Oral Toxicity of Photorhabdus luminescens W14 Toxin Complexes in Escherichia coli

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Abstract
Previous attempts to express the toxin complex genes ofPhotorhabdus luminescens W14 inEscherichia coli have failed to reconstitute their oral toxicity to the model insectManduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity toM. sexta when expression inE. coli is used. Further, when transcription from native toxin complex gene promoters is used, maximal toxicity in E. coli cultures is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologoustcaABC operon) with no recombinant tccChomolog in a different P. luminescensstrain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer thatP. luminescens K122 carries a functionaltccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from bothP. luminescens K122 and E.coli were purified as high-molecular-weight particulate preparations. Transmission electron micrograph (TEM) images of these particulate preparations showed that the expression oftcdAB (either with or without tccC) inE. coli produces visible ∼25-nm-long complexes with a head and tail-like substructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin itself or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.