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Applied and Environmental Microbiology, November 2001, p. 5017-5024, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5017-5024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Oral Toxicity of Photorhabdus
luminescens W14 Toxin Complexes in
Escherichia coli
Nicholas
Waterfield,1
Andrea
Dowling,1
Sadhana
Sharma,1
Phillip J.
Daborn,1
Ursula
Potter,2 and
Richard H.
Ffrench-Constant1,*
Department of Biology and
Biochemistry1 and Centre for Electron
Optical Studies,2 University of Bath, Bath
BA2 7AY, United Kingdom
Received 21 May 2001/Accepted 9 August 2001
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ABSTRACT |
Previous attempts to express the toxin complex genes of
Photorhabdus luminescens W14 in
Escherichia coli have failed to
reconstitute their oral toxicity to the model insect
Manduca sexta. Here we show that the
combination of three genes, tcdA, tcdB,
and tccC, is essential for oral toxicity to
M. sexta when expression in E. coli is used. Further, when
transcription from native toxin complex gene promoters is used, maximal
toxicity in E. coli cultures is
associated with the addition of mitomycin C to the growth medium. In
contrast, the expression of tcdAB (or the homologous
tcaABC operon) with no recombinant tccC
homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that
P. luminescens K122 carries a functional
tccC-like homolog within its own genome, a hypothesis
supported by Southern analysis. Recombinant toxins from both
P. luminescens K122 and E.
coli were purified as high-molecular-weight particulate
preparations. Transmission electron micrograph (TEM) images of these
particulate preparations showed that the expression of
tcdAB (either with or without tccC) in
E. coli produces visible ~25-nm-long
complexes with a head and tail-like substructure. These data are
consistent with a model whereby TcdAB constitutes the majority of the
complex visible under TEM and TccC either is a toxin itself or is an
activator of the complex. The implications for the potential mode of
action of the toxin complex genes are discussed.
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INTRODUCTION |
The toxin complex
(tc) genes of Photorhabdus luminescens
belong to a family of genes found across different members of the Enterobacteriaceae (10).
Photorhabdus spp. are bacteria symbiotic with
entomopathogenic nematodes of the family Heterorhabditidae, and
homologs of the toxin complex genes are also found in a second genus of
bacteria, Xenorhabdus, that are symbiotic with nematodes of
a different family, Steinernematidae (4, 8). As well as
being found in nematode-symbiotic bacteria, toxin complex gene homologs
have recently been reported for Serratia
entomophila (6). In this species, they are
present on a 115-kb plasmid associated with amber disease in the grass
grub Costelytra zealandica, where they cause
cessation of feeding, clearance of the gut, amber coloration, and
eventual insect death (6). We have also located a
tca-like operon in the unfinished genome sequence of the
human pathogen Yersinia pestis (10),
which is the causative agent of bubonic plague (9).
However, the role of this operon in either the insect vector (the flea)
or the mammalian host remains unknown (10).
P. luminescens strain W14 produces several
high-molecular-weight toxin complexes with oral toxicity to insects
(2). Two of these, Tca and Tcd, have previously been shown
to be responsible for the majority of oral toxicity to the lepidopteran
insect Manduca sexta via gene knockout studies
(2). Tca also shows characteristic, midgut-specific
histopathology in M. sexta (1).
Previous attempts to express recombinant toxins by the introduction of
tca and tcd plasmids into Escherichia
coli have failed (2). In these experiments, although the full-length toxin complex polypeptides were produced, they
were not exported from the bacterial cell. In order to define the
essential subset of W14 genes required for oral toxicity and to verify
conditions enhancing the release of active toxin, we expressed
tca, tcd, and tcc gene components in
E. coli and in a second strain of P. luminescens, K122, that shows no oral toxicity to
M. sexta.
Here we report that the coexpression of tccC with the
tcdAB operon is sufficient for oral toxicity in
E. coli XL1Blue. In contrast, the expression of
either tcdAB or the homologous tcaABC operon
alone is sufficient to confer oral toxicity on P. luminescens K122 in the absence of W14-derived
tccC expression. Maximal oral toxicity in E. coli also requires the addition of mitomycin C to the
culture medium. Particulates of high molecular weight purified from
either recombinant bacterial species were toxic when fed to larvae of
M. sexta. Examination of these toxin preparations by transmission electron microscopy (TEM) showed that tcdAB
encodes a visually distinct complex with a head and tail-like
substructure. These data are discussed in relation to a model whereby
tcdAB encodes the visually distinct complex, whose synthesis
and/or release is stimulated by mitomycin C, and tccC
encodes either an actual toxin or an activator of the complex.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
P.
