Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M309859200 on January 5, 2004

J. Biol. Chem., Vol. 279, Issue 15, 14595-14601, April 9, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/15/14595    most recent
M309859200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, S. E.
Right arrow Articles by East, P. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, S. E.
Right arrow Articles by East, P. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Novel Secreted Protein Toxin from the Insect Pathogenic Bacterium Xenorhabdus nematophila*

Susan E. Brown{ddagger}, Anh T. Cao, Eric R. Hines, Raymond J. Akhurst, and Peter D. East

From the CSIRO Entomology, GPO Box 1700, Acton, Australian Capital Territory 2601, Australia

Received for publication, September 5, 2003 , and in revised form, December 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The bacterium Xenorhabdus nematophila is an insect pathogen that produces several proteins that enable it to kill insects. Screening of a cosmid library constructed from X. nematophila strain A24 identified a gene that encoded a novel protein that was toxic to insects. The 42-kDa protein encoded by the toxin gene was expressed and purified from a recombinant system, and was shown to kill the larvae of insects such as Galleria mellonella and Helicoverpa armigera when injected at doses of around 30–40 ng/g larvae. Sequencing and bioinformatic analysis suggested that the toxin was a novel protein, and that it was likely to be part of a genomic island involved in pathogenicity. When the native bacteria were grown under laboratory conditions, a soluble form of the 42-kDa toxin was secreted only by bacteria in the phase II state. Preliminary histological analysis of larvae injected with recombinant protein suggested that the toxin primarily acted on the midgut of the insect. Finally, some of the common strategies used by the bacterial pathogens of insects, animals, and plants are discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The availability of more than 100 microbial genomes, including the benign and pathogenic forms of closely related bacteria, is leading to an improved understanding of the common mechanisms used by pathogenic bacteria to invade and survive in hosts. The current evidence suggests that bacterial pathogens of various hosts share many genes that aid virulence and survival (1, 2). The products of such genes are often called virulence factors and include factors required for host and tissue tropism, cytotoxicity and multiplication within the host (3). These virulence genes are often present on genomic islands that have been shared among pathogens by horizontal gene transfer (4). The complete genomes available to date have a bias toward vertebrate pathogens, with only a few available for plant pathogens, and one for an invertebrate pathogen, the recently released Photorhabdus luminescens strain TT01 genome (5). The study of insect pathogens such as Xenorhabdus nematophila, P. luminescens, or Bacillus thuringiensis may provide insight into pathogen evolution and may also lead to the development of new insecticides (6).

Xenorhabdus and Photorhabdus are Gram-negative bacteria that live in symbiosis with nematodes (7–9). This bacteria-nematode association is highly toxic to many insect species, and in most cases the bacteria alone are highly virulent once they are circulating in the hemocoel of the insect (10). The bacteria and nematode share a complex life cycle, which includes symbiotic and pathogenic stages. During the symbiotic stage, the bacteria are carried in the gut of the nematode, but after infection of an insect host, the nematodes inject the bacteria into the insect hemocoel. Over several days, the combined actions of the nematode and bacteria kill the insect. Within the hemocoel of the insect carcass, the bacteria grow to stationary phase while the nematodes develop and sexually reproduce. The final stage of development is the re-association of the bacteria and nematodes to form non-feeding infective juveniles, which emerge from the insect carcass to find new hosts. The naturally occurring bacterial symbionts found in the gut of the nematodes are called phase I cells. Variant forms, called phase II, are rarely observed in vivo but are often observed under laboratory conditions (7, 9–11) and also in the free living clinical isolates of Photorhabdus (12). The phase II bacteria are still toxic to insects such as Galleria mellonella, despite having many altered properties, including motility and lipase, phospholipase, and protease activities (7, 9–11).

Throughout their life cycle, the bacteria and the nematodes produce a variety of metabolites to enable them to colonize and reproduce in the insect host. These metabolites often have overlapping functions, a strategy that is likely to contribute to the success of the nematode-bacteria association against a variety of insect hosts (9, 13). The metabolites produced include molecules to help evade the insect immune system, enzymes such as proteases, lipases, and phospholipases to maintain a food supply during reproduction (7, 9, 14, 15), and antifungal and antibacterial agents to prevent degradation or colonization of the insect carcass while the bacteria and nematodes reproduce (9, 16). The bacteria and nematodes also produce toxins that are responsible for killing the insect host. Analysis of the genome of P. luminescens identified more predicted toxin genes than any other bacteria sequenced to date, including potential gene products with homology to hemolysin A, chitinase, Rtx (repeats-in-toxin)-like toxin, and {delta}-endotoxin (5). The only toxins studied in detail are the Tc toxins from P. luminescens strain W14 (17–19), although a small amount of work has also been done on a 39-kDa toxin from X. nematophila (20), the large Xin toxin from X. nematophila strain BJ (21), and the PhlA hemolysin from P. luminescens strain TT01 (22). This paper describes the identification, purification, and characterization of a novel insect toxin from X. nematophila strain A24.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Toxin Gene—High molecular weight genomic DNA was isolated from X. nematophila strain A24 (23), partially digested with Sau3AI to generate fragments in the size range 30–50 kb, dephosphorylated, and ligated to linearized cosmid cloning vector "Supercos" (Stratagene) (24). The ligated DNA was packaged in vitro using Gigapack II XL Packaging Extract (Stratagene), transfected into the Escherichia coli strain NM554 (Stratagene), and screened by G. mellonella injection bioassay as described below. One clone, designated cos149, was chosen for further analysis. Deletion analysis of this clone (25) was performed using the Erase-a-Base kit (Promega) and the enzymes BamHI, ClaI, SphI, SacI, and HindIII (Roche Applied Science). Digested DNA was ligated into pGEM7Z(f)+ (Promega) and transformed by electroporation into E. coli strain DH5{alpha}, and the lysates were screened for insecticidal activity using the G. mellonella bioassay. The smallest of the deletion clones that retained insecticidal activity was sequenced on both strands using a combination of vector and gene-specific primers. Similarly, the DNA immediately surrounding the toxin gene and at the 5' and 3' ends of the cosmid clones and the intermediate deletion clones was sequenced using a combination of vector and sequence-specific primers. DNA sequencing was performed on an automated sequencer (Applied Biosystems model 377) using ABI PrismTM di-deoxy dye-terminator sequencing mix (Applied Biosystems). DNA was isolated from strains using a standard alkaline lysis procedure and analyzed by agarose gel electrophoresis (24).

