Showing papers on "Photorhabdus luminescens published in 2011"

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Abstract: Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms We posited that these differences would be reflected in their respective genomes To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004 As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P asymbiotica genomes suggests genomic divergence These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points

Genomic analysis reveals versatile organisms for quorum quenching enzymes: acyl-homoserine lactone-acylase and -lactonase.

Abstract: Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and -acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and -acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation.

The xnp1 P2-like Tail Synthesis Gene Cluster Encodes Xenorhabdicin and is Required for Interspecies Competition

Abstract: Xenorhabdus nematophila, the mutualistic bacterium of the nematode Steinernema carpocapsae, produces the R-type bacteriocin called xenorhabdicin, which is thought to confer a competitive advantage for growth in the insect host. We have identified a P2-like tail synthesis gene cluster (xnp1) that is required for xenorhabdicin production. The xnp1 genes were expressed constitutively during growth and were induced by mitomycin C. Deletion of either the sheath (xnpS1) or fiber (xnpH1) genes eliminated xenorhabdicin production. Production of R-type bacteriocins in a host organism had not been shown previously. We show that xenorhabdicin is produced in the hemocoel of insects infected with the wild type but not with the ΔxnpS1 deletion strain. Xenorhabdicin prepared from the wild-type strain killed the potential competitor Photorhabdus luminescens TT01. P. luminescens was eliminated during coculture with wild-type X. nematophila but not with the ΔxnpS1 strain. Furthermore, P. luminescens inhibited reproduction of S. carpocapsae in insect larvae, while coinjection with wild-type X. nematophila, but not the ΔxnpS1, strain restored normal reproduction, demonstrating that xenorhabdicin was required for killing P. luminescens and protecting the nematode partner. Xenorhabdicin killed X. nematophila from Steinernema anatoliense, demonstrating for the first time that it possesses intraspecies activity. In addition, activity was variable against diverse strains of Xenorhabdus and Photorhabdus and was not correlated with phylogenetic distance. These findings are discussed in the context of the role of xenorhabdicin in the life cycle of the mutualistic bacterium X. nematophila.

Targeting of the actin cytoskeleton by insecticidal toxins from Photorhabdus luminescens.

Abstract: Photorhabdus luminescens produces several types of protein toxins, which are essential for participation in a trilateral symbiosis with nematodes and insects. The nematodes, carrying the bacteria, invade insect larvae and release the bacteria, which kill the insects with their toxins. Recently, the molecular mechanisms of the toxin complexes PTC3 and PTC5 have been elucidated. The biologically active components of the toxin complexes are ADP-ribosyltransferases, which modify actin and Rho GTPases, respectively. The actions of the toxins are described and compared with other bacterial protein toxins acting on the cytoskeleton.

The Regulation of Secondary Metabolism and Mutualism in the Insect Pathogenic Bacterium Photorhabdus luminescens.

Abstract: Photorhabdus is a genus of insect-pathogenic Gram-negative bacteria that also maintain a mutualistic interaction with nematodes from the family Heterorhabditis This complex life cycle, involving different interactions with different invertebrate hosts, coupled with the amenability of the system to laboratory culture has resulted in the development of Photorhabdus as a model system for studying bacterial–host interactions Photorhabdus is predicted to have an extensive secondary metabolism with the genetic potential to produce > 20 different small secondary metabolites Therefore, this system also presents us with a unique opportunity to study the contribution of secondary metabolism to the environmental fitness of the producing organism in its natural habitat (ie, the insect and/or the nematode) In vivo and in vitro studies have revealed that the vast majority of the genetic loci in Photorhabdus predicted to be involved in the production of secondary metabolites appear to be cryptic and, to date, although several have been characterized, only three compounds have been studied in any great detail: 3,5-dihydroxy-4-isopropylstilbene, the β-lactam antibiotic carbapenem, and an anthraquinone pigment In this chapter, we describe how these compounds are made and the role (if any) that they have during the interactions between Photorhabdus and its invertebrate hosts We will also outline recent work on the regulation of secondary metabolism in Photorhabdus and comment on how this has led to an increased understanding of mutualism in this bacterium

Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes.