luminescens strains W14 (3) and K122 (isolated
by D. Clarke from a nematode collected in the Republic of Ireland) were cultured at 30°C with aeration in 2% Proteose Peptone 3 (PP3) and
Luria-Bertani (LB) broth, respectively. When necessary, agar powder was
added to 1.5% (wt/vol). The five plasmids used in this study are
diagrammed in Fig. 1. Plasmid pA1
represents a portion of the tca locus containing the
tcaA, tcaB, and tcaC genes. Plasmid pD1 contains the equivalent genes, tcdA and tcdB,
from the tcd locus. The three remaining plasmids represent
variants of the tcd locus either with the
lysR-like regulator and promoter region alone upstream of
tcdA (pD2) or with different orientations of tccC
downstream (pD3 and pD4). Note that, with the exception of the
tccC gene in pD4, which is transcribed by the pBR322
tet gene promoter, transcription in these plasmids relies
upon native Photorhabdus promoters.
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FIG. 1.
Diagram of toxin complex genes present in the
recombinant plasmids used in this study. These are derived from either
the tca (pA) or the tcd (pD) locus of
P. luminescens W14. Plasmids pA1 and pD1
are Sau3A restriction fragments cloned into the
BamHI site of Bluescript pBCKS(+). Plasmid pD2 is an
SphI-BamHI fragment of pD1 cloned into
pBR322. Plasmids pD3 and pD4 represent the pD2 backbone with a
PCR-generated copy of tccC cloned into the
BamHI site. In pD4, tccC is transcribed
by the tetracycline resistance gene promoter on pBR322.
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Plasmids were transformed into
P. luminescens
K122 using electroporation. For antibiotic selection, 15 µg of
chloramphenicol/ml
was added to the growth medium to select for
plasmids pA1 and
pD1, while 100 µg of ampicillin/ml was used for pD2,
pD3, and
pD4. Briefly, a fresh culture of
P. luminescens K122 was grown
overnight. Cells were harvested
by centrifugation and washed in
an equivalent volume of ice-cold
sterile water. Cells were washed
twice again in equivalent volumes of
ice-cold 10% glycerol before
final resuspension in 1/200 the original
volume of 10% glycerol.
Concentrated plasmid DNA prepared from
E. coli by the Qiagen miniprep
kit protocol was
added to 50 µl of cells in a 0.2-cm electroporation
cuvette. After a
2.1-kV pulse (100
of resistance), the cells
were resuspended in
ice-cold LB medium, incubated at 30°C for
1 h, and then plated
on LB agar supplemented with the relevant
antibiotic. Plates were
incubated for 2 days at 30°C.
Oral bioassays.
For bioassays, 100 µl of either bacterial
culture supernatants or toxin preparations was applied to
1-cm3 disks of artificial wheat germ diet. The
treatment was allowed to soak into the food block and dry for
approximately 20 min under laminar flow. Three first-instar
M. sexta neonate larvae were then placed on each
food block before incubation at 25°C for 7 days. After this time, the
percent mortality of larvae and the weight of surviving larvae were
recorded. Oral toxicity was assessed as the relative weight gain of
animals on the treated diet in comparison to control animals fed
nonrecombinant K122 or E. coli supernatants. Due
to difficulties in assessing and comparing the levels of protein
expression of the different toxin complex components in the various
strains, supernatants were compared using only the relative toxicity of
standardized dilutions. The effective supernatant concentration which
reduced relative weight gain by 50% (EC50) was
calculated using nonlinear regression analysis, and t tests
of EC50s were performed to test for significant differences.
Particulate toxin preparations.
Prior to either
DEAE-Sepharose or CsCl gradient purification of recombinant
toxins, crude preparations of particulate material were made. Briefly,
300 ml of a 3-day-old stationary-phase Photorhabdus culture
(grown at 30°C) in either LB medium or 2% PP3 medium was centrifuged at 8,000 rpm in a GS3 rotor for 30 min at 4°C. The supernatant was decanted to remove the cell pellet, and the
centrifugation was repeated to remove any remaining cells. The
cell-free supernatant was centrifuged at 150,000 × g for 90 min
at 4°C in a Sorvall ultracentrifuge to harvest particulate material.
