Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium Photorhabdus luminescens

Abstract

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 ...

A Phosphopantetheinyl Transferase Homolog Is Essential for Photorhabdus luminescens To Support Growth and Reproduction of the Entomopathogenic Nematode Heterorhabditis bacteriophora

“…Analyses of phase-dependent characteristics and the ability of the cells to support nematode growth and reproduction or to be retained by IJ nematodes were performed as described above. The pathogenicity of bacteria for insects was determined as described previously (11). Positive insect pathogenicity was defined 72 h postinjection as 50% mortality of insect larvae resulting from a dose of less than 30 cells.…”

Analysis of the PixA Inclusion Body Protein of Xenorhabdus nematophila

“…produce two distinct proteinaceous intracellular inclusions during stationary phase in culture and in insects (2,10,11). The sister taxon of Xenorhabdus, Photorhabdus, which engages in mutualistic associations with nematodes of the Heterorhabditidae family (1,7,13), also produces two morphologically distinct proteinaceous inclusion bodies (4,8). The finding that intracellular crystalline inclusions are produced in both Xenorhabdus and Photorhabdus species suggests they are integral to the symbiotic pathogenic life cycles of these bacteria.…”

The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells

“…The high molecular mass (~1 MDa) toxin complexes (Tc's) are dominant secreted virulence factors of the insect pathogen and nematode symbiont Photorhabdus luminescens Bowen & Ensign, 1998). They the tccC-like or [C] components (ffrench-Constant & Waterfield, 2006;Waterfield et al, 2001b).…”