Abstract: In silico analysis of available bacterial genomes revealed the phylogenetic proximity levels of enzymes responsible for biosynthesis of lipopolysaccharide (LPS) of Yersinia pestis, the cause of plague, to homologous proteins of closely related Yersinia spp. and some other bacteria (Serratia proteamaculans, Erwinia carotovora, Burkholderia dolosa, Photorhabdus luminescens and others). Isogenic Y. pestis mutants with single or double mutations in 14 genes of LPS biosynthetic pathways were constructed by site-directed mutagenesis on the base of the virulent strain 231 and its attenuated derivative. Using high-resolution electrospray ionization mass spectrometry, the full LPS structures were elucidated in each mutant, and the sequence of monosaccharide transfers in the assembly of the LPS core was inferred. Truncation of the core decreased significantly the resistance of bacteria to normal human serum and polymyxin B, the latter probably as a result of a less efficient incorporation of 4-amino-4-deoxyarabinose into lipid A. Impairing of LPS biosynthesis resulted also in reduction of LPS-dependent enzymatic activities of plasminogen activator and elevation of LD50 and average survival time in mice and guinea pigs infected with experimental plague. Unraveling correlations between biological properties of bacteria and particular LPS structures may help a better understanding of pathogenesis of plague and implication of appropriate genes as potential molecular targets for treatment of plague.

Photorhabdus luminescens subsp. kleinii subsp. nov. (Enterobacteriales: Enterobacteriaceae)

Abstract: Association between bacteria Photorhabdus and their nematode hosts Heterorhabditis represents one of the emerging models in symbiosis studies. In this study, we isolated the bacterial symbionts of the nematode Heterorhabditis georgiana. Using gyrB sequences for phylogenetic analysis, these strains were shown to be part of the species of Photorhbdus luminescens but with clear separation from currently recognized subspecies. Physiological properties and DNA-DNA hybridization profiles also supported the phylogenetic relationship of these strains. Therefore, a new subspecies, Photorhabdus luminescens subsp. kleinii subsp. nov., is proposed with the type strain KMD37(T) (=DSM 23513 =ATCC =NRRL B-59419).

Virulence of entomopathogenic bacteria Xenorhabdus bovienii and Photorhabdus luminescens against Galleria mellonella larvae.

Abstract: Keeping in view the serious health and environmental apprehensions associated with the use of pesticides, entomopathogenic symbiotic bacteria have the potential to supersede pesticides for the management of various pests. Lab experiments were conducted to test the toxicity of two bacteria Xenorhabdus bovienii and Photorhabdus luminescens at different bacterial concentrations against Galleria mellonella larvae and influence of different abiotic factors viz.: substrates, temperatures and moisture levels were ascertained on the efficacy of these bacteria. P. luminescens and X. bovienii caused the maximum mortality (99 and 90%, respectively) at a concentration of 4 x 107 cells/ml. Mortality caused by P. luminescens was significantly higher than that of X. bovienii. Highest mortality was observed on sand as compared to filter paper. A temperature of 30oC and a moisture level of 20 % were found optimum for the maximum mortality.

Submerged Monoxenic Culture Medium Development for Heterorhabditis bacteriophora and its Symbiotic Bacterium Photorhabdus luminescens: Protein Sources

Abstract: Most medium formulations for improving culture of entomopathogenic nematodes (EPN) based on protein sources have used enriched media like animal feed such as dried egg yolk, lactalbumin, and liver extract, among other ingredients. Most results, however, showed unstable yields and longer production time. Many of the results do not show the detailed parameters of fermentation. Soy flour, cotton seed flour, corn gluten meal, casein powder, soytone, peptone, casein hydrolysates, and lactalbumin hydrolysate as protein sources were tested to determine the source to support optimal symbiotic bacteria and nematode growth. The protein hydrolysates selected did not improve bacterial cell mass compared with the yeast extract control, but soy flour was the best, showing 75.1% recovery and producing more bacterial cell number (1.4×10⁹/ml) than all other sources. The highest yield (1.85×10⁵ IJs/ml), yield coefficient (1.67×10⁶ IJs/g medium), and productivity (1.32×10⁷ IJs/l/day) were also achieved at enriched medium with soybean protein.

The occurrence of photorhabdus-like toxin complexes in Bacillus thuringiensis.

Abstract: Recently, genomic sequencing of a Bacillus thuringiensis (Bt) isolate from our collection revealed the presence of an apparent operon encoding an insecticidal toxin complex (Tca) similar to that first described from the entomopathogen Photorhabdus luminescens. To determine whether these genes are widespread among Bt strains, we screened isolates from the collection for the presence of tccC, one of the genes needed for the expression of fully functional toxin complexes. Among 81 isolates chosen to represent commonly encountered biochemical phenotypes, 17 were found to possess a tccC. Phylogenetic analysis of the 81 isolates by multilocus sequence typing revealed that all the isolates possessing a tccC gene were restricted to two sequence types related to Bt varieties morrisoni, tenebrionis, israelensis and toumanoffi. Sequencing of the ∼17 kb tca operon from two isolates representing each of the two sequence types revealed >99% sequence identity. Optical mapping of DNA from Bt isolates representing each of the sequence types revealed nearly identical plasmids of ca. 333 and 338 kbp, respectively. Selected isolates were found to be toxic to gypsy moth larvae, but were not as effective as a commercial strain of Bt kurstaki. Some isolates were found to inhibit growth of Colorado potato beetle. Custom Taqman® relative quantitative real-time PCR assays for Tc-encoding Bt revealed both tcaA and tcaB genes were expressed within infected gypsy moth larvae.

