Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli

Previous attempts to express the toxin complex genes of Photorhabdus lumine scens W14 in Escherichia coli have failed to reconstitute their oral toxici ty to the model insect Manduca sexta. Here we show that the combination of three genes, tcdA, tcdB, and tccC, is essential for oral toxicity to M. sex ta when expression in E. coli is used. Further, when transcription from nat ive toxin complex gene promoters is used, maximal toxicity in E. coli cultu res is associated with the addition of mitomycin C to the growth medium. In contrast, the expression of tcdAB (or the homologous tcaABC operon) with n o recombinant tccC homolog in a different P. luminescens strain, K122, is s ufficient to confer oral toxicity on this strain, which is otherwise not or ally toxic. We therefore infer that P. luminescens K122 carries a functiona l tccC-like homolog within its own genome, a hypothesis supported by Southe rn analysis. Recombinant toxins from both P. luminescens K122 and E. coli w ere purified as high-molecular-weight particulate preparations. Transmissio n electron micrograph (TEM) images of these particulate preparations showed that the expression of tcdAB (either with or without tccC) in E. coli prod uces visible similar to 25-nm-long complexes with a head and tail-like subs tructure. These data are consistent with a model whereby TcdAB constitutes the majority of the complex visible under TEM and TccC either is a toxin it self or is an activator of the complex. The implications for the potential mode of action of the toxin complex genes are discussed.