FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2001466227> ?p ?o ?g. }

• W2001466227 endingPage "9580" @default.
• W2001466227 startingPage "9571" @default.
• W2001466227 abstract "Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehélin, M. (2003) J. Biol. Chem. 278, 3030–3039). We show here that this toxin, Xenorhabdus α-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C1. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins. Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehélin, M. (2003) J. Biol. Chem. 278, 3030–3039). We show here that this toxin, Xenorhabdus α-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C1. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins. Entomopathogenic bacteria are widely used as crop protection agents. Most studies on these bacteria have focused on the properties of insecticidal toxins, with a view to improving them. These pathogens are able to overcome insect immune responses (1Brehélin M. Drif L. Baud L. Boemare N. Insect Biochem. 1989; 19: 301-307Crossref Scopus (68) Google Scholar, 2Park Y. Kim Y. J. Insect Physiol. 2000; 46: 1469-1476Crossref PubMed Scopus (146) Google Scholar), which parallel those of mammals to some extent. Insects do not have adaptive immunity and are easy to handle, making them a powerful tool for studies of host innate immunity and for the identification of bacterial virulence factors (3Miyata S. Casey M. Frank D.W. Ausubel F.M. Drenkard E. Infect. Immun. 2003; 71: 2404-2413Crossref PubMed Scopus (195) Google Scholar). The genomes of invertebrate pathogens represent a potentially extensive reservoir of virulence genes that have evolved over long periods to overcome the innate immune responses of their hosts. This virulence gene pool may act as a source of virulence factors for transfer into human commensal or pathogenic bacteria (4Waterfield N. Wren B. ffrench-Constant R. Nat. Rev. Microbiol. 2004; 2: 833-884Crossref PubMed Scopus (83) Google Scholar).Xenorhabdus nematophila (Enterobacteriaceae) is a Gram-negative bacterium that is transported into insects by the entomopathogenic nematode Steinernema carpocapsae. Once inside the insect, it secretes various extracellular factors, including antibiotics, lipases, proteases, and toxins, which are involved in insect killing (5Forst S. Nealson K. Microbiol. Rev. 1996; 60: 21-43Crossref PubMed Google Scholar). In particular, X. nematophila grows within the body of the insect and must, therefore, be able to escape the immune response, but little is known about the way in which it does this. Cellular immunity comes into play immediately after the penetration of a foreign body into the insect hemocoel, and X. nematophila must escape these potent cellular reactions. Hemocytes are the immunocytes of insects, and cellular immune reactions against bacteria involve cells of several different lineages. In our insect model, Spodoptera littoralis (Lepidoptera), plasmatocytes build nodules that isolate clumps of bacteria and necrotic insect tissues, and granular hemocytes are the professional phagocytes (6Ribeiro C. Brehélin M. J. Insect Physiol. 2006; 52: 417-429Crossref PubMed Scopus (236) Google Scholar). Cytotoxic factors targeting these immune system cells are good candidates for the molecules mediating immunosuppression. In X. nematophila grown in liquid culture, various cytotoxic activities principally targeting the hemocytes have been identified (7Ribeiro C. Duvic B. Oliveira P. Givaudan A. Palha F. Simoes N. Brehélin M. J. Insect Physiol. 1999; 45: 677-685Crossref PubMed Scopus (42) Google Scholar, 8Brillard J. Ribeiro C. Boemare N. Brehélin M. Givaudan A. Appl. Environ. Microbiol. 2001; 67: 2515-2525Crossref PubMed Scopus (75) Google Scholar, 9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). One of the major cytotoxic activity present in X. nematophila broth growth, C1 (8Brillard J. Ribeiro C. Boemare N. Brehélin M. Givaudan A. Appl. Environ. Microbiol. 