Purification of Recombinant Toxin Protein—All protein purification steps were carried out at 4 °C unless otherwise stated. Expression of the full-length protein from X. nematophila strain A24 was performed in the IMPACT system (New England Biolabs), in which the toxin open reading frame was cloned at the 5' end of a self-splicing intein coding sequence fused to a short DNA sequence encoding a chitin binding domain. Recombinant plasmid containing the X. nematophila strain A24 toxin gene was prepared in the IMPACT vector pCYB3 and transformed into the E. coli strains DH10B or BL21(DE3) by electroporation. Cultures (500 ml) of LB1 broth containing 100 µg/ml ampicillin were grown at 37 °C until the A600 reached 0.5–0.6. Protein production was induced with 1 mM isopropyl-{beta}-thiogalactoside, and the cells were grown overnight at 14 °C. The preparation of bacterial cell extracts, affinity isolation of the fusion proteins on chitin columns, on-column dithiothreitol-mediated cleavage of the fusion proteins, and elution of the purified toxin proteins were all performed according to the manufacturer's instructions, except that 1 mM phenylmethylsulfonyl fluoride was added to the lysis and cleavage buffers. E. coli maltose-binding protein (MBP) was prepared from the IMPACTTM system using the same methods and was used as a negative control for the insect bioassays. Purified proteins were exchanged into phosphate-buffered saline, pH 7.4, and concentrated using Ultrafree spin cartridges (Millipore) with a nominal molecular mass cut-off of 5 or 10 kDa. Protein purity was checked by SDS-PAGE, using either the buffer system of Laemmli (26) with a Bio-Rad Mini-Protean apparatus, or the NuPAGE Novex system (Invitrogen). All gels were stained with Fast Stain (Zoion Research). Protein concentrations were determined by the Bradford assay using the Bio-Rad Protein Assay reagent and BSA (Sigma) as a standard. The identity of the protein was confirmed by mass spectroscopy on a Voyager Elite matrix-assisted laser desorption ionization-time of flight-mass spectrometer (Perseptive Biosystems) for both whole and trypsin-digested protein samples. Mass spectroscopy on the whole protein was performed using sinapinic acid as the matrix and BSA as the standard. The in-gel tryptic digest and subsequent mass spectroscopy were carried out essentially as described previously (27), except that the gel pieces were not treated with dithiothreitol and iodoacetamide. Theoretical molecular mass and trypsin digest patterns were calculated using Protein Prospector (prospector.ucsf.edu/, version 3.4.1).

Bioinformatics—Programs available via BioManager (www.angis.org.au/, release 4.0) were used to analyze the DNA sequences for the presence of open reading frames (Flip). Searches of the non-redundant nucleotide and protein data bases (released Oct. 17, 2003) were performed using the various BLAST programs (version 2.2.6) available via NCBI (www.ncbi.nlm.nih.gov/BLAST/). Motif and fold recognition analysis of the protein sequence was performed using programs such as InterProScan (www.ebi.ac.uk/interpro/, July 22, 2003 (28)), PANAL (mgd.ahc.umn.edu/panal/ (29)), Genequiz (jura.ebi.ac.uk:8765/ext-genequiz/ (30)), and the Structure Prediction Metaserver (bioinfo.pl/Meta/pdb-test.pl/ (31)). The secondary structure was predicted using NPS@ (npsa-pbil.ibcp.fr/ (32)). The amino acid composition of the sequence was compared with bacterial sequences available at EBI (www.ebi.ac.uk/proteome/). The sequence was also analyzed using programs available via ExPASy (kr.expasy.org/tools/), including SignalP (version 1.1) and PSORT (version 6.4).

Toxin Expression in Native Bacteria—Phase I and phase II bacteria from glycerol stocks of X. nematophila strain A24 were streaked on LB agar plates and grown at 28 °C for 2 days. The phase of the bacteria was confirmed by growth on nutrient agar, bromothymol blue, and tetrazolium chloride agar plates (33) and assays for antibiotic and lecithinase activity (34). Single colonies from the LB plates were used to inoculate 5 ml of nutrient broth (13 g/liter, Oxoid), and cultures were grown at 200 rpm for 24 or 48 h at 14, 20, or 28 °C. Cultures (2 ml) were harvested by centrifugation (3000 x g, 10 min), and the supernatant was removed (the soluble secreted material). The cells were resuspended in 1 ml of lysis buffer (20 mM Tris-Cl, 10 mM NaCl, pH 8), and 500 µg of lysozyme was added to 500 µl of this suspension and left on ice for 30 min. After boiling for 2 min, 50 µg of DNase I was added, and the sample was left at 37 °C for 1 h. The resulting suspension was centrifuged (3000 x g, 10 min), and the supernatant was removed (the soluble material from lysis). The final pellet (the insoluble material from lysis) was resuspended in 500 µl of phosphate-buffered saline. The samples were analyzed by SDS-PAGE and Western blot (see below).

