Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.

     

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342^T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337^T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336^T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338^T, isolated from Steinernema rarum.     

Pyrimidine Nucleoside Salvage Confers an Advantage to Xenorhabdus nematophila in Its Host Interactions

Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity.     

Characterization of Xenorhabdus isolates from La Rioja (Northern Spain) and virulence with and without their symbiotic entomopathogenic nematodes (Nematoda: Steinernematidae)

Eighteen Xenorhabdus isolates associated with Spanish entomopathogenic nematodes of the genus Steinernema were characterized using a polyphasic approach including phenotypic and molecular methods. Two isolates were classified as Xenorhabdus nematophila and were associated with Steinernema carpocapsae. Sixteen isolates were classified as Xenorhabdus bovienii, of which fifteen were associated with Steinernema feltiae and one with Steinernema kraussei. Two X. bovienii Phase II were also isolated, one instable phase isolated from S. feltiae strain Rioja and one stable phase from S. feltiae strain BZ. Four representative bacterial isolates were chosen to study their pathogenicity against Spodoptera littoralis with and without the presence of their nematode host. The four bacterial isolates were pathogenic for S. littoralis leading to septicemia 24 h post-injection and killing around 90% of the insect larvae 36 h post-injection, except for that isolated from S. kraussei. After 48 h of injection, this latter isolate showed a lower final population in the larval hemolymph (10⁷ instead of 10⁸ CFU per larvae) and a lower larval mortality (70% instead of 95-100%). The virulence of the nematode-bacteria complexes against S. littoralis showed similar traits with a significant insect larvae mortality (80-90%) 5 days post-infection except for S. kraussei, although this strain reached similar of larval mortality at 7 days after infection.     

Bacteriophage Lambda as a Delivery Vector for Tn10-Derived Transposons in Xenorhabdus bovienii

Xenorhabdus bovienii wild-type strains lack a functional receptor protein (LamB) in the outer membrane and as a result are unable to adsorb coliphage lambda (λ). Introduction of plasmids encoding lamB into X. bovienii T228 results in constitutive expression of LamB in the outer membrane of this organism. LamB-expressing strains of X. bovienii adsorb lambda bacteriophage particles and can be used as hosts for lambda::Tn constructs. A Tn10-derived transposon, element 9 (J. C. Way, D. Davis, D. Morisato, D. E. Roberts, and N. Kleckner, Gene 32:369-379, 1984) was used to construct a variety of insertion mutants of X. bovienii. Mutants that had altered expression of protease, lipase, DNase, dye-binding capability, and hemolytic activity, in addition to a series of auxotrophic mutants, were isolated.     

Differential pathogenicity of two entomopathogenic bacteria,Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle,Tribolium castaneum

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae, respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A₂ activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.     

Multilocus Sequence Analysis of Xanthomonads Causing Bacterial Spot of Tomato and Pepper Plants Reveals Strains Generated by Recombination among Species and Recent Global Spread of Xanthomonas gardneri

Four Xanthomonas species are known to cause bacterial spot of tomato and pepper, but the global distribution and genetic diversity of these species are not well understood. A collection of bacterial spot-causing strains from the Americas, Africa, Southeast Asia, and New Zealand were characterized for genetic diversity and phylogenetic relationships using multilocus sequence analysis of six housekeeping genes. By examining strains from different continents, we found unexpected phylogeographic patterns, including the global distribution of a single multilocus haplotype of X. gardneri, possible regional differentiation in X. vesicatoria, and high species diversity on tomato in Africa. In addition, we found evidence of multiple recombination events between X. euvesicatoria and X. perforans. Our results indicate that there have been shifts in the species composition of bacterial spot pathogen populations due to the global spread of dominant genotypes and that recombination between species has generated genetic diversity in these populations.     

Virulence of entomopathogenic nematodes and their symbiotic bacteria against the hazelnut weevil Curculio nucum

The virulence of different entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae, isolates from Catalonia (NE Iberian Peninsula), and their symbiotic bacteria was assessed with regard to the larvae and adults of the hazelnut weevil, Curculio nucum L. (Coleoptera: Curculionidae). The nematode strains screened included one Steinernema affine, five Steinernema feltiae, one Steinernema carpocapsae, one Steinernema sp. (a new species not yet described) and one Heterorhabditis bacteriophora. The pathogenicity of all the strains of nematodes was tested on larvae and only four of them on adults of the hazelnut weevil. Larval mortality ranged from 10% with S. affine to 79% with Steinernema sp. Adult mortality was higher in S. carpocapsae, achieving 100% adult weevil mortality. The pathogenicity of the symbiotic bacteria Xenorhabdus bovienii, X. kozodoii, X. nematophila and Photorhabdus luminescens was studied in larvae and adults of C. nucum. In the larvae, X. kozodoii showed a LT50 of 22.7 h, and in the adults, it was 20.5 h. All nematodes species except S. affine tested against larvae showed great potential to control the insect, whereas S. carpocapsae was the most effective for controlling adults.     