The supernatant was discarded, and the pellet was washed by gentle
resuspension in 50 ml of 50 mM HEPES buffer (pH 6.8) before a second
centrifugation at 150,000 × g for 90 min at 4°C to pellet
particulate material. The pellet was finally resuspended in 600 µl of
ice-cold phosphate-buffered saline (PBS) and stored at 4°C.
DEAE-Sepharose purification.
As a second step in recombinant
toxin purification, the particulate preparations were further separated
by DEAE-Sepharose chromatography. For DEAE-Sepharose chromatography,
the particulate material recovered after the first high-speed
centrifugation described above was resuspended in 10 ml of ice-cold
PBS, and an equivalent volume of DEAE-Sepharose CL-6B anion exchanger
(in PBS) was added. This mixture was incubated at room temperature for
15 min. The Sepharose resin was harvested by low-speed centrifugation
(3,000 × g), and the supernatant was discarded. The resin was
resuspended in 40 ml of ice-cold PBS and again harvested by
centrifugation. This wash was repeated three more times, and the resin
finally was resuspended in 10 ml of elution buffer (0.5 M NaCl, 50 mM phosphate buffer [pH 7.4]). The resin was removed by centrifugation, and the supernatant containing toxins was centrifuged at 150,000 × g for 90 min at 4°C to pellet particulate material. Particulate material was finally resuspended in 600 µl of ice-cold PBS and stored
at 4°C.
CsCl density gradient column purification.
Particulate
material containing the toxins prepared using the DEAE-Sepharose method
was further fractionated on a CsCl density gradient. A 300-µl portion
of the sample was brought to a volume of 5.4 ml at a density of 1 g of CsCl/ml and centrifuged for 24 h at 14°C in a Beckman VTi65
rotor. Fractions of approximately 200 µl each were sequentially drawn
off the bottom of the column and examined by sodium dodecyl sulfate
(SDS)-10% polyacrylamide gel electrophoresis (PAGE) for protein
content. Fractions containing the same protein profiles were pooled and
dialyzed against 5 liters of PBS at 4°C.
Native gel electrophoresis.
Samples from particulate
preparations were brought to 30 µl with 50 mM Tris-HCl buffer (pH 7).
Native gel electrophoresis was performed as described elsewhere
(3) with modifications. Briefly, samples were separated by
electrophoresis in an agarose gel consisting of a 5-cm-long 1.5%
(wt/vol) agarose "stacking" region in 100 mM Tris-HCl (pH 7) and a
15-cm-long 1.9% agarose "resolving" region in 200 mM Tris-borate
(pH 8.3). The anode and cathode buffers consisted of 1 M Tris-HCl (pH
8.3) and 0.025 M Tris-0.192 M glycine (pH 8.3), respectively. The gel
was run for 4 to 5 h at 120 mA on ice. Duplicate lanes were run on
either side of the gel. Half was then cut off and stained with
Coomassie brilliant blue to visualize the positions of the proteins.
The unstained half of the gel was then laid on top of this portion in
order to localize and excise the unstained native protein complexes from the gel. Protein was electroeluted from these excised agar blocks
by electrophoresis inside dialysis membranes suspended in
Tris-borate-EDTA running buffer (on ice). Electroeluted protein was
concentrated by phenol precipitation for visualization by SDS-10%
PAGE and Coomassie brilliant blue staining.
Electron microscopy.
For TEM, pioloform-covered
300-mesh copper grids, coated with a fine layer of carbon, were used as
substrates for the protein fractions in the microscope. Four different
aqueous negative stains were tested with the protein samples: 1%
uranyl acetate, 3% ammonium molybdate, 3% methylamine tungstate, and
2% sodium silicotungstate. The preferred stain, 3% methylamine
tungstate, produced acceptable contrast and minimum artifacts and was
subsequently used for all samples viewed in the microscope.
The coated grids were exposed to UV light for 16 h immediately
prior to use to ensure adequate wetting of the substrate. A
10-µl
drop of sample was applied to the TEM grid, and the protein
was allowed
to settle for 5 min. The liquid was absorbed with
filter paper from the
edge of the grid and substituted immediately
with 10 µl of filtered
negative stain solution. This drop was
partially removed with filter
paper, and the grid was allowed
to air dry thoroughly before viewingwas
done with a Jeol (Tokyo,
Japan) 1200EX transmission electron microscope
operating at 80
kV.