Antibody Production—Antibody was raised against his6 V16tox,2 a P. luminescens protein closely related to A24tox, following standard procedures at the Institute for Medical and Veterinary Science (Adelaide, Australia). Approximately 400 µg of purified his6V16tox protein was used to immunize each of two New Zealand White rabbits by subcutaneous injection. Each animal received a primary inoculation and three boosts at 3-week intervals. Following the final boost ~40 ml of serum was collected from each animal. The antiserum was used without further purification.

Western Blots—Western blots were performed using a nitrocellulose membrane (Hybond-C Extra, Amersham Biosciences) on a Novablot semi-dry blotter at 0.8 mA/cm2 in transfer buffer (39 mM glycine, 48 mM Tris-Cl, 0.375% SDS). The membrane was processed at room temperature in Tris-buffered saline containing 0.1% Tween 20 using three 5-min washes between all steps. The steps used were a 1-h or overnight block with 3% BSA, a 1-h incubation with the toxin antibody (1:3000 dilution), and a 1-h incubation with anti-rabbit IgG alkaline phosphatase conjugate (Sigma, 1:30,000 dilution). The blots were visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega) in substrate buffer (100 mM Tris-Cl, 5 mM MgCl2, 100 mM NaCl, pH 9.5).

Insects—Helicoverpa armigera (Lepidoptera) were reared until fourth instar on an artificial diet (35). G. mellonella (Lepidoptera) were reared at 27 °C until fourth instar on an artificial diet containing dried rice cereal (500 g), honey/glycerol mix (154 ml of honey, 179 ml of glycerol, 37 ml of water), brewers yeast (30 g), and methyl 4-hydroxybenzoate (Tegasept, 0.2% w/w). Lucilia cuprina (Diptera) were reared at 27 °C until third instar on an artificial liver and meat meal diet.

Bioassays—The activity of crude extracts and purified proteins was determined using an intra-hemocoel injection assay against the insects G. mellonella, H. armigera, and L. cuprina. Larvae were treated with 5–10 µl of either crude or pure sample, which was injected through an intersegmental membrane into the abdominal region. Bioassays were performed on at least 10 larvae that were held at 22 °C, and mortality was recorded daily over 3–6 days. For testing of the cosmid library against G. mellonella, crude extracts were prepared by growing E. coli cultures overnight at 28 °C in LB broth containing 150 µg/ml ampicillin and treating them with 2 mg/ml lysozyme (Amresco) for 15 min. The oral activity of this crude extract was also determined by incorporating 5 ml of crude extract into 95 ml of diet for H. armigera. The temperature dependence of the insecticidal activity was determined using crude extracts of cultures of A24tox in pGEM7Z(f)+/DH5{alpha} that were grown overnight at 37 °C, treated with lysozyme (1 mg/ml), and left at room temperature for 30 min. These extracts were injected into G. mellonella larvae that were kept at 20 and 25 °C. The activity of purified recombinant A24tox was tested against all three insects, using a dose range of 1–1000 ng of protein per larva for G. mellonella and H. armigera and 10–100 ng of protein per larva for L. cuprina. For G. mellonella and H. armigera, data were fitted to the probit curve using POLO-PC (LeOra software, 1987), a program that corrects for natural mortality.

Histology—Fourth instar H. armigera larvae were injected with 10 µl of purified A24tox (100 ng) or MBP (100 ng). At 18 h post-injection, fixative (4% formaldehyde in 100 mM sodium phosphate, pH ~7.2) was injected into three larvae; holes were punctured into the cuticle, and the larvae were transferred to fixative overnight at 4 °C. The larvae were embedded in paraffin wax, sectioned at 6 µm, and the sections stained with hematoxylin and eosin. Images were captured with a ProgRes 3012 digital camera on a Leica Diaplan microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of Toxin Gene—Xenorhabdus and Photorhabdus bacteria are known to be toxic to insects, but to date the only toxins from the bacteria that have been studied in detail are the Tc toxins. In order to identify other insect toxins from the bacteria, crude extracts from a cosmid library constructed from X. nematophila strain A24 were tested for insecticidal activity using a G. mellonella injection bioassay. The library screen identified two clones with insecticidal activity, whereas control lysates prepared from E. coli NM554 cells containing nonrecombinant Supercos vector showed no insecticidal activity. Both cosmid clones appeared to contain the same region of ~35 kb of X. nematophila genomic DNA. Deletion analysis of this cosmid clone, further injection bioassays, and DNA sequencing identified an open reading frame of 1104 bp (368 amino acids) that was required for toxicity (Fig. 1). The DNA sequence has been deposited in GenBankTM (accession number AX029373 [GenBank] ). An analysis of the 5' region of the gene identified possible imperfect –10 and –35 RNA polymerase recognition sites and an imperfect Shine-Dalgarno sequence, suggesting that the open reading frame was likely to be transcribed. Note that it is not possible to say whether the 1.1-kb toxin gene is part of the genome or a megaplasmid, as the cosmid clone had a size of ~35 kb, and X. nematophila strain A24 is known to contain megaplasmids of size 70 and 120 kb (11).



View larger version (107K):
[in this window]
[in a new window]
  		FIG. 1.
The DNA sequence of X. nematophila A24tox, with the amino acid sequence of the open reading frame shown below the DNA sequence. Shaded in gray are amino acids that belong to peptides that were identified by mass spectroscopy on a tryptic digest of A24tox.

