bearing only carboxyl ester bonds, such as Tweens, diacylglycerol, and triolein, were not precipitated by the five compounds. This substrate specificity indicated that the X. nematophilus F1 lecithin precipitate-associated molecules could correspond to a lecithinase that is able to cleave only phosphoryl ester bonds. Such enzymatic activity could theoretically correspond to phospholipase C or D. However, the latter possibility is unlikely since the purified lecithinase reacted with phosphatidic acid, which cannot be hydrolyzed by phospholipase D. In order to probe phospholipase C activity, we used p-nitrophenylphosphorylcholine, which is a specific substrate of phospholipase C (20). The five purified samples were not able to hydrolyze p-nitrophenylphosphorylcholine. This could reflect a default of the substrate, which is quite different from a true phospholipid (30). The five HPLC-purified molecules might represent five isomers, as expected based on their identical adsorption spectra, their common basic properties, and their identical substrate specificities. Although the protease treatments (pronase E treatments) revealed their peptide character, these proteins share some unusual characteristics, such as the lack of absorption at 280 nm and the inability to be stained by Coomassie blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (data not shown), indicating that there may be a lack of aromatic amino acids (10, 12). Because the biochemical properties of the five purified molecules remain unclear, we decided to use the generic name lecithinase to describe a molecule which induces a zone of precipitation when it is applied to phospholipid agarose layers.
Several well-described phospholipases C have been found to be involved in bacterial virulence (27). The consequences may be immediate and direct, as is the action of Clostridium perfringens alpha toxin against erythrocytes, or subtle, as is the reaction of Listeria monocytogenes phosphatidylinositol-specific phospholipase C, which allows bacteria to grow in infected cells (27).
Injections of the five purified lecithinases into the hemocoels of different species of insects did not result in increased mortality. Moreover, these molecules did not exhibit cytolytic activities against sheep erythrocytes or insect hemocytes. These results demonstrate that the X. nematophilus lecithinase had no entomotoxic effects. However, although Tn5-induced X. bovienii lecithinase-deficient mutants still killed the insect larvae, they showed reduced virulence for G. mellonella. This was seen clearly in the results of the 50% lethal dose analysis, which showed that the G. mellonella mortality rate was significantly lower with the Tn5-induced lecithinase mutant than with wild-type parent strain T228/1 (22). The Xenorhabdus lecithinase may participate in the virulence of the nematobacterial complex by allowing intracellular bacterial growth in insects in the same way that phospholipase C acts in B. thuringiensis virulence (11).
It is commonly accepted that the symbiotic bacteria Xenorhabdus spp. provide some nutrients to their nematode hosts by releasing digestive exoenzymes into insect cadavers (15). Lipidic nutrition of the nematodes is considered a key factor for the reproduction and development of the nematode larvae (33). Most Xenorhabdus spp. possess both a lipase specific for the nonpolar lipids and a lecithinase which hydrolyzes the polar phospholipids. The bacterial activity of the latter may reflect a high level of adaptation of the Xenorhabdus spp. to their nematode hosts. Dunphy and Webster (13) showed that in vitro production of Steinernema carpocapsae on lipid-containing media involves the consumption of choline, a nutrient which is liberated after hydrolysis of phosphatidylcholine by phospholipase C. This fact strongly suggests that the lecithinase of X. nematophilus may play a role in the lipidic metabolism of its nematode host, S. carpocapsae, by providing the choline nutrient to it.
Future work on lecithinases produced by X. nematophilus will focus on enzymatic characterization of the purified molecule in which radiolabeled substrates and protein sequencing are used. Gnotobiological experiments on lipid-rich media involving both purified enzyme and lecithinase mutants should greatly help workers to evaluate the role of this bacterial enzyme in the lipidic metabolism of S. carpocapsae.
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ACKNOWLEDGMENTS |
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We thank Jean Luciani for assistance with the insect pathogenesis assays and Michel Brehélin for assistance with the insect hemocyte experiments. We also thank Trevor Jackson (Agresearch, Lincoln, New Zealand) and Carey Smith (CSIRO-CILBA, Montpellier, France) for revising the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Pathologie Comparée, Université Montpellier II, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique (URA INRA-CNRS no. 2209), C.P. 101, 34095 Montpellier Cedex 05, France. Phone: 33-4-67-14-37-40. Fax: 33-4-67-14-46-79. E-mail: boemare{at}ensam.inra.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Appl Environ Microbiol, July 1998, p. 2367-2373, Vol. 64, No. 7
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