The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat-and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study. The mutualistic association of the enteric entomopathogenic bacterium Photorhabdus luminescens and its nematode vectorHeterorhabditis bacteriophora is a potentially useful model for studies of vector-borne disease and use as biological control agents for arthropod vectors of disease and agricultural insect pests. The two partners, acting in concert, are voracious pathogens to a wide variety of insect larvae (24, 28; for a recent review, see reference 15). The dauer juvenile (DJ) stage of the nematode carries in its intestine a monoculture of symbiotic P. luminescens (13,20,21,25). The nematode locates a susceptible insect victim, enters the hemocoel, and releases its charge of bacteria, and death of the insect follows, usually within 24 h (15,24,28). Insect mortality is primarily attributable to the extreme virulence of P. luminescens, where the 50% lethal dose for some insects is less than 30 cells when injected into the hemocoel (24,25,29). The bacteria produce potent orally active insecticidal protein toxins that are being developed as biological control agents (5, 6, 18).During growth inside the insect cadaver, the DJ develops into a reproductively mature adult hermaphrodite. The nematode offspring feed on the P. luminescens cells and are highly specific in requiring their specific strain of P. luminescens for growth and reproduction (1,12,16). Following several rounds of reproduction, lasting approximately 2 weeks and in apparent response to impending nutrient limitation, severa...

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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 ...

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The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P. luminescens cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P. luminescens were screened for the loss of the ability to support growth and reproduction of H. bacteriophora nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of P. luminescens to support nematode growth and reproduction are discussed.Photorhabdus luminescens (Enterobacteriaceae) bacteria are symbiotic with entomopathogenic rhabditid nematodes of the family Heterorhabditidae, with which they cooperate in infecting a wide variety of insect larvae (38, 45; for reviews, see references 25 and 26). The nematode requires P. luminescens for insect pathogenicity (34), while the bacteria depend on the nematodes for transmission between insect prey. The infective juvenile (IJ)-stage nematodes specifically retain symbiotic P. luminescens cells in their gut mucosa, and transmission of the bacteria is a requisite for insect pathogenicity (31,32,34). The nematodes require P. luminescens cells as a substrate for growth and reproduction (2,21,22,30). It was suggested previously that symbiotic P. luminescens cells provide favorable nutritional conditions for Heterorhabditis bacteriophora nematodes to grow and reproduce (45).During prolonged laboratory culture, P. luminescens strains show a tendency to undergo an apparent phase variation phenomenon (8, 9, 36). The native form of the bacteria, termed primary phase, is isolated from the IJ stage of the nematode. The secondary-phase variants appear at high frequency during prolonged culturing, while more rare is the generation of primary-phase cells from secondary phase (6). The secondaryphase cells differ from the primary-phase cells in colony morphology, cell size, and dye uptake characteristics (6, 7, 9, 52). Also, typical primary-phase characteristics such as bioluminescence, pigment synthesis, phospholipase and siderophore activities, and production of intracellular crystalline inclusion proteins are depressed or absent in secondary-phase cells. The mechanism and role of phase variati...