 
Purification and Characterization of Toxin Protein—Biochemical characterization of the toxin encoded by the cloned gene from X. nematophila strain A24 required recombinant expression of the full-length protein. Initial attempts to overproduce the protein from commonly used E. coli vectors and strains, such as pGEM7Zf(+) in DH10B, resulted in the expression of very small amounts of protein, most of which was insoluble. Similarly, the toxin was mostly insoluble when expressed as a glutathione S-transferase fusion protein using the vector pGEX (Amersham Biosciences). Eventually, a useful amount of soluble protein without N- or C-terminal modifications was obtained by expressing the toxin as a fusion protein in the IMPACT system. After expression and purification according to the manufacturer's instructions, the A24tox construct produced a major soluble protein product of the expected size of ~42 kDa, as determined by SDS-PAGE (Fig. 2A) and mass spectroscopy (see below). The final preparations contained 90% or more of the toxin protein, along with contaminants that varied slightly depending on the cleavage and wash conditions used during the purification process. Western blotting with an antibody known to be highly specific for the toxin indicated that some of these contaminants were related to A24tox, in particular the uncleaved fusion protein that runs at about 90 kDa on SDS-PAGE (Fig. 2B). E. coli GroEL (~57–60 kDa) can also co-purify in small amounts with the target protein (www.neb.com/neb/faqs/impact.html). After purification and concentration, the yield of A24tox was ~0.4 mg per liter of E. coli culture. The identity of the protein was confirmed by mass spectroscopy, where an analysis of the tryptic digest of A24tox identified peptides that covered 77% of the predicted open reading frame, including peptides from both the N and C termini (Fig. 1). The measured molecular weight (41,485) was within instrumental error (0.05%) of the calculated molecular weight (41,502). Both the whole protein and tryptic digest spectra suggested that the N-terminal methionine residue of the recombinant protein had not been processed.



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 2.
A, NuPAGE of A24tox protein purified from the IMPACT system. Samples are A24tox at ~0.5 (lane 1) and 0.25 µg (lane 2), with the molecular mass marker positions shown in kDa on the left. B, Western blot of the gel shown in A using the toxin antibody, with the molecular mass marker positions shown in kDa on the left.

 
Biological Activity of the Toxin—The biological activity of the recombinant purified protein was tested by an intra-hemocoel injection bioassay, a method that delivers the bacteria directly into the hemolymph and thus mimics the release of the bacteria into the hemolymph that occurs soon after a nematode infects the insect host (7, 8). The bioassay results indicated that purified A24tox killed a high percentage of G. mellonella, H. armigera, or L. cuprina larvae after a single injection of at least 10 ng of toxin per larva. MBP purified from the IMPACT system was used as a control for these experiments and did not cause significant larval mortality (0–5%) at any of the quantities tested. This confirmed that A24tox was responsible for the observed toxicity and not any of the minor contaminants present in the toxin purification. With H. armigera, the larvae ceased feeding almost immediately and were dead after 2–3 days, and for G. mellonella, the larvae were unable to spin cocoon silk and were dead after 6 days. Mortality was dependent on the concentration of the toxin, as shown in Fig. 3A for G. mellonella and H. armigera. The LD50 values were calculated by probit analysis (Fig. 3A) and are listed with the 95% confidence limits shown in parentheses. G. mellonella was killed effectively by A24tox (LD50 = 26 ng (11–48 ng)), as was H. armigera (LD50 = 5.2 ng (1.4–9.0 ng)). Because the average larval mass at the time of injection was ~140 mg for H. armigera and ~800 mg for G. mellonella, this indicated that the LD50 values were in the range 30–40 ng/g. The A24tox protein was also active by injection against L. cuprina larvae, with an observed mortality of 60, 100, and 100% at doses of 10, 50, and 100 ng, respectively, compared with a MBP control mortality of 5–10%. Crude extracts of A24tox from E. coli strains, which were known to be active by injection, did not have any oral activity when incorporated into the H. armigera diet (data not shown). The activity of an E. coli crude extract against G. mellonella larvae was found to be temperature-dependent (Fig. 3B), with the toxin causing 100% mortality in approximately half the time at 25 °C as compared with 20 °C.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 3.
The biological activity of A24tox. A, dose-response curve for the injection bioassay of recombinant A24tox (1 ng to 1 µg) against G. mellonella ({blacktriangleup}) and H. armigera () larvae. The experimental points are shown as shapes, and the fitted lines were calculated using a cumulative distribution function. B, the temperature dependence of the activity of lysed extracts of cultures of A24tox in pGEM7Z(f)+/DH5{alpha} against G. mellonella. pGEM7Z(f)+ at 20 °C (+) and 25 °C (x), and A24tox/pGEM7Z(f)+ at 20 °C ({blacksquare}) and 25 °C ({diamondsuit}).

 
It is clear that a major target of the toxin is the caterpillar midgut (Fig. 4). Control animals injected with MBP showed the expected histology for healthy H. armigera larvae (Fig. 4a). At 18 h after the injection of toxin, we observed disruption of the basement membrane and intercellular plasma membrane connective structures, with numerous cells sloughed into the gut lumen, and a large amount of unidentified material appearing in the gut lumen (Fig. 4b). The damage was consistent throughout the midgut, with no damage seen to the fore- or hindgut. Some damage was also observed in the fat body.2



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 4.
Histological analysis of A24tox-treated H. armigera larvae. The images are of longitudinal sections through the anterior region of the midgut of fourth instar larvae 18 h after injection with 100 ng of MBP (a) or A24tox (b). The field of view is 310 µm. BM, basement membrane; E, midgut epithelium; L, midgut lumen. An open arrowhead indicates spaces between cells of the gut epithelium; a solid arrowhead indicates breakdown of the basement membrane, a white asterisk indicates cells sloughed into the midgut lumen, and a x indicates rafts of unidentified material observed only in midgut lumen of toxin-treated larvae.