2001; 67: 2515-2525Crossref PubMed Scopus (75) Google Scholar), targets the phagocytes. C1 is also hemolytic for sheep red blood cells (SRBC) 4The abbreviations used are: SRBC, sheep red blood cells; αX, α-xenorhabdolysin; Xax, Xenorhabdus α-xenorhabdolysin; RRBC, rabbit red blood cells; PBS, phosphate-buffered saline; ORF, open reading frame; HA, hemagglutinin; TUNEL, terminal dUTP nick-end labeling; DAPI, 4′,6-diamidino-2-phenylindole;OD,opticaldensity;z-,benzyloxycarbonyl;fmk,fluoromethylketone;HU, hemolytic units. 4The abbreviations used are: SRBC, sheep red blood cells; αX, α-xenorhabdolysin; Xax, Xenorhabdus α-xenorhabdolysin; RRBC, rabbit red blood cells; PBS, phosphate-buffered saline; ORF, open reading frame; HA, hemagglutinin; TUNEL, terminal dUTP nick-end labeling; DAPI, 4′,6-diamidino-2-phenylindole;OD,opticaldensity;z-,benzyloxycarbonyl;fmk,fluoromethylketone;HU, hemolytic units. but is not hemolytic for rabbit red blood cells (RRBC). C1 lyses cells via a mechanism involving protein toxins, as it is susceptible to heat and trypsin (8Brillard J. Ribeiro C. Boemare N. Brehélin M. Givaudan A. Appl. Environ. Microbiol. 2001; 67: 2515-2525Crossref PubMed Scopus (75) Google Scholar), and a peptide cytotoxin called α-xenorhabdolysin (αX) has been purified from a culture medium with C1 activity (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).We report here the molecular characterization of this cytotoxin produced by X. nematophila and show that it has both necrotic and apoptotic activities in insect hemocytes and mammalian cells. The genes encoding this toxin were also identified in various plant and human pathogens.EXPERIMENTAL PROCEDURESBacterial Strains, Production and Purification of Toxin—The strains and plasmids used in this study are listed in Table 1. X. nematophila (strain F1, laboratory collection) were grown in Luria Bertani broth at 28 °C. In these conditions cytotoxic C1 production was maximal in 20-h-old cultures (8Brillard J. Ribeiro C. Boemare N. Brehélin M. Givaudan A. Appl. Environ. Microbiol. 2001; 67: 2515-2525Crossref PubMed Scopus (75) Google Scholar). The cytotoxin was prepared as described (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) with a modification of the final step (reverse phase high performance liquid chromatography (HPLC)) that was replaced by a HPLC gel filtration on 60 cm × 7.5-mm inner diameter column (TSKgel, G3000SW TosoHaas) with elution in 0.25 ml/min phosphate buffer (20 mm, pH7) containing 300 mm NaCl. Under these conditions positive fractions were eluted in a single peak. These fractions were used as purified cytotoxin. The cytotoxin was named Xenorhabdus α-xenorhabdolysin (Xax).TABLE 1Strains and plasmids used in this studyStrain or plasmidDescriptionReferenceE. coliSURE 2e14-(McrA-)Δ(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuCΔTn5 (Kanr) uvrC [F′ proAB lac1qZ Ω M15 Tn10 (Tetr)]StratageneTop10F- mcrA.(mrr-hsdRMS-mcrBC) Δ 80lacZ M15.lacX74 deoR recA1 araD139(araA-leu)7697 galU galK rpsL endA1 nupGInvitrogenCFP201MC4100 sheA::Tn5-2.1 null mutantRef. 10del Castillo F.J. Leal S.C. Moreno F. del Castillo I. Mol. Microbiol. 1997; 25: 107-115Crossref PubMed Scopus (90) Google ScholarX. nematophilaF1X. nematophila, wild typeLaboratory collectionxaxAB9F1 xaxA::Ω CmThis workPlasmidpJQ200KSGmr sacRB mob oriV (p15A replicon)S. ForstpHP45-Ω CmApr Cmr interposon Ω CmRef. 11Fellay R. Frey J. Krisch H. Gene (Amst.). 1987; 52: 147-154Crossref PubMed Scopus (555) Google ScholarpBBR1MCS-5Medium copy mobilizable vector; GmRRef. 12Kovach M.E. Elzer P.H. Hill D.S. Robertson G.T. Farris M.A. Roop R.M. Peterson K.M. Gene (Amst.). 