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RESULTS |
Expression of toxin genes in E.
coli.
The expression of different combinations of
genes from the P. luminescens W14 tcd
and tcc loci shows that two factors are necessary and
sufficient for high levels of oral toxicity to M. sexta. First, coexpression of tccC with
tcdAB (plasmid pD3 or pD4) in E. coli
is necessary to confer oral toxicity, while the expression of
tcdAB alone (plasmid pD2) is not sufficient (Fig. 2a). Second, in addition to the
coexpression of all three genes, to achieve full toxicity, mitomycin C
must be added to the growth medium (Fig. 2b); alternatively, in its
absence, cultures must be allowed to stand for more than 48 h for
toxicity to become apparent. The expression of tccC alone in
the arabinose-inducible expression plasmid pBAD30 (5) was
insufficient to confer oral toxicity.
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FIG. 2.
Effect of the presence of TccC and mitomycin C induction
on the oral toxicity of recombinant P.
luminescens K122 and E.
coli. (a) Histogram showing relative weight gain (mean
and standard error for six larvae per treatment) of M.
sexta fed on a diet treated with whole cultures of
strains containing plasmids with or without tccC. Note
that plasmids containing tccC alone [E.
coli(pBADtccC)] do not inhibit weight
gain, whereas plasmids carrying tcdAB in
P. luminescens K122 [K122(pD2)] or
tcdAB and tccC in E.
coli [E. coli(pD3) and
E. coli(pD4)] inhibit growth to 10% or
less of the control weight. (b) Relative weight gain of larvae fed on
supernatants grown in the presence (dark shading) or absence (light
shading) of mitomycin C. Note that although some toxicity can be
observed without mitomycin C [e.g., the 20% reduction seen with
E. coli(pD4)], toxicity is substantially
increased (~90% weight reduction) by the addition of mitomycin C to
cultures containing plasmids coexpressing both tcdAB and
tccC [E. coli(pD3) and
E. coli(pD4)]. The dagger represents a
sample in which the mortality of the insects was 100%.
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Interestingly, low levels of oral toxicity to
M. sexta are seen in exponentially growing cultures but are
putatively cell
associated (i.e., absent from the growth medium).
Further, oral
toxicity is not increased via physical lysis (sonication)
of cells
from these cultures. The fact that purified Tcd particles are
still orally toxic after sonication (data not shown) suggests
that
release from the cytoplasm is not limiting toxicity in these
cultures.
One possible explanation for these observations is that
the toxin
complexes are surface associated and that either aging
of the culture
or the addition of mitomycin C promotes their detachment
(either
directly or via disruption of the cell
membranes).
Expression of toxin genes in P.
luminescens K122.
Both the tcaABC and
the tcdAB expression plasmids (pA1 and pD1, respectively)
conferred the oral toxicity of the supernatants on recombinant K122,
with relative weight gain EC50s of 0.23- and
0.003-fold, respectively (Fig. 3).
Notably, the EC50 for K122 expressing
tcdAB (pD1) was significantly lower (P > 0.001) than that for the original strain, W14, itself (0.003- versus
0.05-fold). Further, supernatants from K122 expressing tcdAB
(pD1) were 38.6 times more potent than the equivalent supernatants from
K122 expressing tcaABC (pA1). These comparisons do not take
into account the specific amount of toxin complex protein produced by
each recombinant strain; however, they do illustrate that it is
possible to make recombinant P. luminescens
strains which are more orally toxic than the original P. luminescens strain, W14, itself.
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FIG. 3.
Relative weight gain of M.
sexta larvae fed on different dilutions (1× to 0.002×)
of P. luminescens supernatants. Strains
used were orally toxic P. luminescens W14
and P. luminescens K122 (lacking oral
toxicity) expressing recombinant Tca or Tcd from the W14-derived
recombinant plasmid pA1 or pD1 (Fig. 1), respectively. Note that the
expression of Tcd alone in strain K122 produces a more orally toxic
supernatant than does the expression of strain W14 (which contains a
mixture of Tca and Tcd). Weights (mean and standard error for nine
larvae) are relative to the mean weight for larvae fed on a supernatant
from nonrecombinant K122 (which is set at 1.0). The means and standard
errors for three independent experiments are shown. The estimated
EC50s are as follows: W14, 0.05×; Tca, 0.23×; and Tcd,
0.003× (see Discussion).