 
Expression of the toxin in laboratory cultures of the native X. nematophila strain A24 bacteria was examined by Western blot using the antibody raised against the his6V16tox protein (Fig. 5). The toxin was detected in the cell lysate of both phase I and II bacteria and in the extracellular secreted fraction of phase II but not phase I bacteria. However, the toxin detected in the cell lysate was insoluble, and soluble toxin was detected only in the secreted material of phase II bacteria. A24tox was expressed under all growth conditions (24 or 48 h, and 14, 20, or 28 °C), but the greatest amount of soluble toxin was produced when the cultures were grown at 20 °C for 48 h. Note that a Western blot showed that toxin protein overproduced from E. coli cultures (A24tox in pGEM7Z(f)+/DH10B) was not secreted.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 5.
Western blot of toxin production by the native X. nematophila strains A24/1 (phase I) and A24/2 (phase II), which were grown for 24 h in nutrient broth media at 20 °C. The lane markings are as follows: se, material secreted into the culture broth; so, soluble material after lysis of the cell pellet; in, insoluble material after lysis of the cell pellet; IMP, IMPACT purified protein. Samples are A24/1 secretion supernatant (lane 1), A24/1 soluble material after lysis (lane 2), A24/1 insoluble material after lysis (lane 3), A24 IMPACT purified (lane 4), A24/2 secretion supernatant (lane 5), A24/2 soluble material after lysis (lane 6), A24/2 insoluble material after lysis (lane 7), and marker (lane 8).

 
Bioinformatic Analysis of Toxin—Bioinformatic analysis of the A24tox gene was performed to gain some idea of the biological function of the toxin. Searches of the non-redundant nucleotide and protein data bases were performed for the A24tox nucleotide and protein sequences using the blastp, blastx, and tblastx programs. The only statistically significant similarity was to a hypothetical protein with no annotation in the related bacterium P. luminescens (plu2326, expect value 10–131). Detailed analysis of the amino acid sequence using methods such as similarity searches and motif and fold recognition also showed no significant matches. Thus it appeared that the toxin had no detectable homologs in the current data bases and therefore was a novel protein with a potentially novel function.

An analysis of the amino acid composition of A24tox, as compared with the bacteria E. coli K12, Yersinia pestis, and Salmonella typhi, suggested that the protein is rich in polar amino acids such as Lys, Asn, Asp, and Gln, poor in amino acids such as Ala, Gly, and Val, and contains no Cys residues. The secondary structure prediction suggested that the protein was likely to be predominantly {alpha}-helical, containing 40% helix and 7% strand. The protein was predicted to contain no signal peptide cleavage sites (SignalP) and to possibly be located on the bacterial inner membrane (PSORT).

Information about the genetic context of the toxin gene was also obtained by sequencing the DNA surrounding the toxin gene. The sequenced region was 3270 bp, including 1014 bp upstream and 1149 bp downstream of the A24tox gene. Single sequence reads (~500 bp) were also obtained from the original cosmid clones or the intermediate deletion clones that were produced when isolating the toxin gene. The exact distance of these DNA segments from the toxin gene was not always known, but the sequences must be within 35 kb of the toxin gene and thus could still provide some information about the broader genetic context of the toxin. The sequenced DNA was subject to blastx and tblastx searches to identify any homologous proteins, and the results are summarized in Table I. The 3270-bp region was also analyzed by the program Flip, which predicted a possible open reading frame just downstream of the toxin gene, corresponding to the ygfY/ygfX gene product from E. coli that was identified by the tblastx search (Table I). No open reading frames were predicted for the region upstream of the toxin gene. The results (Table I) allowed three observations to be made about the genetic context of the toxin gene. First, the number of homologs identified was quite low when considering the length of DNA sequence that was obtained. Second, of the few homologs that were identified (Table I), the matching proteins were frequently found in other bacteria, including pathogens and extremophiles, were often marked as imported or carried on plasmids, and were often implicated in virulence (glycine dehydrogenase, single-stranded DNA exonuclease, and cobyrinic acid a,c-diamide synthase). Finally, the average GC content of the 3270-bp region was about 35%.


View this table:
[in this window]
[in a new window]
  		TABLE I
Homologous genes found from blastx and tblastx searches on the DNA sequences immediately surrounding the A24tox toxin gene and in the original cosmid clones and deletion clones

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A24tox Is a Novel Toxin—We have identified a toxin gene from a cosmid DNA library of the insect-pathogenic bacterium X. nematophila strain A24 (Fig. 1). Sequence similarity searches showed that the A24tox protein corresponding to the open reading frame of the toxin gene was homologous to a hypothetical protein identified from the recently sequenced genome of P. luminescens strain TT01. This suggested that the toxin was present in other related bacteria, which is consistent with our observation that the toxin is widespread in various Photorhabdus and Xenorhabdus strains.2 Detailed bioinformatic analyses of the A24tox protein, including similarity searches, motif recognition, or fold recognition, produced no significant matches to proteins with known structure or function. This suggested that A24tox is a novel virulence factor, with no currently discernible relationship to known toxins.