Suppressive effects of metabolites from Photorhabdus and Xenorhabdus spp. on phytopathogens of peach and pecan

Abstract

Our objective was to determine the suppressive abilities of bacterial metabolites derived from Xenorhabdus and Photorhabdus spp. on Glomerella cingulata, Phomopsis sp., Phytophthora cactorum, and Fusicladosporium effusum, which are fungal or oomycete pathogens of pecan, and Monilinia fructicola, a fungal pathogen of peach. In the first set of in vitro assays, when metabolites were compared based on initial bacterial cell count, X. bovienii (SN) metabolites generally exhibited the greatest suppression of phytopathogens and Xenorhabdus sp. (355) the least with Photorhabdus luminescens (Hb) and Xenorhabdus nematophila (All) being intermediate. In a second set of in vitro assays, in which metabolites were compared at 50 mg per ml acetone, P. luminescens (VS) exhibited greater suppression than P. luminescens (Hb), Photorhabdus sp. (MX4), X. bovienii (SN), and Xenorhabdus sp. (3 – 8b). In in vivo tests, 6 or 12% dilutions of X. bovienii (SN) or P. luminescens (Hb) metabolites caused 90 – 100% suppression of P. cactorum lesions on pecan leaves with only slight phytotoxicity. No phytotoxic effects were observed in detached peach leaves at dilutions up to 25%. Metabolite treatments, derived from X. bovienii (SN) and P. luminescens (Hb), were also tested for suppression of F. effusum sporulation in detached pecan shoots. Reductions in sporulation caused by bacterial metabolites were similar to those following treatment with two chemical fungicides, dodine and fenbuconazole; a third chemical triphenyltin hydroxide had no effect. Further research is warranted to determine if fungal or oomycete incited diseases in pecan and peach can be controlled with metabolites of Xenorhabdus spp. and Photorhabdus spp.     

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.     

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.     

Role of Secondary Metabolites in Establishment of the Mutualistic Partnership between Xenorhabdus nematophila and the Entomopathogenic Nematode Steinernema carpocapsae

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace''s insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.     

In-silico analysis of genomic distribution and functional association of hipBA toxin-antitoxin (TA) homologs in entomopathogen Xenorhabdus nematophila

Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila.     

Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae

Abstract Xenorhabdus nematophila, a Gram‐negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells of X. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis (PAGE). Seven polypeptides ranging 50～250 kDa were well separated from the protein complex (named Xnpt) by sodium dodecyl sulfate (SDS)‐PAGE, five of which are identified as XptA2, xptC1, XptB1, GroEL and hypothetical protein by matrix‐assisted laser desorption‐time‐of‐flight mass spectrometry (MALDI‐TOFMS). Xnpt showed high oral virulence to larvae of diamondback moth (DBM), Plutella xylostella L. (Lepidoptera, Plutellidae) as its median lethal concentration (LC₅₀) against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnpt‐fed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.     

A toxin protein from Xenorhabdus nematophila var. pekingensis and insecticidal activity against larvae of Helicoverpa armigera

Xenorhabdus nematophila var. pekingensis, which is highly virulent for many insects, is a symbiotic bacterium of Steinernema carpocapsae isolated from Beijing soil in China. Previous studies demonstrated that the bacterium had high antifeedant activity against larvae of Helicoverpa armigera, Plutella xylostella and Spodoptera exigua. Herein, we report the purification, molecular cloning and antifeedant activity of an intracellular toxic protein from the bacterium. The purified protein displayed a single band and a relative molecular weight of over 212 kDa determined by SDS-PAGE. We designated the protein as XnAFP2. Peptide segments were obtained by MALDI-TOF and covered 40% of the amino acid sequence of a toxin protein from X. nematophilus PMFI1296. The full cDNA sequence encoding for XnAFP2 (Genbank accession number FJ222606) was amplified from X. nematophlia var. pekingensis and consists of 7575 bp. The gene showed homology with up to 99% identity to the A2 gene from X. nematophila strain BP (GenBank accession number AY282763) and 92% identity to the insecticidal toxin xptA2 gene from X. nematophila PMFI 1296 (GenBank accession number AJ308438). The protein caused a rapid cessation in feeding and reduction in larval weight of H. armigera. When fed to third instar larvae of H. armigera in an artificial diet at 6.0 µg/g (w/w) toxin protein, growth reduction reached 97.9%. The insecticidal protein greatly decreased fourth instar larval weight, lengthened larval stage, and reduced pupation and emergence rates. The antifeedant rate in choice and no-choice leaf disk tests against fifth instar larvae was 78.4 and 87.6% in 24 h, respectively.