1995; 166: 175-176Crossref PubMed Scopus (2677) Google ScholarPBBxaxAB2558-bp region overlapping xaxAB cloned into the XbaI-SalI sites of pBBR1MCS-5This workpAB6pBBxaxAB with a 1-kilobase deletion overlapping the xaxA and xaxB coding regionThis workpAB73.5-kilobase BamHI fragment from pHP45-Ω Cm cloned into pAB6 (contains xaxAB::Ω Cm)This workpAB95.3-kilobase SalI-XbaI fragment from pAB7 cloned into pJQ200KS (contains xaxAB::Ω Cm)This workpBBxaxA1455-bp region overlapping xaxA cloned into the XbaI-SalI sites of pBBR1MCS-5This workpBBxaxB1358-bp region overlapping xaxB cloned into the XbaI-SalI sites of pBBR1MCS-5This workpBAD-B-Myc-HisLow copy vector derived from pBR322; AmpRInvitrogenpXaxA-Myc-HisPCR product of 1224 bp cloned into the NcoI-XbaI of pBAD-B-Myc-HisThis workpHB6-HA-HisHigh copy vector, AmpRRoche Applied SciencepXaxB-HA-HisPCR product of 1050 bp cloned into the HindIII-NotI of pHB6-HA-HisThis work Open table in a new tab In some experiments bacterial extracts (cytosolic fraction) were produced by producing lysates of Escherichia coli (3 freeze-thaw cycles), which were then centrifuged (16,000 × g for 20 min), and the resulting supernatant was filtered (0.22 μm). The toxin was also purified as described above from these E. coli extracts.Tandem Mass Spectrometry—Nanoelectrospray mass spectrometry was performed offline on a quadrupole time-of-flight mass spectrometer (QSTAR Pulsar-i, Applied Biosystems, Foster City, CA) fitted with a Protana nanospray inlet system (Protana, Odense, Denmark). Spectra were recorded with Analyst QS software (Applied Biosystems). Parameters were adjusted as follows: ion spray voltage, 900 V; curtain gas, 25; declustering potential, 45–75 V; focusing potential (FP), 265 V; declustering potential 2, 15 V. Peptides were fragmented in the collision cell using nitrogen gas on the doubly charged ions detected, with an individually optimized collision energy profile (30–55 V). Capillaries (Protana, Odense, Denmark) were loaded with the samples according to the following procedure; each aliquot after trypsin cleavage was solubilized in 5 μl of 1% formic acid, desalted on Poros 20 R2 (Applied Biosystems), packed into a gel-loader pipette tip, and eluted with 1.5 μl of 50:50:1 methanol/water/formic acid (13Wilm M. Mann M. Anal. Chem. 1996; 68: 1-8Crossref PubMed Scopus (1691) Google Scholar). The loaded capillaries were placed in the source tip holder. Molecules were identified by manual partial interpretations of spectra and by means of MASCOT searches (in-house server, Swissprot and Trembl merged data base).Plasmid Derivatives and Gene Cloning—E. coli Sure 2 (Stratagene) was used as an intermediate host for cloning experiments. The oligonucleotide primer sets used (Table 2) were designed from an alignment of several hemolysin loci from other bacteria including Photorhabdus luminescens, X. nematophila, and Yersinia enterocolitica. Standard PCR with each primer set was performed in a 50-μl reaction volume with a Gene Amp 2400 thermocycler system (PerkinElmer Life Sciences). For cloning we generated xaxAB, xaxA, and xaxB fragments with flanking XbaI and SalI sites by PCR. These fragments were inserted into pBBR1MCS-5 to generate pBBxaxAB, pBBxaxA, and pBBxaxB, respectively (Table 1). All constructs were checked by DNA sequencing (MilleGen, Toulouse, France).TABLE 2Primer sets used in this studyPrimerSequenceConstructBsal1fwGCAGTCGACTTAATGATGCGCTGAAGCTGPBBxaxBBxba1revGCATCTAGAGCGGTATATCCGTTGGAGAGpBBxaxB/ABABsal1fwGCAGTCGACTGCCACTAATATAGGTGGTGATGpBBxaxA/AB/pAB6AF1xba1revGCATCTAGACTGCCTGATTCATGGCTTTApBBxaxAEAXBam-revCGGGATCCTTTCAGCCAGCTGCTTATCAGpAB6AMHnco1fwGCACCATGGAGAACGATATGTCATCAAATpXaxA-Myc-HisAMHxba1revGCATCTAGAATGTGATAGAGTTTTTTATATTCAGCCpXaxA-Myc-HisB6HAHind3fwGCAAAGCTTGATGTCTGACAATACCCTCTCApXaxB-HA-HisB6HANot1revGCAGCGGCCGCCGTTTCAGCTTATTGTACpXaxB-HA-His Open table in a new tab Nucleotide Sequence Accession Number—The sequence of the X. nematophila xaxAB locus has been assigned GenBank™ accession number DQ249320.Construction of the xaxAB-null Strain, xaxAB9—Deletion of a 1-kilobase region overlapping xaxA and xaxB ORFs was realized in two steps. The first one was to amplify the first 480 bp of xaxA from pBBxaxAB (Table 1) as the template and by using primers ABsal1fw and EAXBam-rev (Table 2), which contain, respectively, SalI and BamHI restriction sites. The PCR fragment, digested with SalI and BamHI, was then cloned into the corresponding sites of the pBBxaxAB, which contain only the last 770 bp of xaxB and yields plasmid pAB6. Then a chloramphenicol-resistant Ω cassette (11Fellay R. Frey J. Krisch H. Gene (Amst.). 