An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila
2008; Elsevier BV; Volume: 283; Issue: 42 Linguagem: Inglês
10.1074/jbc.m804416200
ISSN1083-351X
AutoresMohan C. Joshi, Animesh Sharma, Sashi Kant, Ajanta Birah, Gorakh Prasad Gupta, Sharik R. Khan, Rakesh Bhatnagar, Nirupama Banerjee,
Tópico(s)Nematode management and characterization studies
ResumoXenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ∼58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ∼3.6 μg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed α-chitin binding activity with Kd ∼ 0.64 μm and Bmax ∼ 4.68 μmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ∼8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity. Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ∼58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ∼3.6 μg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed α-chitin binding activity with Kd ∼ 0.64 μm and Bmax ∼ 4.68 μmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ∼8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity. Xenorhabdus nematophila, a Gram-negative bacterium, resides as symbiont in the gut of a soil nematode of the genus Steinernema (1Herbert E.E. Goodrich-Blair H. Nat. Rev. Microbiol. 2007; 5: 634-646Crossref PubMed Scopus (164) Google Scholar, 2Forst S. Nealson K. Microbiol. Rev. 1996; 60: 21-43Crossref PubMed Google Scholar, 3Akhurst R.J. Dunphy G.B. Beckage N. Thompson S. Federici B. Parasites and pathogens of Insects. 2. Academic Press Inc., New York, NY1993: 1-23Crossref Scopus (118) Google Scholar). The bacteria-nematode association is highly toxic to many insect species, causing rapid larval death. The bacterium has a complex life cycle, encompassing symbiotic and pathogenic stages. The symbiotic phase is spent in the nematode gut, whereas pathogenicity is manifested in the insect larval body. The bacterium is released in the insect hemocoel (3Akhurst R.J. Dunphy G.B. Beckage N. Thompson S. Federici B. Parasites and pathogens of Insects. 2. Academic Press Inc., New York, NY1993: 1-23Crossref Scopus (118) Google Scholar) or gut (4Sicard M. Brugirard-Ricaud K. Pages S. Lanois A. Boemare N.E. Bréhelin M. Givaudan A. Appl. Environ. Microbiol. 2004; 70: 6473-6478Crossref PubMed Scopus (103) Google Scholar), where it produces a variety of effector molecules including toxic proteins to kill the prey. The larval carcass provides a nutrient-rich environment for growth and development of both the nematode and the bacteria. The bacterium alone is also able to kill the insect host when grown axenically in the laboratory medium. Earlier X. nematophila was shown to produce outer membrane vesicle (OMV) 3The abbreviations used are: OMV, outer membrane vesicle; XnGroEL, X. nematophila GroEL; BBMV, brush border membrane vesicle; LacNAc, N-acetyllactosamine; EcGroEL, E. coli GroEL; kb, kilobase(s); PBS, phosphate-buffered saline. during growth in the broth culture (5Khandelwal P. Banerjee-Bhatnagar N. Appl. Environ. Microbiol. 2003; 69: 2032-2037Crossref PubMed Scopus (75) Google Scholar). The OMVs contained a number of proteins and were orally toxic to neonatal larvae of Helicoverpa armigera (6Khandelwal P. Bhatnagar R. Choudhury D. Banerjee N. Biochem. Biophys. Res. Commun. 2004; 314: 943-949Crossref PubMed Scopus (23) Google Scholar). Growing concern of development of resistance in the crop pests to crystal protein toxins of Bacillus thuringiensis has initiated vigorous research world-wide to discover orally active insecticidal proteins. The proteins associated with the OMV complex of X. nematophila provided a pool of potential insecticidal molecules for investigation. Analysis of the OMV proteins led to the identification of a ∼58-kDa GroEL homolog (XnGroEL) as a major component of the complex. The GroEL protein belongs to a highly conserved family of molecular chaperones, which facilitate folding of nascent nonnative