Influence of the Photorhabdus luminescens Phosphomannose Isomerase Gene, manA, on Mannose Utilization, Exopolysaccharide Structure, and Biofilm Formation

Abstract: Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Transcriptional Analysis of a Photorhabdus sp. Variant Reveals Transcriptional Control of Phenotypic Variation and Multifactorial Pathogenicity in Insects

Abstract: Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis. In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01α. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01α. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins (pit and pirB) or putative insecticidal toxins (xnp2) were less transcribed in VAR* than in the TT01α. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ΤΤ01α, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses (sodA, katE, and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H(2)O(2) and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.

Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects

Abstract: The oral toxicity of 5 Photorhabdus spp. strains collected in different regions of Korea was determined in the larvae of Plodia interpunctella, Galleria mellonella, Lucilia caesar, and Culex pipiens pallens. When diet or water containing culture media containing 1 of the 5 different strains was ingested by immature insects, the first instar larvae of both G. mellonella and L. caesar and young larvae of C. pipiens pallens died within 3–5 days after treatment. However, mortality of P. interpunctella neonate larvae was slightly slower and reached 94.4%–100% within 7 days after treatment. The mortality rate of a control group given a diet containing water, the medium without cultured bacteria, or Escherichia coli culture medium was not affected. The mortality rates were 100%, 45.3%, 2.8%, and 0% for Galleria, Lucilia, Plodia, and Culex, respectively, in another control group given a culture medium of Photorhabdus luminescens ssp. laumondii (TT01). In addition, culture media containing Photorhabdus strains significantly inhibited molting of third instar Plodia larvae by as much as 88% 7 days after treatment, whereas molting inhibition was reduced by 0%, 4%, and 20% following treatments with water, E. coli, or TT01 culture media, respectively. Our results suggest that the oral administration of Photorhabdus bacterial medium was highly effective for controlling various immature insects.

Biopesticidal affect of photorhabdus luminescens against galleria mellonella larvae and subteranean termite (termitidae: macrotermis)

Abstract: Abstarct Photorhabdus luminescens was isolated from the haemolymph of Galleria mellonella larvae infected with Heterorhabditis bacteriophora isolate 1743. Different experiments on penetration of cells, time of exposure, cell suspension in broth and different doses of bacterial cell were conducted. P. luminescens symbiotic bacterial suspension containing 4.0×10 4 bacterial cell/ml showed penetration power within 9 and 12 min in G. mellonella and Macrotermis spp., respectively. Suspension of P. luminescens in broth at different doses caused 95 and 98 % mortality of G. mellonella and Macrotermis spp., respectively. The LD 50 value of bacterial doses was found to be190.4 , 27.4 bacterial cell /ml, while probit regression equation were calculated as 3.42 + 0.72 x, 3.69 + 0.74x against G. mellonella and Macrotermis spp., respectively. The LT 50 value of G. mellonella on bee hives was found to be 24 hrs. In case of Macrotermis spp., the LT 50 value was calculated as 17.1 hrs. Maximum mortality (after 24hrs) of cell free suspension was 65 and 80% while bacterial pallets showed 97 % mortality of G. mellonella and Macrotermis spp., respectively. The roundworms of the Phylum Nematoda play an important role in the natural control of many insect populations. They have been found parasitizing species in the orders Hemiptera, Diptera, Hymenoptera, Lepidoptera, Orthoptera, Coleoptera, Thysanoptera, Siphonaptera, as well as Isoptera (Nickle & Welch, 1984). Four families of nematodes viz., the Mermithidae, Allantonematidae, Steinernematidae and Heterorhabditidae have shown promise for use in insects control programmes (Popiel & Hominick, 1992), with most research focusing on species in the latter two (Kaya & Gaugler, 1993). These entomopathogenic nematodes (so-named because they are vectors of pathogenic bacteria of the genus Xenorhabdus and Photorhabdus) have a number of characteristics that make them especially suitable for biological control and for commercial production as microbial insecticides: a broad host range, especially among soildwelling pests; exemption from registration in the USA; ease of production, storage and application; a high degree of safety to vertebrates, plants and other non-target organisms; and amenability to genetic selection (Kaya & Gaugler, 1993; Kaya et al., 1993).

The cloning , expression and characterisation of bacterial chitin-binding proteins from pseudomonas aeruginosa , serratia marcescens, photorhabdus luminescens and photorhabdus asymbiotica.