1987; 52: 147-154Crossref PubMed Scopus (555) Google Scholar) with transcriptional and translational terminators was inserted into the unique BamHI site within the disrupted xaxAB operon of pAB6 to yield plasmid pAB7. The xaxAB region carrying the Cm-interposon was, therefore, purified by digestion of plasmid pAB7 with SalI and XbaI and cloning into the corresponding sites of the pJQ200KS, resulting in plasmid pAB9 (Table 1). Experiment mating and exconjugant selection were done as previously described (14Givaudan A. Lanois A. J. Bacteriol. 2000; 182: 107-115Crossref PubMed Scopus (102) Google Scholar).Production and Purification of Tagged Peptides—For the production of recombinant hemolysin, we generated xaxA and xaxB fragments with flanking HindIII, NcoI, NotI, and Xbal sites by PCR and inserted them into pBAD-B-Myc-His (Invitrogen) or pHB6-HA-His (Roche Applied Science) in-frame with the Myc-His tag or the HA-His tag, respectively, creating XaxAMyc-His and XaxBHA-His (Tables 1 and 2). All constructs were checked by DNA sequencing (MilleGen, Toulouse, France) before being used to transform the E. coli TOP10 and XL1blue strains. Recombinant polyhistidine-tagged proteins were purified on a Protino Ni-IDA 2000 column according to the manufacturer's instruction (Macheray-Nagel, Germany) (see supplemental Fig. S2).Hemolytic Activity and Titration of the Toxin Solutions—SRBC and RRBC were obtained from BioMérieux (France) as a 50% suspension. They were extensively washed in pH 7.2 phosphate-buffered saline (PBS) and were diluted in this buffer to give a 5% suspension. We tested the hemolytic activity of bacteria grown on trypticase soy agar supplemented with 5% SRBC. Hemolysis was observed as a clear zone surrounding colonies. Hemolysis was also assessed in 5% SRBC or RRBC suspensions as described (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The titer of a toxin solution can be calculated from the absorbance value obtained according to the following formula, deduced from numerous absorbance determinations with serial dilutions of toxin (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar): titer (in HU) = 2 × 10(OD – 0.72).In some experiments we assessed osmotic protection by adding polyethylene glycol 6000 to the toxin solution at a final concentration of 30 mm before incubation with the SRBC. In other experiments, before testing for hemolysis we incubated the toxin solutions alone for 1 h at 60 °C or for 1 h at 37 °C with trypsin (30 units) or with SRBC ghosts as described (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).In a series of experiments, SRBC were incubated for 1 h at 4 °C in toxin solutions. The mixture was centrifuged at 4 °C, and the absorbance of the supernatant was measured. We then added 50 μl of a SRBC suspension to this supernatant, incubated the mixture at 37 °C for 1 h, centrifuged it at 10,000 × g, and determined the absorbance at 540 nm of this second supernatant. The pellet obtained after incubation at 4 °C was rapidly rinsed, suspended in PBS, and incubated at 37 °C for 1 h, and its absorbance was measured. Protein concentration was determined by the Bradford method.Immunoprecipitation and Immunoblotting—Recombinant purified epitope-tagged XaxAMyc-His and XaxBHA-His were incubated in Nonidet P-40 buffer (150 mm NaCl, 1% Nonidet P-40, 50 Mm Tris-HCl, pH 8) at 4 °C with monoclonal mouse anti-c-Myc (clone 9E10, Upstate Biotechnology) or monoclonal anti-HA (clone F-7, Santa Cruz Biotechnology) antibodies at 4 °C for 2 h. As controls, XaxAMyc-His was incubated with CifHA-His, a recombinant polyhistidine HA-tagged irrelevant protein (15Marchès E. Ledger T.N. Boury M. Ohara M. TU X. Goffaux F. Mainil J. Rosenshine I. Sugai M. De Rycke J. Oswald E. Mol. Microbiol. 