proteins in the cell (7Fink A.L. Physiol. Rev. 1999; 79: 425-449Crossref PubMed Scopus (873) Google Scholar). The large chaperon assembly is produced by two heptameric rings of 7 identical subunits each, stacked back to back making a large double-ringed cylinder of ∼800 kDa, enclosing a central cavity (8Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar). It requires a 10-kDa co-chaperone GroES, Mg2+, and ATP to carry out the chaperoning activity (9Braig K. Curr. Opin. Struct. Biol. 1998; 8: 159-165Crossref PubMed Scopus (41) Google Scholar). The 548-amino acid-long GroEL polypeptide chain folds into three distinctive domains. A well ordered equatorial domain (residues 6–133 from the N terminus and 409–523 from the C terminus) forms a solid base around the middle of the assembly and provides most of the residues for intersubunit contacts. A considerably less ordered apical domain (residues 191–376) surrounding the opening at the ends of the central cavity shows local flexibility within the domain as well as en bloc movement around a hinge connecting it to the intermediate domain. The intermediate domain (residues 134–190 of the N terminus and 377–408 of the C terminus) is much smaller and links the equatorial domain to the apical domain (10Sigler P.B. Xu Z. Rye H.S. Burston S.G. Fenton W.A. Horwich A.L. Annu. Rev. Biochem. 1998; 67: 581-608Crossref PubMed Scopus (476) Google Scholar). Earlier, a toxic GroEL was been described in another symbiotic bacterium, Enterobacter aerogenes (11Yoshida N. Oeda K. Watanabe E. Mikami T. Fukita Y. Nishimura K. Komai K. Matsuda K. Nature. 2001; 411: 44Crossref PubMed Scopus (99) Google Scholar). The protein was secreted by the bacterium in the saliva of parasitic antlions, which kills its insect prey by causing paralysis. The protein was shown to paralyze cockroaches when injected in their hemocoel (11Yoshida N. Oeda K. Watanabe E. Mikami T. Fukita Y. Nishimura K. Komai K. Matsuda K. Nature. 2001; 411: 44Crossref PubMed Scopus (99) Google Scholar). The GroEL proteins from several pathogenic bacteria are major antigens and are highly expressed under stressful conditions (12Ranford J.C. Henderson B. Mol. Pathol. 2002; 55: 209-213Crossref PubMed Scopus (78) Google Scholar). It is shown to be essential for growth and viability of bacterial cells (13Fayet O. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1989; 171: 1379-1385Crossref PubMed Scopus (546) Google Scholar, 14Dukan S. Nystrom T. J. Biol. Chem. 1999; 274: 26027-26032Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In endosymbiotic bacteria, GroEL is expressed at a higher level under normal growth conditions compared with free living species and is reported to protect against harmful effects of accumulated mutations and preserve fitness of the species (15Fares M.A. Barrio E. Sabater-Munoz B. Moya A. Mol. Biol. Evol. 2002; 19: 1162-1170Crossref PubMed Scopus (65) Google Scholar). In this study we describe some of the unique properties of XnGroEL, secreted by the insecticidal bacterium X. nematophila. OMV Purification and Identification of XnGroEL—X. nematophila strain 19061 was grown in Luria Bertaini medium at 28 °C for 18 h with shaking at 180 rpm. OMVs were prepared from cell-free culture supernatant as described earlier (5Khandelwal P. Banerjee-Bhatnagar N. Appl. Environ. Microbiol. 