Transgenic plants expressing Bacillus thuringiensis (Bt) toxins are currently being deployed for insect control. In response to concerns about Bt resistance, we investigated a toxin secreted by a different bacterium Photorhabdus luminescens, which lives in the gut of entomophagous nematodes. In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver. The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd. Both tca and tcd encode complexes with high oral toxicity to Manduca sexta and therefore they represent potential alternatives to Bt for transgenic deployment.

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Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramidshaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid-to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to Galleria mellonella larvae.Photorhabdus luminescens is a bioluminescent gram-negative, rod-shaped bacterium that was first isolated from a lightemitting insect that had been infected by entomogenous nematodes of the family Heterorhabditidae (22,29). Biochemical tests and analysis of the 16S rRNA revealed that P. luminescens is related to members of the Enterobacteriaceae in the gamma subdivision of purple bacteria (13,31,32).The bacteria reside in the intestinal tract of the infective juvenile (IJ) stage of the nematode, which is the vector for transmission of the bacteria between insect prey. The IJ penetrates the insect, releasing the bacteria into the hemolymph. The bacteria multiply rapidly, killing the insect within 24 to 72 h, at which time the dead insect is visibly bioluminescent (23,25,29). A 50% lethal dose (LD 50 ) of fewer than 5 cells per insect has been reported for Galleria mellonella (wax moth) larvae (15). The bacterium produces potent insecticidal toxins during growth in the insect as well as in laboratory culture (9, 21). The nematode completes several rounds of reproduction while feeding on the bacteria in the insect carcass. Within 10 to 20 days several thousand IJ progeny, each carrying an inoculum of P. luminescens cells, migrate out of the cadaver in search of new insect prey.Cells of P. luminescens growing in insect larvae and in culture medium produce phase-bright inclusion proteins within the cytoplasm (7, 23). Bacteria of the related genus Xenorhabdus, associated with entomogenous nematodes of the family Steinernematidiae, also produce two cytoplasmic inclusion proteins (11). The genes encoding two inclusion proteins, cipA and cipB, of P. luminescens strain NC1 have been cloned and characterized (5). The genes are present at separate loci and show little nucleotide sequence similarity to each other. Blast searches using the nucleotide or amino acid sequences of ...

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed ϳ2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.Photorhabdus luminescens is an insect-pathogenic gram-negative proteobacterium that forms a "symbiosis of pathogens" with insect-pathogenic nematodes (52). In this symbiosis the bacteria are carried in the guts of entomopathogenic nematodes belonging to the family Heterorhabditidae (members of a different group of bacteria, Xenorhabdus spp., are carried in the guts of members of a different group of nematodes, the Steinernematidae). Upon invasion of an insect host by a nematode, the bacteria are released from the gut directly into the open blood circulatory system of the insect, the hemocoel (52). Here the bacteria are thought to release a wide variety of potential virulence factors, including high-molecular-weight toxin complexes (Tc), lipopolysaccharide (LPS), proteases, lipases, and a range of different antibiotics (52). Inferences concerning the involvement of these factors in killing of the insect or in overcoming the insect immune system, however, often result merely from documentation of secretion of the factors into bacterial culture supernatants. Studies examining the precise role of virulence factors during the infection process in insects have not been performed, and studies of Photorhabdus mutants are rare. As a prelude to genetic analysis of potential virulence factors in P. luminescens, we were interested in obtaining a sample sequence of strain W14 in order to document the classes of genes present and to begin to design suitable experiments for analysis of the genes based on a likely idea of their functions.

Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect. Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes. One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease. PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors. The metalloprotease also shows a calcium-and temperature-dependent autolysis. The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh). PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system. Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.

Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the classInsecta.

The current strategy of using transgenic crops expressing insecticidal protein toxins is placing increasing emphasis on the discovery of novel toxins, beyond those already derived from the bacterium Bacillus thuringiensis. Here we review the cloning of four insecticidal toxin complex (tc) encoding genes from a different bacterium Photorhabdus luminescens and of similar gene sequences from Xenorhabdus nematophilus. Both these bacteria occupy the gut of entomopathogenic nematodes and are released into the insect upon invasion by the nematode. In the insect the bacteria presumably secrete these insecticidal toxins, as well as a range of other antimicrobials, to establish the insect cadaver as a monocultural breeding ground for both bacteria and nematodes. In this review, the protein biochemistry and structure of the tc encoding loci are discussed in relation to their observed toxicity and histopathology. These toxins may prove useful as alternatives to those derived from B. thuringiensis for deployment in insect-resistant transgenic plants.

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescenstoxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.