2003; 50: 1553-1567Crossref PubMed Scopus (166) Google Scholar), and XaxBHA-His was incubated with recombinant polyhistidine Myc-tagged Cif (CifMyc-His). 5R. Zumbihl, unpublished data. Immune complexes were then collected with protein-G-Sepharose (GE Healthcare) and washed four times with Nonidet P-40 buffer. Immunoprecipitates were mixed with 2× Laemmli buffer and separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted using anti-Myc antibodies after immunoprecipitation with monoclonal anti-HA or immunoblotted with polyclonal anti-HA (Sigma H6908: rabbit anti-HA tag, affinity-isolated antibody) after immunoprecipitation with anti-Myc antibodies followed by mouse or rabbit secondary antibodies conjugated with horseradish peroxidase. Signals were generated by the enhanced chemiluminescence reaction (GE Healthcare) and detected using x-ray film.Insects, Hemocyte Monolayer Preparation, Human Cell Lines, and Test for Cytotoxic Activity—Insect (larvae of S. littoralis) rearing and hemocyte monolayer preparation were described (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). HeLa cells were cultured at 37 °C in a 5% CO2 atmosphere in RPMI supplemented with 10% fetal calf serum. Cytotoxic activity was tested on hemocyte monolayers and human cell lines on 12-mm glass coverslips (105 cells/coverslip) in a moist chamber at 23 °C (insect) or 37 °C (human) for 10 min to 2 h. Cell mortality was checked by adding 2 μl of trypan blue dye (0.04% final in PBS) and 5 min more of incubation. Specimen preparation for transmission electron microscopy observation was described in Ribeiro et al. (9Ribeiro C. Vignes M. Brehélin M. J. Biol. Chem. 2003; 278: 3030-3039Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Test for Apoptosis—We assessed apoptosis in target cells by TUNEL staining (kit from Roche Applied Science), after incubating cells (hemocyte monolayers or HeLa cell line) for 12 h with toxin solution (5 × 10–3 HU) in modified Eagle's medium and RPMI, respectively. We checked that TUNEL-positive cells were actually apoptotic by adding z-VAD-fmk (a pan-caspase inhibitor) or z-DEVD-fmk (caspase 3-specific inhibitor) to the medium and comparing the proportion (after angular transformation) of TUNEL-positive cells with that in experiments carried out in the absence of inhibitor.RESULTSPurification of the Toxin and Peptide Sequence Determination—Early stationary phase X. nematophila C1 supernatants (8Brillard J. Ribeiro C. Boemare N. Brehélin M. Givaudan A. Appl. Environ. Microbiol. 2001; 67: 2515-2525Crossref PubMed Scopus (75) Google Scholar) were collected, and the toxin was purified as described above, with fractions displaying cytolytic activity against insect hemocytes and hemolytic activity against SRBC eluted in a single peak. When SRBC were incubated at 4 °C with purified toxin (see “Experimental Procedures”), all hemolytic activity remained in the supernatant (Fig. 1). Surprisingly, when active fractions from gel filtration were analyzed by C18 reversephase chromatography (0–95% acetonitrile gradient and 0.01% trifluoroacetic acid), no peak was detected, and no hemolytic or cytolytic activity was detected even in the void volume. Similarly, no ion was detected when positive fractions were analyzed by matrix-assisted laser desorption ionization or quadrupole time-of-flight in the absence of proteolytic digestion (data not shown).After trypsin digestion, several apparently doubly charged signals were selected in the BM031219Q-B2 sample for collision-induced decomposition. Only the peak at m/z 536.74 led to an identification (score 45; under our conditions, the significance threshold was 43), using the MS/MS ion search subroutine