2003; 69: 2032-2037Crossref PubMed Scopus (75) Google Scholar). OMV proteins were resolved by SDS-PAGE, and a ∼58-kDa band was cut and sequenced by Edman degradation. Purification of the Native XnGroEL from Culture Supernatant (Extracellular) and Cell Lysate (Intracellular)—XnGroEL was purified from both the pellet and supernatant of X. nematophila culture. The proteins in the culture supernatant were precipitated with 70% ammonium sulfate and resolved on a 25-ml Q-Sepharose column. XnGroEL was eluted with 100 ml of NaCl gradient (0.3–1.0 m). The partially purified protein was concentrated and further purified by Superose 12 gel filtration column in a fast protein liquid chromatography system. The cell pellet was used for isolation of intracellular XnGroEL. Cells were disrupted by sonication, and the cell lysate was further purified as described earlier for extracellular protein. The proteins were resolved by SDS-PAGE, and purity was checked by silver staining of the gel. For purification of native GroEL protein from Escherichia coli, cells from 5 liters of LB culture were subjected to heat stress at 42 °C for 6 h and further treated as described above for intracellular protein of X. nematophila. Cellular Fractionation and Localization of XnGroEL and XnGroES Proteins—Different cellular fractions of X. nematophila were prepared as described earlier (5Khandelwal P. Banerjee-Bhatnagar N. Appl. Environ. Microbiol. 2003; 69: 2032-2037Crossref PubMed Scopus (75) Google Scholar, 16Wai S.N. Lindmark B. Soderblom T. Takade A. Westermark M. Oscarsson J. Jass J. Richter D.A. Mizunoe Y. Uhlin B.E. Cell. 2003; 115: 25-35Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar), and the proteins were resolved by SDS-PAGE and detected with antibodies of GroEL or GroES (Stressgen). Pronase Treatment of OMV Proteins—OMVs were incubated with 1 unit of Pronase (Roche Applied Science) at 37 °C for 30 min in buffer A (50 mm sodium phosphate, pH 7.2). The reaction was stopped by 1× protease inhibitor mixture (Roche Applied Science), and the proteins were subjected to SDS-PAGE. Biochemical Characterization of XnGroEL—ATPase activity and in vitro porcine lactate dehydrogenase refolding assay was performed as described previously (17Vashisht A.A. Pradhan A. Tuteja R. Tuteja N. Planta J. 2005; 44: 76-87Crossref PubMed Scopus (93) Google Scholar, 18Clarke A.R. Waldman A.D. Hart K.W. Holbrook J.J. Biochem. Biophys. Acta. 1985; 829: 397-407Crossref PubMed Scopus (40) Google Scholar). Construction of Genomic DNA Library and Cloning of groEL Gene—Genomic DNA was digested with different restriction enzymes and probed with a 45-bp nucleotide fragment, derived from the N-terminal amino acid sequence of the protein. A DNA fragment reacting with the probe in the EcoRI-digested DNA was cloned in pUC18 cloning vector (pMJ plasmid) and transformed in E. coli DH5α cells producing the MJ strain. Using the cloned fragment as template and primers from the 5′ and 3′ ends of the groEL coding sequence, a 1.7-kb DNA was amplified by PCR. The amplified DNA was cloned in pGEMTeasy vector producing pMJ1 plasmid. The sequences encoding apical domain (558 bp) and apical-intermediate domains (876 bp) of XnGroEL and GroES protein were PCR-amplified using specific primers, and the products were cloned in pGEMTeasy vector producing pMJ2, pMJ3, and pMJ4 plasmids, respectively. Expression and Purification of Recombinant XnGroEL and Domain Proteins—The 1.7-kb fragment from pMJ1 plasmid was ligated in pET28a expression vector (pMJ5) and transformed in the E. coli BL21DE3-producing MJ5 strain. MJ5 cells were grown in LB medium containing 50 μg/ml kanamycin at 37 °C to exponential phase and induced with 1 mm isopropyl thiogalactopyranoside for 3–4 h. The cells were washed and lysed by sonication, and the cell-free supernatant was purified by nickel-nitrilotriacetic acid-agarose affinity matrix in cold using standard protocol. The recombinant apical domain (∼21 kDa), apical-intermediate domain (∼30 kDa), and GroES protein (∼10 kDa) were also expressed and purified as above, producing plasmids pMJ6, pMJ7, and pMJ8, respectively. Site-directed Mutagenesis—Point mutations in the full-length XnGroEL and apical domain were done using cloned pMJ5 and pMJ6 constructs as template with the site-directed mutagenesis kit (Stratagene) as per the manufacturer's instructions. Five polar substitutions on the outer surface of the apical domain (Tyr-219, Ser-244, Asn-297, Thr-347, and