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In contrast to the results obtained in the experiments with
E. coli, both pA1 and pD1 lack a copy of
tccC but can still confer
oral toxicity on
P. luminescens strain K122. This result suggests
that a
functional
tccC homolog may be present already in
P. luminescens strain K122. Support for this
hypothesis was obtained by Southern
analysis of K122 DNA with a
tccC probe derived from W14 by PCR.
This blot reveals the
presence of several
tccC-like sequences
(Fig.
4a), supporting the hypothesis that
P. luminescens K122
indeed carries
tccC-like homologs. A similar Southern analysis
also
provides evidence for
P. luminescens K122
carrying
tcdA- and/or
tcdB-like genes as well
(Fig.
4b). These results infer
that, in these experiments, oral
toxicity is conferred on
P. luminescens K122
specifically by the
tcdA and
tcdB genes from
strain W14 but
that the
tccC-like gene function is provided
by the genetic background
of K122 itself.
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FIG. 4.
Southern analysis supports the hypothesis that
P. luminescens K122 carries homologs of
W14 tccC-like and tcdA-like genes. The
blots were probed with the highly conserved core region of the
tccC gene (10) (a) and the
P. luminescens W14 tcdAB
operon (b). Probes were derived by PCR from the W14 genome. The DNA was
digested and electrophoresed on 0.7% agarose gels and blotted using
standard techniques. Probe hybridization was visualized using the
Boehringer digoxigenin system. The locations of 1-kb marker DNA
fragments are shown between the two gels (from top to bottom,
10, 8, 6, 5, 4, 3, and 2 kb). Digests were as follows: E,
EcoRI; P, PstI; and H,
HindIII.
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Purification of recombinant toxins.
In order to determine the
particulate nature of the recombinant toxin complexes and as a prelude
to electron microscopy, we enriched the toxin complexes via DEAE column
chromatography. SDS-PAGE analysis of these particulate preparations was
then used to confirm the presence of novel polypeptides associated with
the recombinant toxin complexes. Particulate preparations of host
strain P. luminescens K122 show two predominant
species, of ~30 and 60 kDa. Expression of either the
tcaABC or the tcdAB operon results in the
appearance of additional protein bands putatively corresponding to
cleaved toxin complex fragments (Fig.
5a). The identities of these recombinant fragments were confirmed by Western blotting with anti-TcdA and anti-Tca antibodies (Fig. 5b). Note that the cleaved Tca fragment highlighted by the anti-Tca antibody is the same as that identified in
the particulate preparation from P. luminescens
W14.
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FIG. 5.
(a) Denaturing gel electrophoresis (SDS-PAGE) of
particulate preparations from P.
luminescens wild-type K122 and K122 expressing
recombinant Tca (pA1) and Tcd (pD1). Note that expression from the pA1
and pD1 plasmids leads to the production of additional protein species,
presumably toxin components. (b) Western analysis of P.
luminescens K122 expressing recombinant Tca with an
anti-Tca antibody. Note the detection of an ~60-kDa cross-reacting
species (arrowhead), putatively TcaBii. Note also that the
antibody recognizes the same species in a preparation from
P. luminescens W14.
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To further examine the compositions of the recombinant toxin complexes,
we used native nondenaturing gel electrophoresis.
Native gel analysis
of particulate preparations from host strain
K122 resolves two
complexes. However, recombinant expression of
either
tcaABC
or
tcdAB causes the resulting particulate preparation
to migrate as a single native complex with a mobility different
from that seen in the K122 parent strain (Fig.
6a). This result
suggests that
heterologous expression of
tca and
tcd genes from
strain W14 produces gene products that coassemble with complexes
from
host strain K122 or that alter the synthesis of certain native
K122
polypeptides. Subsequent analysis of these complexes by denaturing
SDS-PAGE confirmed the composition of each native complex. Excision
of
the predominant native band associated with recombinant
tca expression (Fig.
6a), electroelution, and reanalysis by SDS-PAGE
(Fig.
6b) confirmed that it consists of a specific subset of Tca-derived
polypeptides clustered at ~60 kDa (compare Fig.
