title: Oral toxicity of Photorhabdus luminescens W14 toxin complexes in Escherichia coli.
abstract: Previous attempts to express the toxin complex genes of Photorhabdus luminescens W14 in Escherichia coli have failed to reconstitute their oral toxicity 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. sexta when expression in E. 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 homologous tcaABC operon) with no recombinant tccC homolog in a different P. luminescens strain, K122, is sufficient to confer oral toxicity on this strain, which is otherwise not orally toxic. We therefore infer that P. luminescens K122 carries a functional tccC-like homolog within its own genome, a hypothesis supported by Southern analysis. Recombinant toxins from both P. 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 of tcdAB (either with or without tccC) in E. coli produces visible approximately 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.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom.
authors.forename: N
authors.initials: N
authors.lastname: Waterfield
authors.forename: A
authors.initials: A
authors.lastname: Dowling
authors.forename: S
authors.initials: S
authors.lastname: Sharma
authors.forename: P J
authors.initials: PJ
authors.lastname: Daborn
authors.forename: U
authors.initials: U
authors.lastname: Potter
authors.forename: R H
authors.initials: RH
authors.lastname: Ffrench-Constant
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018402:Manduca / Q000187:drug effects* / Q000502:physiology; D008854:Microscopy, Electron; D009876:Operon; D020637:Photorhabdus / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D011994:Recombinant Proteins