Ser-356) and four on the outer surface of the equatorial domain (Ser-126, Lys-133, Asn-474, and Thr-481) were mutated to alanine. The mutated proteins were expressed and purified as the wild type proteins. CD spectra of all the proteins were recorded to compare their secondary structure. Evaluation of Oral Insecticidal Activity—The insect bioassay was carried out in 12- or 24-well flat-bottom plates (NUNC). Different concentrations of the proteins were diluted in 10 mm sodium phosphate buffer, pH 7.5, and each group contained 24–30 neonates. The proteins were applied on the surface of artificial diet and allowed to percolate down. One neonatal larva (24 h old) of H. armigera was released on the surface of the diet in each well (19Khandelwal P. Choudhury D. Birah A. Reddy M.K. Gupta G.P. Banerjee N. J. Bacteriol. 2004; 186: 6465-6476Crossref PubMed Scopus (36) Google Scholar), and the plates were incubated at 25 °C (16-h-day-length periods) with 80% relative humidity. Mortality and larval weight were recorded periodically over the entire larval period. The dose of protein shown in the results was the amount of protein added to the diet. The bioassays were performed more than three times. Heat-inactivated XnGroEL, bovine serum albumin, GroEL homologue from E. coli K-12, and buffer were used as controls. Contribution of different domains of XnGroEL was evaluated by linear regression analysis of percent mortality at equimolar protein concentrations. The 50% lethal concentration (LC50) was determined by Probit analysis, and statistical analysis of the data was done using R package, a web-based tool for statistical computing. Preparation of Brush Border Membrane Vesicles (BBMV) and Binding of XnGroEL—BBMV were prepared from dissected gut of fourth and fifth instar larvae by MgCl2 precipitation, as described previously (20Cioffi M. Wolfersberger M.G. Tissue Cell. 1983; 15: 781-803Crossref PubMed Scopus (50) Google Scholar). For binding assay, 20 μg of BBMV protein was incubated with 10 μg of XnGroEL or variant proteins in a total volume of 30 μl and incubated at 4 °C for 30 min followed by centrifugation at 12,000 × g for 5 min in cold to remove the unbound protein. The pellet was washed with 50 μl of 1× PBS twice and resuspended in 20 μl of 1× PBS. The samples were boiled in Laemmli sample dye for 5 min and resolved by SDS-PAGE. The proteins were transferred on nitrocellulose membrane and blotted with anti-GroEL antibodies diluted 1:20,000. The Western blots were scanned, and the integrated density value of each band representing bound protein was determined. Different substances like soluble chitin (21Guo X.F. Kikuchi K. Matahira Y. Sakai K. Ogawa K. J. Carbohydr. Chem. 2002; 21: 149-161Crossref Scopus (19) Google Scholar), chitosan, and crystalline cellulose were used to test protein binding to BBMV. XnGroEL was incubated with different concentrations of the above compound at 4° for 30 min and centrifuged, and the supernatant containing the unbound protein was added to BBMV and incubated as above, and the membrane-bound protein was estimated as above. To determine the specificity of binding of XnGroEL, competitive inhibition by sugar derivatives like GalNAc, N-acetylneuraminic acid, GlcNAc, N-acetyllactosamine, glucose, mannose, chito-oligosaccharides-N,N′-diacetyl chitobiose, N,N′,N′-triacetyl chitotriose, and hexa-N-acetyl chitohexaose (Dextra Laboratories) were also tested in the binding assay. The sugars were preincubated with the proteins at 4 °C for 30 min followed by incubation with BBMV as described above. To investigate the nature of binding of XnGroEL with the gut epithelium, the BBMVs were treated with different proteases or chitinase from Serratia marcescens (Sigma) at 37 °C for 20 min followed by binding and detection of the protein as described above. Chitinase digestion was also carried out in the presence of 1× protease inhibitor mixture (Roche Applied chemicals). Activity of aminopeptidase N, a protein exposed on the surface of the epithelial membrane, was measured (22Jurat-Fuentes J.L. Adang M.J. Eur. J. Biochem. 