5 with Fig.
6).
It
was not possible to determine exactly which toxin complex polypeptide
subfragments are represented without N-terminal protein sequencing
of
each fragment.
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FIG. 6.
(a) Native gel of particulate preparations from
P. luminescens K122 and K122 expressing
recombinant Tca (pA1) and Tcd (pD1). Note that preparations from the
acceptor strain P. luminescens K122
migrate as two clearly separable complexes, whereas coexpression of W14
Tca or Tcd results in a single native complex being produced by
recombinant P. luminescens K122. The
asterisk represents the recombinant Tca complex excised from the gel
and reanalyzed by SDS-PAGE. (b) Denaturing gel electrophoresis
(SDS-PAGE) of the same K122 and K122-Tca preparations as those analyzed
in panel a. Note that the native Tca complex excised from the gel in
panel a contains three predominant polypeptides (arrowheads). Note also
that this native complex forms visible particles under TEM examination
(see Fig. 8d).
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SDS-PAGE analysis of recombinant Tcd particulate preparations from
E. coli shows that mature Tcd components are more
abundant
in recombinant
E. coli than in
P. luminescens K122. Thus, large
polypeptides,
presumably corresponding to unprocessed TcdA and
TcdB, can clearly be
seen in recombinant
E. coli preparations
(Fig.
7) but are not visible in similar
preparations from host
strain
P. luminescens K122
(Fig.
5a). Importantly, TcdA and TcdB
appear to be produced at similar
levels either in the presence
(pD4) or in the absence (pD2) of TccC.
However, as noted above
(Fig.
2b), the
tcdA and
tcdB gene products become orally toxic
only after
coexpression with
tccC. These findings confirm that
TcdA and
TcdB are necessary but not sufficient for toxicity and
that TccC is
required to confer full oral toxicity to
M. sexta.
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FIG. 7.
Denaturing gel electrophoresis (SDS-PAGE) of particulate
preparations from E. coli expressing
recombinant TcdAB from three plasmids, pD2, pD3, and pD4. Species
corresponding to TcdA and TcdB are indicated by arrowheads.
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Electron microscopy.
To identify the supramolecular structures
associated with native and recombinant toxin complexes, we performed
TEM on particulate preparations from both recombinant E. coli and P. luminescens K122
containing toxin complex gene-carrying plasmids and also from the
original P. luminescens strain, W14. Particulate
preparations from E. coli carrying
tcd-containing plasmids show the presence of distinct
supramolecular complexes (Fig. 8a and b),
which are absent from control preparations of E. coli lacking the recombinant plasmids. These complexes are
~25 nm long and have a distinct head and protruding tail-like
substructure. Similar structures are also present in particulate
preparations from P. luminescens strain W14 (Fig.
8c) and in preparations from recombinant P. luminescens strain K122 (Fig. 8d). These complexes are
visible both in strains expressing tcdAB (Fig. 8a) and in
strains coexpressing tcdAB together with tccC
(Fig. 8b). These results suggest that only the tcdA and
tcdB gene products are necessary to form the visibly
distinct complexes. Further, coexpression of tccC does not
change the visible structure of the complexes (at this resolution) but
does confer on recombinant bacterial strains expressing all three genes
oral toxicity to M. sexta. These data suggest the
alternative hypotheses that either TccC activates the TcdA-TcdB complex
or TccC is itself a toxic component required for high levels of oral
toxicity to M. sexta.
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FIG. 8.
Transmission electron micrographs of particulate
preparations from wild-type and recombinant strains of
E. coli and P.
luminescens showing the supramolecular structure of the
toxin complexes. (a) E. coli expressing
TcdAB (pD2). Note the presence of visible complexes (circled);
however, these preparations are not orally toxic (Fig. 2a). (b)
E. coli coexpressing TcdAB and TccC. Note
the presence of the same visible particles, which are now orally toxic
(Fig. 2a). (c) Preparation from wild-type P.
luminescens W14 showing visible toxin complexes. (d)
Native gel preparation (asterisk in Fig. 6a) from K122 expressing
TcaABC, again showing the same particles. Bar, 50 nm. These data show
that TcdA and TcdB together are sufficient to produce visible particles
but that coexpression of TccC is essential for oral toxicity to
M. sexta (see Discussion).
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DISCUSSION |
Three genes are required for oral toxicity.