from MASCOT. This signal was identified as peptide IIESQDVIR from the Q7N5I7_PHOLL protein. Alternatively, the MQ(I/L)D partial sequence could be obtained by manually interpreting the collision-induced decomposition spectrum resulting from the peak at m/z 553.75. Using the sequence query search subroutine of MASCOT, this signal was identified as the DVMQIDTER peptide from the same protein.Identification of Putative Hemolysin Loci in Different Bacterial Pathogens—The 342-amino acid protein containing both peptide sequences from Xax is a putative protein encoded by gene plu1961 (Fig. 2) in Photorhabdus luminescens TT01 (16Duchaud 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. Kunst F. Nat. Biotechnol. 2003; 21: 1307-1313Crossref PubMed Scopus (479) Google Scholar), a bacterium closely related to Xenorhabdus. Because the entire genome sequence of P. luminescens has been described, the genomic sequences flanking plu1961 showed that plu1962 was found immediately upstream from plu1961, strongly suggesting that these genes are cotranscribed. This putative hemolysin locus is located downstream from a gene encoding a tRNA for lysine in the TT01 genome. Another operon encoding similar proteins (plu3075–3076) (61 and 49% identity to plu1961 and plu1962, respectively) was found elsewhere in the P. luminescens genome, adjacent to the nuoA to nuoN region encoding putative NADH dehydrogenases. The genomic organization of this second putative hemolysin locus was found to be conserved in the genome of a clinical isolate of Photorhabdus asymbiotica isolated from patients in the United States (US3105/77) (www.sanger.ac.uk/Projects/P_asymbiotica/). In the completed genome sequences of bacteria, Plu1961 and Plu1962 display blastP matches (E value between 3e-7 to 3e-33) with two pairs of putative proteins (Psyr_3990, Psyr_3989 and PSTPO4287, PSTP04571) predicted from the genome sequences of two pathovars of the plant pathogenic bacteria, Pseudomonas syringae pv. syringae (Fig. 2) and pv. tomato, respectively (17Feil H. Feil W. Chain P. Larimer F. DiBartolo G. Copeland A. Lykidis A. Trong S. Nolan M. Goltsman E. Thiel J. Malfatti S. Loper J. Lapidus A. Detter J. Land M. Richardson P. Kyrpides N. Ivanova N. Lindow S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11064-11069Crossref PubMed Scopus (346) Google Scholar). Recently, Vodovar et al. (18Vodovar N. Vallenet D. Cruveiller S. Rouy Z. Barbe V. Acosta C. Cattolico L. Jubin C. Lajus A. Segurens B. Vacherie B. Wincker P. Weissenbach J. Lemaitre B. Medigue C. Boccard F. Nat. Biotechnol. 2006; 24: 673-679Crossref PubMed Scopus (196) Google Scholar) reported the genome sequence of an insect pathogen bacterium belonging to the genus Pseudomonas, P. entomophila, and they also showed that this bacterium secretes a diffusible hemolysin activity on sheep blood agar contrary to the other Pseudomonads. Using the Microbial Genome Annotation System (www.genoscope.cns.fr/agc/mage/wwwpkgdb/), two closely linked genes encoding predicted proteins PSEEN4370 and PSEEN 4369 similar to XaxA and XaxB family (supplemental Fig. S1), respectively, were detected in the genome sequence of P. entomophila. Both genes are located at a synteny break point between the genomes of P. entomophila and the other Pseudomonas spp. We also searched for homologues of this pair of genes in the bacterial genomes that are currently being sequenced. Significant similarities were found in TblastN searches between Plu1961 and Plu1962 and two predicted proteins from Y. enterocolitica (E = 1.5e-43 and 4.1e-83, respectively) (Fig. 2) and from Proteus mirabilis HI4320 (E = 6.3e-9 and 7.5e-57) (www.sanger.ac.uk/). Finally, we detected homologues to this pair of genes (plu1961–1962), as expected, in the incomplete genome sequence of a X. nematophila strain (E = 5e-96 and 1e-130) (xenorhabdus.danforthcenter.org/). As shown in Fig. 2, the putative hemolysin loci, containing two closely