2004; 271: 3127-3135Crossref PubMed Scopus (222) Google Scholar) to evaluate the effect of chitinase treatment on the membrane surface. Detection of Binding of XnGroEL by Immunofluorescence—Fourth to fifth instar larvae of H. armigera were starved for 12 h and dissected to take out the gut. The latter were washed in 1× MET buffer (50 mm Tris-HCl, pH 7.0, 100 mm mannitol, 1× protease inhibitor mixture, and 1 mm EGTA) and fixed in fixing solution (1% formaldehyde + 50 mm phosphate buffer, pH 7.0) for 16 h at 4 °C. The samples were embedded in paraffin blocks, and 6-μm-thick sections were cut and placed on glycerol-coated slides. For detection of protein binding, the sections were deparaffinated with xylene for 10 min at room temperature followed by sequential washing with ethanol (100, 80, 70, 50, and 20%), distilled water, and 1× PBS. Twenty μg of the proteins (XnGroEL or double mutant) were laid over the gut sections and incubated for 2 h at 4 °C. The slides were washed 3 times with wash buffer (1× PBS + 0.01% Tween 20) followed by blocking with 3% bovine serum albumin in 1× PBS for 2 h at 4 °C. The sections were incubated with anti-XnGroEL antibodies (1:10,000) for 16 h at 4 °C followed by extensive washing with wash buffer. The sections were incubated with anti-rabbit ALEXA 488 secondary antibodies (Molecular Probes, OR) at a dilution of 1:1500 and incubated for 2 h at 4°C. The samples were washed extensively with wash buffer, and coverslips were placed on the sections with anti-fade agent (Bio-Rad) and viewed in a Fluorescence microscope (Nikon ECLIPSE TE 2000-U) under blue light at a magnification of 20×. Chitin Binding Assay—Binding of the wild type and mutant proteins with α-chitin (from Crab shells, Sigma Aldrich) was evaluated as described earlier (23Vaaje-Kolstad G. Huston D.R. Riemen A.H.K. Eijsink V.G.H. van Alten D.M.F. J. Biol. Chem. 2005; 280: 11313-11319Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). A 20 mg/ml stock suspension of the substrate α-chitin was prepared in buffer (50 mm Tris-HCl, pH 7.0). For studying the time course of binding, a 500-μl reaction mixture contained 0.5 mg of substrate and 50 μg of protein in the above buffer. The tubes were incubated on a rotary shaker at room temperature, and samples were taken at intervals (5, 10, 15, 30, and 60 min). The suspension was centrifuged for 5 min at 13,000 × g, and optical density of the supernatant was measured at 280 nm to determine the amount of unbound protein. To determine the binding constants of XnGroEL variant proteins, the assay procedure was as follows; each protein variant was diluted to a range of concentrations (10–300 μg/ml) in 50 mm Tris-HCl, pH 7.0, from a stock solution of known strength, and A280 of each dilution was measured to create a standard curve of the individual variants. For chitin binding assay, 0.5 mg/ml α-chitin was added to different concentrations of XnGroEL variants (10, 20, 50, 100, 200, and 300 μg/ml), in a total volume of 1 ml, and the tubes were mixed gently on a rotary shaker at 60 rpm for 16 h at room temperature. Subsequently the samples were centrifuged at 13,000 × g for 5 min, A280 of the supernatants was measured, and protein concentrations were calculated from the standard curves. All the values below 20 μg/ml were verified by protein estimation by Bradford reagent. A suitable blank containing 0.5 mg/ml α-chitin in buffer was used in all the experiments, performed in triplicate. The dissociation constant Kd and substrate binding capacities Bmax were determined by fitting the binding isotherms to the one-site binding equation, Pbound = Bmax[Pfree]/Kd + [Pfree], where Pbound denotes protein specifically bound to