Other toxin proteins isolated and characterized from bacteria related to Xenorhabdus are the Tc toxins from P. luminescens strain W14 (17–19), a 39-kDa toxin from X. nematophila (20), the large Xin toxin from X. nematophila strain BJ (21), and the PhlA hemolysin from P. luminescens strain TT01 (22). Of these, only the Tc toxins have been studied in any detail. The 39-kDa toxin was shown to be secreted, active by hemolymph injection against G. mellonella larvae, and could be purified by anion-exchange chromatography (20). However, the limited information available for this protein makes it impossible to determine whether it is the same toxin that we have identified here. There is also limited information available about the Xin toxin, which has a molecular mass of greater than 1000 kDa, is expressed intercellularly, and is orally toxic to H. armigera larvae (21). The PhlA hemolysin was shown not to be a major virulence determinant (22). The Tc toxins from P. luminescens W14 form a large protein complex that is toxic to insects by either ingestion or injection (17–19). The high molecular mass complex consists of about 10 polypeptides, ranging in size from 30 to 200 kDa, which is secreted from the bacteria and requires proteolytic processing for activity. Multiple tc genes have been identified in several Photorhabdus species (5, 36, 37), and tc-like genes have been identified in other insect-associated bacteria (X. nematophila (36, 38), Serratia entomophila (39), and Y. pestis (36)), as well as in Pseudomonas syringae pv. tomato, Fibrobacter succinogenes, and Treponema denticola (40). The toxin we have characterized in this work is quite different from the Tc toxins. It appears to be an isolated gene that produces a single 42-kDa protein. It does not seem to require proteolytic processing to be toxic. Moreover, in the insects tested it is only active by intra-hemocoel delivery. Because we can not make any significant annotation for the A24tox gene or protein, and it is different to the known insect toxins, we must conclude that it is a novel toxin.

Mode of Action of A24tox—A24tox has insecticidal activity (Fig. 3A), causing larvae to cease feeding almost immediately after toxin injection. The larvae then take several days to die and have a mortality rate that is dependent on temperature (Fig. 3B). The dose of A24tox required to kill insects (30–40 ng/g of larvae) is of the same order of magnitude as that of other insecticidal toxins, such as the Tc toxins from Photorhabdus against M. sexta larvae (18), and the insecticidal crystal proteins of B. thuringiensis against Helicoverpa zea (41) and Leptinotarsa decemlineata (42). Injection or ingestion of the Tca complex of P. luminescens strain W14 by M. sexta larvae causes damage to the midgut cells, resulting in a shedding of the midgut epithelium into the gut lumen followed by lysis of the epithelium (43). This histopathology was similar to that observed with the {delta}-endotoxins from B. thuringiensis and cholesterol oxidase from Streptomyces (43), although it is worth noting that similar histopathologies do not necessarily imply the same mode of action. In comparison, although the mode of action of the A24tox protein is not yet known, it also causes significant damage to the midgut of toxin-treated H. armigera larvae (Fig. 4), a histopathology that is consistent with the observations that the larvae cease feeding almost immediately after toxin injection. However, the observed pathology of the midgut2 (cells being sloughed into the gut lumen, disruption of the basement membrane and intercellular attachments, a large amount of unidentified material appearing in the gut lumen, and no evidence of cell lysis) is different from that observed with either the Tc or Bt toxins.

X. nematophila Strain A24 Has an Active Secretion System for Toxins—When the native X. nematophila bacteria were cultured under laboratory conditions, soluble A24tox was detected only in the extracellular secreted material of phase II bacteria (Fig. 5). These results indicated that X. nematophila had an operational secretion system. Because there are no identifiable secretion signal peptides at the N terminus of the toxin, it is possible that A24tox is secreted by a type III secretion system. These results are consistent with reports that Xenorhabdus and Photorhabdus secrete a large number of proteins, including the Tc toxins (37), and the genomic analysis of the related P. luminescens that predicted proteins with homology to the Yersinia type III secretion machinery (5, 37). It was also interesting that the toxin was secreted only in the phase II cultures. Phase I bacteria are the active bacteria in the nematode gut, whereas phase II bacteria are most commonly observed under laboratory conditions and as yet have an unknown role in vivo. Both phase I and phase II bacteria are known to be toxic to insects (33). In general, phase changes are known to assist bacteria in responding to environmental changes, and there is a suggestion that different bacterial phases may be important for evading host defense systems (7, 9, 10).

A24tox Is Part of a Genomic Island—Analysis of the DNA near the toxin gene showed that the genetic region immediately adjacent to the toxin gene had relatively low homology to sequences in the current data bases. This was also seen for the region surrounding the Tc toxins from P. luminescens strain W14, where a high proportion (15 of 40) of putative open reading frames near the tc genes showed no homology to sequences in the current data bases (36). These results suggest that there are significant differences between Xenorhabdus and Photorhabdus and other bacteria. Furthermore, the few homologs that were identified in the analysis of the sequenced DNA of X. nematophila (Table I) suggested that the bacterium had shared genetic material with other bacteria, that the toxin genes were associated with other genes possibly involved in virulence, that the 35% GC content of the DNA region containing the toxin was different from the 43–44% estimated for the whole genome (5, 44), and that mobile genetic factors such as transposons were present. These are all indicators of pathogenicity islands or genomic islands (4), suggesting that A24tox is part of a genomic island that is involved in pathogenicity. This is consistent with observations that the P. luminescens genome contains several genomic islands that are involved in functions such as symbiosis and pathogenicity (45).

Common Virulence Strategies of Pathogenic Bacteria—It is interesting to compare the virulence strategies used by insect pathogens such as Xenorhabdus and Photorhabdus to those used by other pathogens (2, 46). Our analysis of the A24tox gene and surrounding DNA indicated that the bacteria had shared genetic material with other bacteria and that the A24tox gene was likely to be part of a genomic island involved in pathogenicity. Similar observations were also made for the tc-like genes from P. luminescens, X. nematophila, and other bacteria (36, 38–40) and the mcf gene from P. luminescens (47). We also showed that X. nematophila had an active secretion system, and others (5, 37) have shown that the genome of P. luminescens contains many proteins predicted to be involved in secretion systems. The Tc toxins in P. luminescens have a high level of redundancy, where knockout of any one of the tc gene products causes a reduction but not a loss of toxicity (5, 19). Many homologs of adhesins, invasins, and fimbria proteins have been found in P. luminescens and X. nematophila (5, 10, 37, 48) and are often used to aid colonization of the host (2). These four examples, acquisition of genetic material by horizontal transfer, the use of secretion systems for virulence factors, the production of virulence factors with a high level of redundancy, and the use of pili and fimbriae to aid colonization, clearly indicate that the insect-pathogenic bacteria have much in common with other animal and plant bacterial pathogens (2). There is still some debate on the species classification of Xenorhabdus and Photorhabdus, but because the invertebrates evolved before vertebrates, it is possible that these insect pathogens are the ancestors of the animal pathogens. It is also clear that insect pathogens such as Xenorhabdus and Photorhabdus may be a potentially rich resource for the discovery of additional virulence mechanisms and proteins with other novel bioactivities.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 61-2-6246-4053; Fax: 61-2-6246-4173; E-mail: sue.brown{at}csiro.au.

