Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae)

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would a llow the detection of viable bacteria in vivo without the necessity for dis ruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into differen t strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was nece ssary to use older cultures (96-160 h) in the stationary phase of X. nemato philus for mating with relatively small differences (<2-fold) in transconju gant yield for the different strains and phases of X. nematophilus, All tra nsconjugants emitted high levels of light with optimum bioluminescence at 2 7 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity , and temperature conditions were similar to those encountered by the bacte ria in the hemolymph of the larvae of Galleria mellonella. Plasmids were de tected in the transconjugants after 6 months of subculturing the bacteria w ithout antibiotic selection. Aside from light emission, luminescent transco njugants had the same physiological properties as the nonluminescent parent al strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacteria l-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readi ly be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interaction s, but also as a potential agent to follow the infection and death of the i nsect population.