the substrate and Pfree is unbound protein in the supernatant, by nonlinear regression using the Graph Pad Prism software 3.0 (San Diego, CA). To analyze the nature of molecular interactions between XnGroEL and α-chitin, the binding assay was also performed in the presence of 10–100 mm sodium chloride. Sequence and Structure Analysis—NCBI BLAST (24Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Anang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) was used to find out the closest homologous sequence to XnGroEL. Global alignment of X. nematophila GroEL sequence with E. coli GroEL was done using Needle program from EMBOSS (25Rice P. Longden I. Bleaseby A. Trends Genet. 2000; 16: 276-277Abstract Full Text Full Text PDF PubMed Scopus (6541) Google Scholar). SWISS-MODEL (26Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4537) Google Scholar) was used to generate the three-dimensional structure, which was viewed and analyzed with CHIMERA (27Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comp. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (29006) Google Scholar). Energy calculations of proteins were done using NAMD and VMD (28Phillips J.C. Braun R. Wang W. Gumbart J. Tajkhorshid E. Villa E. Chipot C. Skeel D.R. Kale L. Schulten K. J. Comp. Chem. 2005; 26: 1781-1802Crossref PubMed Scopus (13350) Google Scholar). Multiple sequence alignment and phylogenetic analysis of proteins were done using ClustalW2 (29Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56003) Google Scholar). Nucleotide Sequence and Accession Number—GroESL operon sequence has been submitted to GenBank™ (accession number AY184491). Identification and Purification of Native XnGroEL Protein—The SDS-PAGE profile of OMV proteins prepared from culture supernatant of X. nematophila contained multiple proteins ranging from 10 to 200 kDa (5Khandelwal P. Banerjee-Bhatnagar N. Appl. Environ. Microbiol. 2003; 69: 2032-2037Crossref PubMed Scopus (75) Google Scholar). The N-terminal sequence of a predominant protein band of ∼58 kDa identified it as a homolog of E. coli heat shock protein GroEL. The XnGroEL protein eluting between 0.5 and 0.7 mm NaCl from the ion-exchange column contained minor impurities (Fig. 1A, lane 2). The partially purified protein was passed through Superose-12 size fractionation column, and XnGroEL was eluted in the void volume in oligomeric form. The void volume fractions contained pure homogeneous native XnGroEL protein (Fig. 1A, lane 3), also seen by silver staining (Fig. 1A, lane 4). No lipopolysaccharide was found associated with the protein (data not shown). The final yield of purified protein was ∼2 mg per liter of culture supernatant. EcGroEL protein was also purified from 5 liters of cell lysate, and homogeneity was examined by silver staining. Biochemical Characterization of XnGroEL Protein—Elution of XnGroEL in the void volume of Superdex-200 column indicated that the purified protein existed as high molecular (>600 kDa) oligomer (data not shown), as reported for EcGroEL. XnGroEL was able to hydrolyze γ-P32-labeled ATP with the release of inorganic phosphate. Synthesis of the protein by X. nematophila was higher compared with E. coli at 28 °C and was further enhanced when subjected to heat shock at 37 °C. The protein was able to function as chaperone in an in vitro lactate dehydrogenase folding system (data of the above experiments not shown). Export of XnGroEL Protein—Because the protein was found in culture supernatant, we examined whether the protein was actually secreted or was present due to cell lysis. Different cellular fractions, e.g. cytosolic, inner membrane (IM), periplasmic, outer membrane (OM), and extracellular proteins, were subjected to SDS-PAGE analysis followed by Western blotting (Fig. 2A). The presence of XnGroEL was observed in all the above fractions, whereas HNS (histone-like nucleoid structuring protein), a