Construction of a Pseudomonas sp. derivative carrying the cry9Aa gene from Bacillus thuringiensis and a proposal for new standard criteria to assess entomocidal properties of bacteria

An isolate of Pseudomonas sp. (16S rDNA sequence 98% homologous to P. graminis and P. lutea) was isolated from the phyllosphere of black pine in northern Italy and used as a host for the gene encoding the Cry9Aa entomocidal toxin from Bacillus thuringiensis subsp. galleriae. An expression system featuring a synthetic highest-consensus promoter specifically tailored for the regulated induction of cloned genes over a broad range of Gram-negative bacteria was used to drive the production of the introduced toxin. The construct showed effective toxicity toward larvae of the greater wax moth (Galleria mellonella), which was also used as a model insect for establishing a number of newly proposed toxicity indices (LC50 cellular efficiency, toxin cellular efficiency, GMO efficiency, lethal cellular intake). These were devised in order to express toxicities of entomocidal bacteria in a standard fashion enabling the fine tuning of biocontrol treatments as well as the comparative evaluation of different reports.