High levels of
recombinant Tcd-based oral toxicity to M. sexta
require the presence of three distinct genetic elements, i.e., TcdA,
TcdB, and TccC. Further, the data presented here suggest that
tccC itself is an essential part of this high-level oral toxicity. We note that in the plasmids used here, the copy of tccC was derived from the original (first-described)
tcc locus. However, it is important to realize that our
recent extended sequencing of the original four toxin complex loci
shows that further tccC-like sequences are also present both
upstream and downstream of the original tcd locus
(10). This result suggests that physical linkage in the
genome between tcdAB genes and tccC-like elements is not necessary in order for them to confer oral toxicity when cloned
together on the same recombinant plasmid. This hypothesis is supported
by our recent isolation of a P. luminescens W14
cosmid that encompasses tcdA, tcdB, and one of
the neighboring tccC-like elements and that confers oral
toxicity on the E. coli strain that carries it
(unpublished results).
The suggestion that three genetic elements are necessary for high
levels of oral toxicity to insects is consistent with the
work of
others on toxin complex gene homologs in the free-living
bacterium
S. entomophila. In this species, all three of the
equivalent
genes,
sepA,
sepB, and
sepC, are necessary and sufficient to confer
the amber
disease phenotype (which includes attack of the insect
gut) in the New
Zealand grass grub on recombinant
E. coli
(
6).
Further, Morgan and others, working with further
toxin complex
gene homologs from the nematode symbiont
Xenorhabdus nematophilus,
recently indicated that
high-level oral toxicity of recombinant
E. coli
also requires
tcdA,
tcdB, and
tccC
homologs (
xptA1,
xptB1,
and
xptC1)
(
8). Interestingly, however, these workers also
reported
that high-level expression of the
tcdA homolog alone
can
confer lower levels of oral toxicity on
E. coli.
In our experiments,
however, plasmids encoding
P. luminescens W14
tcdA but lacking
tcdB
could not be recovered due to consistent rearrangements in
the
resulting plasmid constructs. To date, we have therefore been
unable to
test the level of oral toxicity to
M. sexta
associated
with recombinant expression of
tcdA alone.
When either the
tcdAB or the
tcaABC operon was
expressed in a different
P. luminescens host,
strain K122, the presence of
W14
tccC was not required for
oral toxicity. This result suggests
that a
tccC-like homolog
already present in
P. luminescens K122
is able to
confer oral toxicity on recombinant W14 TcdAB. This
hypothesis is
supported by Southern analysis, which shows that
K122 carries
tcdA- and/or
tcdB-like sequences (Fig.
4b). If,
as
proposed,
P. luminescens K122 therefore
carries both
tcd- and
tccC-like gene homologs,
either they are not expressed in this
strain under the conditions
tested or they do not confer oral
toxicity to
M. sexta. As oral toxicity can be conferred on
P. luminescens K122 simply by the addition of either the
tcdAB or
the
tcaABC operon from strain W14, these
genes may themselves
encode the factors essential for lepidopteran gut
toxicity. Further,
these data also raise the formal possibility that
toxin complex
homologs in other
P. luminescens
strains that (like strain K122)
show no observable oral toxicity to
M. sexta may not be effective
on the lepidopteran
gut.
Finally, the observations presented here also have potential
implications for the putative mechanism(s) of secretion (and/or
display) of the toxin complex proteins from bacteria. First, oral
toxicity of the
P. luminescens W14 supernatant is
seen only in
stationary phase when the bacteria are cultured in vitro
(
3).
Second, and similarly, the oral toxicity of
Tcd-producing
E. coli supernatants increases
either with the age of the bacterial culture
or with the addition of
mitomycin C. One potential explanation
for these observations is that
recombinant toxin complexes are
cell associated and are released into
the culture supernatant
only when mitomycin C is added (presumably
resulting in increased
cell lysis and associated membrane disruption).
As the total level
of toxicity (combined cell- and
supernatant-associated oral toxicity)
also increases with the addition
of mitomycin C, this compound
also may stimulate increased Tcd
production via stress-induced
activation of the
tcd promoter
itself. At this stage, it is still
not clear how such large toxin
complexes are exported from the
bacterial cell without any obvious
secretion device. However,
the hypotheses presented above can be tested
directly and should
allow researchers to gain an understanding of the
relative contributions
of bacterial cell lysis and culture stress in
the production and
release of toxin complex
proteins.
