Insecticidal Toxicity of Yersinia frederiksenii Involves the Novel Enterotoxin YacT

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.

Most of the insecticidal toxins used in agriculture come from a single bacterium Bacillus thuringiensis or 'Bt'. Here we review our work on the array of toxins produced by Photorhabdus and Xenorhabdus bacteria that are symbiotic with entomopathogenic nematodes, and discuss their potential for use in agriculture as alternatives to Bt. Despite the fact that both Photorhabdus and Xenorhabdus are introduced directly into the insect blood stream by their nematode vectors, they produce a range of toxins with both oral and injectable insecticidal activity. The toxin complexes (Tc's) are large orally active toxins that are displayed on the outer surface of the bacterium. They require three components (A-C) for full toxicity and one 'A' component has been successfully expressed in transgenic Arabidopsis to confer insect resistance. One such group of Tc's, the PirAB binary toxins, have oral activity against mosquitoes and some caterpillar pests. Their mode of action is not known but they show significant sequence similarity to a recently described neurotoxin beta-leptinotarsin-h isolated from the blood of the Colorado potato beetle. Other toxins such as 'makes caterpillars floppy' (Mcf) and proteins encoded by the 'Photorhabdus virulence cassettes' (PVCs) only show injectable activity. Mcf1 promotes apoptosis in a wide range of cells and appears to mimic mammalian BH3 domain-only proteins in the mitochondrion whereas the mode of action of the PVCs remains undetermined. The likely biological reasons for the massive functional redundancy in Photorhabdus insecticidal toxins are discussed.