linked genes similar to plu1961–62, were found in the same orientation in different genomic contexts in the chromosome of Xenorhabdus, Photorhabdus, P. syringae, and Yersinia. However, these potential virulence genes are not located on or in the vicinity of mobile genetic elements.FIGURE 2Predicted genetic organizations of xaxAB hemolysin loci in the genomic sequences of various Gram-negative bacteria. All genes in the chromosomal sections are indicated by gray arrows, except for xaxAB homologues, which are shown in dark gray. Genomic organizations were compared using genomic data from P. luminescens laumondii (strain TT01) (genolist.pasteur.fr/PhotoList/), P. syringae pv. syringae B728a (img.jgi.doe.gov), Y. enterocolitica 8081 (www.sanger.ac.uk/Projects/Y_enterocolitica), and X. nematophila ATCC19061 (maizeapache.ddpsc.org/xeno_blast/). Because X. nematophila sequencing has not yet been completed, ORFs were detected using ORF finder (www.ncbi.nlm.nih.gov/gorf/gorf.html), and deduced amino acid sequences were then compared using the blastP algorithm at the NCBI. Locus tag numbers and predicted products are mentioned below and above the arrows, respectively. The numbers in the margins indicate the coordinates on the chromosome. Peptide sequences obtained after sequence searches using MASCOT are indicated together with matches with the deduced amino acid sequence of Plu1961 from P. luminescens TTO1 (Q7N5I7). RNB, exoribonuclease II.View Large Image Figure ViewerDownload Hi-res image Download (PPT)XaxAB from X. nematophila F1 Is a Functional Hemolysin/Cytolysin—To demonstrate that this locus encoded Xax, we first cloned the potential hemolysin locus from X. nematophila F1 and expressed recombinant xaxAB in E. coli (see “Experimental Procedures”). No E. coli transformants were obtained when xaxAB was inserted into high copy number plasmids, such as pUC19. The region encompassing the xaxAB genes was amplified and cloned under control of the plac promoter in a medium copy number plasmid, pBBR1MCS-5 resulting in pBBxaxAB, which was then transferred to the E. coli SURE strain. As expected, in the presence of 0.2 mm isopropyl 1-thio-β-d -galactopyranoside, the E. coli SURE (pBBxaxAB) strain displayed strong hemolytic activity on sheep blood agar plate, whereas the clone containing vector alone (pBBR1MCS-5) did not (Fig. 3). This suggests that this locus is involved in hemolytic activity. However, several studies have shown that the overproduction of heterologous regulators (19Westermark M. Oscarsson J. Mizunoe Y. Urbonaviciene J. Uhlin B. J. Bacteriol. 2000; 182: 6347-6357Crossref PubMed Scopus (88) Google Scholar) or the presence of prophagic inserts containing a holin locus from X. nematophila (20Brillard J. Boyer-Giglio M.-H. Boemare N. Givaudan A. FEMS Microbiol. Lett" @default.
• W2001466227 created "2016-06-24" @default.
• W2001466227 creator A5005654858 @default.
• W2001466227 creator A5012649715 @default.
• W2001466227 creator A5027648388 @default.
• W2001466227 creator A5033037635 @default.
• W2001466227 creator A5070180686 @default.
• W2001466227 creator A5070746297 @default.
• W2001466227 creator A5070951004 @default.
• W2001466227 creator A5090674717 @default.
• W2001466227 date "2007-03-01" @default.
• W2001466227 modified "2023-10-11" @default.
• W2001466227 title "The xaxAB Genes Encoding a New Apoptotic Toxin from the Insect Pathogen Xenorhabdus nematophila Are Present in Plant and Human Pathogens" @default.
• W2001466227 cites W1974058535 @default.
• W2001466227 cites W1992780831 @default.
• W2001466227 cites W1996382396 @default.
• W2001466227 cites W1998712064 @default.
• W2001466227 cites W2004274040 @default.
• W2001466227 cites W2026609654 @default.
• W2001466227 cites W2031195396 @default.
• W2001466227 cites W2031884770 @default.
• W2001466227 cites W2036917159 @default.
• W2001466227 cites W2039275110 @default.
• W2001466227 cites W2049497114 @default.