cytosolic protein, or DsbA, a periplasmic marker protein, was not detected in the culture supernatant (data not shown), suggesting a specific secretion pathway for export of XnGroEL outside the cell. GroES, the canonical co-chaperone, was present in the cytoplasmic fraction but absent in culture supernatant or OMVs of X. nematophila (Fig. 2B). Treatment of OMV preparations with Pronase degraded XnGroEL completely, indicating its location on the surface, possibly in association with the outer membrane of the bacteria (Fig. 2C). Cloning, Expression, and Purification of Recombinant Proteins—Southern blot analysis of genomic DNA after restriction digestion identified a ∼4-kb fragment containing the gene in the EcoRI digest. The partial library produced with 3–5 kb of EcoRI-digested DNA fragments in pUC18 vectors led to the isolation of a positive clone with an ∼3.8-kb insert (pMJ1). The sequence of the 3.8-kb DNA fragment showed that it contained GroESL operon spanning ∼2.4 kb preceded by two hypothetical open reading frames and part of aspartate ammonium lyase A. The nucleotide sequence of GroESL operon of X. nematophila was very similar to closely related Photorhabdus spp. (85%) and E. coli (84%). The translated amino acid sequence of XnGroEL was (89%) similar to the EcGroEL sequence. The recombinant XnGroEL protein was obtained from the pET28a construct (pMJ2); it was purified to homogeneity by nickel-nitrilotriacetic acid column (Fig. 1A, lane 5). The purified recombinant XnGroEL was obtained as oligomeric complex, which remained stable at pH 8.8 (Fig. 1B). The protein showed ATPase activity and in vitro chaperoning activity like native XnGroEL. The 21-kDa apical domain and the 30-kDa apical-intermediate domains existed as monomer and had no ATPase activity (data not shown). Toxicity of XnGroEL in H. armigera Larvae—Both the native and recombinant XnGroEL proteins retarded growth of H. armigera neonates at 1–20 μg/g diet when fed orally. Reduction in the average weight of the surviving larvae was 50–55% on sixth day of the larval period. A dose-dependent effect on larval mortality was observed at concentrations 5–15 μg/g (Fig. 3A). Heat-inactivated XnGroEL, EcGroEL, and bovine serum albumin used as control, caused low mortality (∼5%) of the insect larvae. The 50% lethal concentration (LC50) of native protein purified from cell lysate (intracellular) and culture supernatant (extracellular) were ∼3.8 μg/g, and ∼3.6 μg/g, respectively. The recombinant full-length protein was toxic with LC50 ∼ 4.8 μg/g. The apical domain of XnGroEL was also toxic to insect neonates when fed orally in a dose-dependent manner (LC50 ∼ 5.8 μg/g). The relationship between larval death and protein concentration was evaluated by linear regression analysis. The values of the slope (m) representing toxicity were 0.006, 0.01, and 0.02 for apical domain, apical-intermediate domain, and full-length protein, respectively, indicating that the apical domain retained ∼1/3 that of the toxicity of the full-length protein. The toxicity was increased to ∼1/2 that of the full-length protein by the addition of intermediate domain (apical-intermediate domain) (analysis of variance, p < 0.001) (Fig. 3B). These results suggested that although the apical domain of XnGroEL alone was able to manifest toxicity, the intermediate and the equatorial domains too have some role in toxicity of the protein. Reduced toxicity of the domains could also be attributed to their inability to oligomerize, which could be necessary for providing strength by cooperative binding. Protein Sequence and Structural Analysis—To investigate the basis of interaction of XnGroEL at molecular levels, pairwise alignment of primary amino acid sequence with non-toxic E. coli protein was performed (Fig. 4A). In addition, the GroEL sequences of free-living and symbi
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