1 The abbreviations used are: LB, Luria-Bertani; BSA, bovine serum albumin; MBP, maltose-binding protein. Back

2 S. Brown, A. Cao, P. Dobson, E. Hines, R. Akhurst, P. East, manuscript in preparation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Keen, N., Staskawicz, B., Mekalanos, J., Ausubel, F., and Cook, R. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8752–8753[Free Full Text]
  2. Cao, H., Baldini, R. L., and Rahme, L. G. (2001) Annu. Rev. Phytopathol. 39, 259–284[CrossRef][Medline] [Order article via Infotrieve]
  3. Shea, J. E., Santangelo, J. D., and Feldman, R. G. (2000) Curr. Opin. Microbiol. 3, 451–458[CrossRef][Medline] [Order article via Infotrieve]
  4. Hacker, J., and Kaper, J. B. (2000) Annu. Rev. Microbiol. 54, 641–679[CrossRef][Medline] [Order article via Infotrieve]
  5. Duchaud, E., Rusniok, C., Frangeul, L., Buchrieser, C., Givaudan, A., Taourit, S., Bocs, S., Boursaux-Eude, C., Chandler, M., Charles, J. F., Dassa, E., Derose, R., Derzelle, S., Freyssinet, G., Gaudriault, S., Medigue, C., Lanois, A., Powell, K., Siguier, P., Vincent, R., Wingate, V., Zouine, M., Glaser, P., Boemare, N., Danchin, A., and Kunst, F. (2003) Nat. Biotechnol. 21, 1307–1313[CrossRef][Medline] [Order article via Infotrieve]
  6. Hilder, V. A., and Boulter, D. (1999) Crop Prot. 18, 177–191[CrossRef]
  7. Forst, S., Dowds, B., Boemare, N., and Stackebrandt, E. (1997) Annu. Rev. Microbiol. 51, 47–72[CrossRef][Medline] [Order article via Infotrieve]
  8. Burnell, A. M., and Stock, S. P. (2000) Nematology 2, 31–42
  9. Akhurst, R. J., and Dunphy, G. B. (1993) in Parasites and Pathogens of Insects (Beckage, N. E., Thompson, S. N., and Federici, B. A., eds) pp. 1–23, Academic Press, Inc., San Diego, CA
  10. Forst, S., and Nealson, K. (1996) Microbiol. Rev. 60, 21–43[Free Full Text]
  11. Smigielski, A. J., and Akhurst, R. J. (1994) J. Invertebr. Pathol. 64, 214–220
  12. Peel, M. M., Alfredson, D. A., Gerrard, J. G., Davis, J. M., Robson, J. M., Mcdougall, R. J., Scullie, B. L., and Akhurst, R. J. (1999) J. Clin. Microbiol. 37, 3647–3653[Abstract/Free Full Text]
  13. Collmer, A., Lindeberg, M., Petnicki-Ocwieja, T., Schneider, D. J., and Alfano, J. R. (2002) Trends Microbiol. 10, 462–469[CrossRef][Medline] [Order article via Infotrieve]
  14. Bowen, D., Blackburn, M., Rocheleau, T., Grutzmacher, C., and Ffrench-Constant, R. H. (2000) Insect Biochem. Mol. Biol. 30, 69–74[CrossRef][Medline] [Order article via Infotrieve]
  15. Thaler, J. O., Duvic, B., Givaudan, A., and Boemare, N. (1998) Appl. Environ. Microbiol. 64, 2367–2373[Abstract/Free Full Text]
  16. Akhurst, R. (1993) in Nematodes and the Biological Control of Insect Pests (Kaya, H., Bedding, R., and Akhurst, R., eds) pp. 127–135, CSIRO Australia, East Melbourne, Australia
  17. Guo, L. N., Fatig, R. O., Orr, G. L., Schafer, B. W., Strickland, J. A., Sukhapinda, K., Woodsworth, A. T., and Petell, J. K. (1999) J. Biol. Chem. 274, 9836–9842[Abstract/Free Full Text]
  18. Bowen, D. J., and Ensign, J. C. (1998) Appl. Environ. Microbiol. 64, 3029–3035[Abstract/Free Full Text]
  19. Bowen, D., Rocheleau, T. A., Blackburn, M., Andreev, O., Golubeva, E., Bhartia, R., and Ffrench-Constant, R. H. (1998) Science 280, 2129–2132[Abstract/Free Full Text]
  20. Ryu, K. G., Bae, J. S., Yu, Y. S., and Park, S. H. (2000) Biotechnol. Bioprocess Eng. 5, 141–145
  21. Pan, Y. H., Jian, H., Zhang, J., Liu, Z., Chen, Z. Y., Yang, X. F., Yang, H. W., and Huang, D. F. (2002) Prog. Nat. Sci. 12, 310–312
  22. Brillard, J., Duchaud, E., Boemare, N., Kunst, F., and Givaudan, A. (2002) J. Bacteriol. 184, 3871–3878[Abstract/Free Full Text]
  23. Marmur, J. (1961) J. Mol. Biol. 3, 208–218
  24. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  25. Henikoff, S. (1984) Gene 28, 351–359[CrossRef][Medline] [Order article via Infotrieve]
  26. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  27. Campbell, P. M., Harcourt, R. L., Crone, E. J., Claudianos, C., Hammock, B. D., Russell, R. J., and Oakeshott, J. G. (2001) Insect Biochem. Mol. Biol. 31, 513–520[CrossRef][Medline] [Order article via Infotrieve]
  28. Zdobnov, E. M., and Apweiler, R. (2001) Bioinformatics 17, 847–848[Abstract/Free Full Text]