• W2001466227 cites W2055766814 @default.
• W2001466227 cites W2056274611 @default.
• W2001466227 cites W2057294829 @default.
• W2001466227 cites W2065824991 @default.
• W2001466227 cites W2069577561 @default.
• W2001466227 cites W2077033667 @default.
• W2001466227 cites W2078767021 @default.
• W2001466227 cites W2083968635 @default.
• W2001466227 cites W2097512519 @default.
• W2001466227 cites W2100693791 @default.
• W2001466227 cites W2120053454 @default.
• W2001466227 cites W2128625079 @default.
• W2001466227 cites W2129455687 @default.
• W2001466227 cites W2132048333 @default.
• W2001466227 cites W2132590114 @default.
• W2001466227 cites W2136521365 @default.
• W2001466227 cites W2137475137 @default.
• W2001466227 cites W2146615689 @default.
• W2001466227 cites W2152940141 @default.
• W2001466227 cites W2163647890 @default.
• W2001466227 cites W2168069647 @default.
• W2001466227 cites W2169643092 @default.
• W2001466227 doi "https://doi.org/10.1074/jbc.m604301200" @default.
• W2001466227 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17229739" @default.
• W2001466227 hasPublicationYear "2007" @default.
• W2001466227 type Work @default.
• W2001466227 sameAs 2001466227 @default.
• W2001466227 citedByCount "95" @default.
• W2001466227 countsByYear W20014662272012 @default.
• W2001466227 countsByYear W20014662272013 @default.
• W2001466227 countsByYear W20014662272014 @default.
• W2001466227 countsByYear W20014662272015 @default.
• W2001466227 countsByYear W20014662272016 @default.
• W2001466227 countsByYear W20014662272017 @default.
• W2001466227 countsByYear W20014662272018 @default.
• W2001466227 countsByYear W20014662272019 @default.
• W2001466227 countsByYear W20014662272020 @default.
• W2001466227 countsByYear W20014662272021 @default.
• W2001466227 countsByYear W20014662272022 @default.
• W2001466227 countsByYear W20014662272023 @default.
• W2001466227 crossrefType "journal-article" @default.
• W2001466227 hasAuthorship W2001466227A5005654858 @default.
• W2001466227 hasAuthorship W2001466227A5012649715 @default.
• W2001466227 hasAuthorship W2001466227A5027648388 @default.
• W2001466227 hasAuthorship W2001466227A5033037635 @default.
• W2001466227 hasAuthorship W2001466227A5070180686 @default.
• W2001466227 hasAuthorship W2001466227A5070746297 @default.
• W2001466227 hasAuthorship W2001466227A5070951004 @default.
• W2001466227 hasAuthorship W2001466227A5090674717 @default.
• W2001466227 hasBestOaLocation W20014662271 @default.
• W2001466227 hasConcept C104317684 @default.
• W2001466227 hasConcept C159047783 @default.
• W2001466227 hasConcept C24527192 @default.
• W2001466227 hasConcept C2776460866 @default.
• W2001466227 hasConcept C2777367657 @default.
• W2001466227 hasConcept C2777612826 @default.
• W2001466227 hasConcept C54355233 @default.
• W2001466227 hasConcept C59822182 @default.
• W2001466227 hasConcept C86803240 @default.
• W2001466227 hasConcept C89423630 @default.
• W2001466227 hasConceptScore W2001466227C104317684 @default.
• W2001466227 hasConceptScore W2001466227C159047783 @default.
• W2001466227 hasConceptScore W2001466227C24527192 @default.
• W2001466227 hasConceptScore W2001466227C2776460866 @default.
• W2001466227 hasConceptScore W2001466227C2777367657 @default.
• W2001466227 hasConceptScore W2001466227C2777612826 @default.
• W2001466227 hasConceptScore W2001466227C54355233 @default.
• W2001466227 hasConceptScore W2001466227C59822182 @default.
• W2001466227 hasConceptScore W2001466227C86803240 @default.
• W2001466227 hasConceptScore W2001466227C89423630 @default.
• W2001466227 hasIssue "13" @default.
• W2001466227 hasLocation W20014662271 @default.
• W2001466227 hasLocation W20014662272 @default.
• W2001466227 hasLocation W20014662273 @default.

• next