Abstract: It is well recognised that most proteins are subject to post translational modifications and that these modifications can have specific effects on the biological properties and functions of these proteins. The majority of proteins secreted by cells are modified by the attachment of oligosaccharide chains. This glycosylation event has been shown to impact correct protein folding, protein stability, solubility, to aid in cell recognition and to help regulate cell processes. In order to gain a deeper understanding into the impact of altered glycosylation patterns on cellular processes and cell recognition it is necessary to develop new technologies to profile the glycan species displayed on the surface of protein molecules. The present study was dedicated to the development of prokaryotic chitin-binding proteins as novel carbohydrate-binding molecules. Prokaryotic chitin-binding proteins from Serratia marcescens, Pseudomonas aeruginosa, Photorhabdus asymbiotica and Photorhabdus luminescens were cloned, over-expressed in E. coli and purified to homogeneity via (His)6 affinity tags. The activity and specificity of these proteins was tested using a number of insoluble substrates; chitin, chitosan and crystalline cellulose. The ability of these proteins to bind to protein linked glycans was tested using Enzyme linked lectin assays (ELLAs). None of the proteins exhibited any ability to bind glycoproteins in this assay format. A novel N-acetylglucosamine binding assay was developed using CBP21 and the ability to immobilise active CBP21 on a sepharose surface was also demonstrated. Sugar inhibition studies indicated that CBP21 may be capable of binding to mannan and galactan polymers. A site-directed mutagenesis of CBP21 was carried out on the putative binding domain residues to alter the affinity of CBP21. Residues Y54, E55, P56, Q57, S58, E60, T111, H114 and D182 were mutated to alanine, expressed, purified and characterised. The mutation H114A was shown to negatively impact on β-chitin affinity, the Q57A mutant had an increased affinity for chitosan with the proteins Y54A, T111A and D182A displaying an increased affinity for Cellulose. Furthermore it was shown that the putative C-terminal binding domain of CbpD is a chitin-binding domain and that the putative chitin-binding proteins CbpA and CbpL are capable of binding to both α- and β-chitin.

Bioluminescent high‐throughput assay for the bacteria adherence to the tissue culture cells

Abstract: The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.

Antibacterial methods based on bioluminescent bacteria

Abstract: Bioluminescent bacteria are common in salt water environment, but there is the only one genus of soil bacteria with natural bioluminescence – Photorhabdus sp. These symbiotic bacteria reside the gut of entomopathogenic nematodes which are obligate insect parasites with the increasing importance as biological control agent. Isolated Photorhabdus luminescens was used separately for determination of pathogenity to insects. Except P. luminescens the artificial bioluminescent bacterium was used - genetically modified Escherichia coli K12 that carries Photorhabdus genes for bioluminescence. Both of these G- bacteria can be used for antibacterial assays based on their bioluminescence ability. Bioluminescence reaction is mediated by bacterial enzyme luciferase which catalyses the oxidation of long-chain aldehyde (substrate) and reduces flavin mononucleotide with emission of light. This emission can be immediately measured by the luminometer. Bioluminesence is directly connected to kinetics of bacterial growth (the more living bacteria the higher luminescence signal). Immune systems of both vertebrates and invertebrates content number of antibacterial peptides; moreover cooperating with complement cascade and myeloperoxidase activity in vertebrates. After addition of sample (e. g. insect hemolymph, vertebrate blood, plasma or serum) we observed decreasing viability of bacteria. The time required for 50% viability of bacteria was evaluated using kinetic curves corresponding to antibacterial activity of samples. Using different conditions of bioluminescence assay and inhibitors of particular immune effectors, we optimised assay for antibacterial activity measurements of specific parts of immune system both in vertebrates and invertebrates. Our research is supported by grant from Grant Agency of Czech Republic (GA206/09/P470).

Development of a Freeliving Nematode Panagrellus redivivus in Saccharomyces cerevisiae with cip Genes

Abstract: CipA and CipB are two types of intracellular crystalline inclusion proteins produced by Photorhabdus luminescens bacteria,which are symbionts of entomopathogeic Heterorhabditis nematodes.To understand the biological function of these proteins for free living Panagrellus redivivus nematodes,recombinant Saccharomyces cerevisiae expression system of Cip proteins were constructed and the resulting yeast cells were used to feed the sterile J1 juveniles of P.redivivus.In recombinant yeast cells with CipA and/or CipB,the nematodes developed about 24 h faster than those in the yeast cells without Cipproteins.This promotion was reflected in two aspects: to short the cycle time and to enhance the reproductive ability of P.redivivus nematode.It means that the nutrient sources from entomopathogeic nematodes are acceptable by this free-living nematode.

Insecticidal Toxin Complex Proteins from Xenorhabdus

Abstract: 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. The30-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.