Ice nucleation active (INA) bacteria are the most potent heterogeneous ice nuclei in nature, which have become an important biological resource for diverse applica-tions. Many researches have proved that INA bacteria can raise the supercooling points (SCPs) of insect pests, then reduce their cold hardiness. However, INA bacteria抯 ineffi-cient colonization on the surface or in the guts of insects, and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling in-sect pests in agricultural fields. In this study, we constructed a recombinant plasmid mob-Tn5-iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. In addition, Ent. cloacae strains stably carrying iceA and expressing high ice nucleation activity (INA), even in the absence of antibi-otic pressure, were constructed through conjugal mobiliza-tion and Tn5 transposition. Ent. cloacae strains have been reported to be able to efficiently colonize in the guts of in-sects, but have weak plant epiphytic ability. Therefore, these transgenic Ent. cloacae may be promising candidates for control of insect pests in agricultural fields. Many researches have proved that INA bacteria can raise the supercooling points (SCPs) of insect pests, then However, INA bacteria ’s ineffi-cient colonization on the surface or in the guts of insects, and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling in-sect pests in agricultural fields . In this study, we constructed a recombinant plasmid mob-Tn5-iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of iceA into chromosomal DNA of many gram- negative bacteria by Tn5 transposition. In addition, Ent. Cloacae stably carrying iceA and expressing high ice nucleation activity (INA), even in the absence of antibi-otic pressure, were constructed through conjugal mobilization and Tn5 transposition. Ent. Cloacae strains have been reported to be able to efficiently colonize in the guts of in-sects, but have weak plant epiphytic ability. Thus, these transgenic Ent. Cloacae may be promising candidates for control of insect pests in agricultural fields .