Sequence Analysis of Insecticidal Genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC₅₀] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P_L promoter were shown to have insecticidal activity (LC₅₀ of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Interactions of insecticidal toxin gene products from Xenorhabdus nematophilus PMFI296

Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.     

Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

昆虫病原线虫共生菌BP品系杀虫毒素基因簇中各基因与杀虫活性的关系

昆虫病原线虫共生菌Xenorhabdus nematophilus BP的多个杀虫毒素基因集中在一起形成一个约40kb的基因簇。为研究这个基因簇中各基因与杀虫活性的关系,对该共生菌粘粒文库中5个粘粒克隆XnBP76、XnBP83、XnBP203、XnBPp378 和XnBP414及XnBP83的3个亚克隆插入DNA片段的基因结构和它们对棉铃虫的杀虫活性进行了比较,结果显示,xptB1, xptC1和xptA2 3个基因或后两者的联合表达产物具有最强的杀虫效果,缺失其中的任何1个或2个会使杀虫活力大幅度地下降或完全消失;而xptD1和xptA1的缺失对毒素基因簇的表达产物的杀虫活力影响很小;杀虫毒素的物理混合没有明显的增效作用。     

Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation

Toxin complexes from Xenorhabdus and Photorhabdus spp. bacteria represent novel insecticidal proteins. We purified a native toxin complex (toxin complex 1) from Xenorhabdus nematophilus. The toxin complex is composed of three different proteins, XptA2, XptB1, and XptC1, representing products from class A, B, and C toxin complex genes, respectively. We showed that recombinant XptA2 and co-produced recombinant XptB1 and XptC1 bind together with a 4:1:1 stoichiometry. XptA2 forms a tetramer of ～1,120 kDa that bound to solubilized insect brush border membranes and induced pore formation in black lipid membranes. Co-expressed XptB1 and XptC1 form a tight 1:1 binary complex where XptC1 is C-terminally truncated, resulting in a 77-kDa protein. The ～30-kDa C-terminally cleaved portion of XptC1 apparently only loosely associates with this binary complex. XptA2 had only modest oral toxicity against lepidopteran insects but as a complex with co-produced XptB1 and XptC1 had high levels of insecticidal activity. Addition of co-expressed class B (TcdB2) and class C (TccC3) proteins from Photorhabdus luminescens to the Xenorhabdus XptA2 protein resulted in formation of a hybrid toxin complex protein with the same 4:1:1 stoichiometry as the native Xenorhabdus toxin complex 1. This hybrid toxin complex, like the native toxin complex, was highly active against insects.     

Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Transformation of Xenorhabdus nematophilus

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Transformation of Xenorhabdus nematophilus.

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

致病杆菌属和光杆状菌属细菌杀虫毒素蛋白

致病杆菌属和光杆状菌属细菌是一类分别与斯氏线虫属和异小杆属线虫共生的昆虫病原细菌 ,属肠杆菌科 ,此类细菌产生的杀虫毒素蛋白是近年来发现的一类高效、杀虫谱广的新型杀虫蛋白。此类毒素蛋白对多种昆虫具有注射和口服毒性 ,在同一菌株中有多个杀虫基因 ,各杀虫蛋白基因之间具有协同毒力效应 ,杀虫蛋白基因在大肠杆菌和植物中表达的毒素蛋白对多种害虫具有口服毒性。     

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.     

Characterization of Tn5-Induced Mutants of Xenorhabdus nematophilus ATCC 19061

A negative-selection vector, pHX1, was constructed for use in transposon mutagenesis of Xenorhabdus nematophilus ATCC 19061. pHX1 contains the Bacillus subtilis levansucrase gene which confers sucrose sensitivity. In addition, various Tn5-containing plasmids with different replication origins were transferred by conjugation from Escherichia coli into X. nematophilus ATCC 19061, and one of these plasmids, pGS9, yields Tn5 insertion mutants of X. nematophilus ATCC 19061. By using these two delivery vehicles, more than 250 putative Tn5 insertion mutants of X. nematophilus ATCC 19061 were isolated and were then characterized. Mutants that were altered in bromothymol blue adsorption, ability to lyse sheep erythrocytes, production of antibiotics on a variety of media, and virulence for Galleria mellonella were found.     

Isolation and Entomotoxic Properties of the Xenorhabdus nematophilus F1 Lecithinase

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C₁₈ reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.     

Biochemical Characterization and Agglutinating Properties of Xenorhabdus nematophilus F1 Fimbriae

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus.     

Inactivation of a Novel Gene Produces a Phenotypic Variant Cell and Affects the Symbiotic Behavior of Xenorhabdus nematophilus

Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.     

Survival of Xenorhabdus nematophilus and Photorhabdus luminescens in water and soil

Strains of Xenorhabdus nematophilus and Photorhabdus luminescens were genetically marked with kanamycin resistance and the xylE gene to aid theirdetection in water and soil. Following release in river water, cells declined to undetectable levelsin 6 d. In sterile river water, this decline was enhanced with cells detectable for only 2 d. In sterileMilli-Q purified water, the decline was slower than in either sterile or non-sterile river water.Survival in soil was also restricted with cells only detectable for 7 d. These experiments indicatedthat both X. nematophilus and P. luminescens have limited survival orcompetitive abilities in these environments. The faster decline of populations in sterile river waterwas unexpected, and the possible formation of specialized survival stages was investigated. Insterile water, a non-culturable but viable population of cells was detected, indicating that cellsmay survive longer than anticipated in the environment and remain undetectable using standardmicrobiological methods. The implications of this work to the use of these strains in biologicalcontrol and the release of genetically-modified micro-organisms is discussed.     

Xenorhabdus nematophila BP杀虫毒素基因的序列分析及其杀虫活性

XnBP83是从Xenorhabdus nematophila BP基因组粘粒文库中筛选出的一个对棉铃虫有较强口服杀虫活性的克隆．采用亚克隆结合primer-walking DNA测序技术对粘粒XnBP83的插入片段进行序列测定．该插入片段全长38939bp,其中包括5个与杀虫活性相关的tc类基因xptA1、xptB1、xptC1、xptA2、xptD1．序列分析显示：a．插入片段中的xptD1不完整,与X．nematophila PMFI296 XptD1相应氨基酸序列有99％的相似性．b．BP xptA1读码框全长7569bp,编码2520个氨基酸,与PMFI296的XptA1氨基酸序列有98％的相似性,两者在第2200-2223氨基酸区域连续有23个氨基酸不同．c．BP xptB1读码框全长3051bp,编码1016个氨基酸,与PMFI296 XptB1氨基酸序列有98％的相似性,在第620-650氨基酸之间有28个氨基酸差异．d．BP xptC1读码框全长4225bp,编码1408个氨基酸,与PMFI296的XptC1氨基酸序列有96％的相似性．在BP的第232氨基酸后插入了一个TAQRYLAK的氨基酸序列,在第627-646氨基酸区域内,有18个氨基酸不同．e．BP xptA2读码框全长7574bp,编码2524个氨基酸,与PMFI296的XptA2氨基酸序列有90％的相似性,在BP品系XptA2的第788-855氨基酸和第1630～1784氨基酸有两个明显变异区．将XnBP83培养物上清和沉淀饲喂棉铃虫、甜菜夜蛾、斜纹夜蛾和粉纹夜蛾,结果表明XnBP83对所测昆虫有广谱杀虫活性．     

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.