  29. Silverstein, K. A., Kilian, A., Freeman, J. L., Johnson, J. E., Awad, I. A., and Retzel, E. F. (2000) Bioinformatics 16, 1157–1158[Abstract/Free Full Text]
  30. Andrade, M. A., Brown, N. P., Leroy, C., Hoersch, S., de Daruvar, A., Reich, C., Franchini, A., Tamames, J., Valencia, A., Ouzounis, C., and Sander, C. (1999) Bioinformatics 15, 391–412[Abstract/Free Full Text]
  31. Bujnicki, J. M., Elofsson, A., Fischer, D., and Rychlewski, L. (2001) Bioinformatics 17, 750–751[Abstract/Free Full Text]
  32. Combet, C., Blanchet, C., Geourjon, C., and Deleage, G. (2000) Trends Biochem. Sci. 25, 147–150[CrossRef][Medline] [Order article via Infotrieve]
  33. Akhurst, R. J. (1980) J. Gen. Microbiol. 303–309
  34. Boemare, N. E., and Akhurst, R. J. (1988) J. Gen. Microbiol. 134, 751–761
  35. Akhurst, R. J., James, W., Bird, L. J., and Beard, C. (2003) J. Econ. Entomol. 96, 1290–1299[Medline] [Order article via Infotrieve]
  36. Waterfield, N. R., Bowen, D. J., Fetherston, J. D., Perry, R. D., and Ffrench-Constant, R. H. (2001) Trends Microbiol. 9, 185–191[CrossRef][Medline] [Order article via Infotrieve]
  37. Ffrench-Constant, R. H., Waterfield, N., Burland, V., Perna, N. T., Daborn, P. J., Bowen, D., and Blattner, F. R. (2000) Appl. Environ. Microbiol. 66, 3310–3329[Abstract/Free Full Text]
  38. Morgan, J. A. W., Sergeant, M., Ellis, D., Ousley, M., and Jarrett, P. (2001) Appl. Environ. Microbiol. 67, 2062–2069[Abstract/Free Full Text]
  39. Hurst, M. R. H., Glare, T. R., Jackson, T. A., and Ronson, C. W. (2000) J. Bacteriol. 182, 5127–5138[Abstract/Free Full Text]
  40. Ffrench-Constant, R., Waterfield, N., Daborn, P., Joyce, S., Bennett, H., Au, C., Dowling, A., Boundy, S., Reynolds, S., and Clarke, D. (2003) FEMS Microbiol. Rev. 26, 433–456[CrossRef][Medline] [Order article via Infotrieve]
  41. Karim, S., Gould, F., and Dean, D. H. (2000) Curr. Microbiol 41, 214–219[CrossRef][Medline] [Order article via Infotrieve]
  42. Haffani, Y. Z., Cloutier, C., and Belzile, F. J. (2001) Biotechnol. Prog. 17, 211–216[CrossRef][Medline] [Order article via Infotrieve]
  43. Blackburn, M., Golubeva, E., Bowen, D., and Ffrench-Constant, R. H. (1998) Appl. Environ. Microbiol. 64, 3036–3041[Abstract/Free Full Text]
  44. Heungens, K., Cowles, C. E., and Goodrich-Blair, H. (2002) Mol. Microbiol. 45, 1337–1353[CrossRef][Medline] [Order article via Infotrieve]
  45. Waterfield, N. R., Daborn, P. J., and Ffrench-Constant, R. H. (2002) Trends Microbiol. 10, 541–545[CrossRef][Medline] [Order article via Infotrieve]
  46. Donnenberg, M. S. (2000) Nature 406, 768–774[CrossRef][Medline] [Order article via Infotrieve]
  47. Daborn, P. J., Waterfield, N., Silva, C. P., Au, C. P. Y., Sharma, S., and Ffrench-Constant, R. H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10742–10747[Abstract/Free Full Text]
  48. Brehelin, M., Cherqui, A., Drif, L., Luciani, J., Akhurst, R., and Boemare, N. (1993) J. Invertebr. Pathol. 61, 188–191[CrossRef]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
E. E. Herbert Tran and H. Goodrich-Blair
CpxRA Contributes to Xenorhabdus nematophila Virulence through Regulation of lrhA and Modulation of Insect Immunity
Appl. Envir. Microbiol., June 15, 2009; 75(12): 3998 - 4006.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
J. Banerjee, J. Singh, M. C. Joshi, S. Ghosh, and N. Banerjee
The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin
J. Bacteriol., November 15, 2006; 188(22): 7957 - 7962.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
S. E. Brown, A. T. Cao, P. Dobson, E. R. Hines, R. J. Akhurst, and P. D. East
Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria
Appl. Envir. Microbiol., February 1, 2006; 72(2): 1653 - 1662.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
P. Khandelwal, D. Choudhury, A. Birah, M. K. Reddy, G. P. Gupta, and N. Banerjee
Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila
J. Bacteriol., October 1, 2004; 186(19): 6465 - 6476.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/15/14595    most recent
M309859200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, S. E.
Right arrow Articles by East, P. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, S. E.
Right arrow Articles by East, P. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement