Yersinia pestis, the causative agent of bubonic plague, is transmitted from mammal to mammal primarily by the bites of infected fleas. Although the molecular pathogenesis of plague in the mammal has been well studied, the molecular and genetic mechanisms that enable Y.pestis, uniquely among the Enterobacteriaceae, to utilize a blood-sucking insect for transmission have not been investigated. We are studying the interaction of Y.pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild type or specific Y.pestis mutants. We seek to identify Y.pestis genes that are required for the bacteria to infect the flea midgut and to produce blockage of the flea foregut that is required for biological transmission. We have termed these genes transmission factors (analogous to virulence factors, the genes required for pathogenesis in the mammal). The strategy entails first identifying bacterial genes that are differentially expressed at a temperature typical of the cold- blooded flea host (20C) as opposed to the temperature of the warm-blooded mammalian host (37C). Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors will allow further studies into the molecular mechanisms of the bacterial infection of the flea vector. The work provides a model that can be applied to other arthropod-borne bacterial pathogens.