In summary, here we have shown via TEM analysis that both recombinant
TcdAB complexes from
E. coli and native complexes
(of
unknown composition) from
P. luminescens W14
are ~25 nm long and
have a distinct head and tail-like substructure.
Coexpression
of TccC with TcdAB does not change the visual nature of
the complex
in TEM analysis but is required for full levels of oral
toxicity
to our model insect,
M. sexta. At
present, we are uncertain about
the significance of either the
structure of the toxin complexes
or the molecular role of the genetic
component
tccC. However,
we note two salient features that
may form the basis for further
investigation. First, the observation of
phage-related holin-lysin
genes within some toxin complex loci
(
10) raises the possibility
that they are released from
the bacteria via cell lysis. Second,
although the mode of action of the
toxin complex proteins remains
obscure, we note that the two TccC
homologs presently identified
for
Y. pestis both
contain putative active sites for protein tyrosine
phosphatases
(
10), enzymes that are absent from bacteria and
that are
typically associated with bacterial effectors, such as
SptP
(
7).
| |
ACKNOWLEDGMENTS |
This work was supported by a grant to R.F.-C. from the BBSRC.
Nucleotide sequencing was performed by P. Wilkinson with an ABI3700
sequencer supported by a Wellcome/JIF grant to R.F.-C.
We thank D. Bowen and T. Rocheleau, formerly in the laboratory of
R.F.-C. at the University of Wisconsin
Madison, for providing plasmids.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology and Biochemistry, University of Bath, Bath BA2 7AY, United
Kingdom. Phone: 44 1225 826261. Fax: 44 1225 826779. E-mail:
bssrfc{at}bath.ac.uk.
| |
REFERENCES |
| 1.
|
Blackburn, M.,
E. Golubeva,
D. Bowen, and R. H. ffrench-Constant.
1998.
A novel insecticidal toxin from Photorhabdus luminescens: histopathological effects of toxin complex A (Tca) on the midgut of Manduca sexta.
Appl. Environ. Microbiol.
64:3036-3041[Abstract/Free Full Text].
|
| 2.
|
Bowen, D.,
T. A. Rocheleau,
M. Blackburn,
O. Andreev,
E. Golubeva,
R. Bhartia, and R. H. ffrench-Constant.
1998.
Insecticidal toxins from the bacterium Photorhabdus luminescens.
Science
280:2129-2132[Abstract/Free Full Text].
|
| 3.
|
Bowen, D. J., and J. C. Ensign.
1998.
Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens.
Appl. Environ. Microbiol.
64:3029-3035[Abstract/Free Full Text].
|
| 4.
|
ffrench-Constant, R., and D. Bowen.
1999.
Photorhabdus toxins: novel biological insecticides.
Curr. Opin. Microbiol.
2:284-288[CrossRef][Medline].
|
| 5.
|
Guzman, L.-M.,
D. Belin,
M. J. Carson, and J. Beckwith.
1995.
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
J. Bacteriol.
177:4121-4130[Abstract/Free Full Text].
|
| 6.
|
Hurst, M. R.,
T. R. Glare,
T. A. Jackson, and C. W. Ronson.
2000.
Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens.
J. Bacteriol.
182:5127-5138[Abstract/Free Full Text].
|
| 7.
|
Kaniga, K.,
J. Uralil,
J. B. Bliska, and J. E. Galan.
1996.
A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium.
Mol. Microbiol.
21:633-641[CrossRef][Medline].
|
| 8.
|
Morgan, J. A.,
M. Sergeant,
D. Ellis,
M. Ousley, and P. Jarrett.
2001.
Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296.
Appl. Environ. Microbiol.
67:2062-2069[Abstract/Free Full Text].
|
| 9.
|
Perry, R. D., and J. D. Fetherston.
1997.
Yersinia pestisetiologic agent of plague.
Clin. Microbiol. Rev.
10:35-66[Abstract].
|
| 10.
|
Waterfield, N. R.,
D. J. Bowen,
J. D. Fetherston,
R. D. Perry, and R. H. ffrench-Constant.
2001.
The toxin complex genes of Photorhabdus: a growing gene family.
Trends Microbiol.
9:185-191[CrossRef][Medline].
|
Applied and Environmental Microbiology, November 2001, p. 5017-5024, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5017-5024.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
