Objective    To construct a vector plasmid which facilitates generating precise deletion in the genes of gram-negative bacteria.     Methods    The vector was designed to encode following fragments; the replication origin of the π protein-dependent origin of plasmid R6K (not replicate in bacteria other than π protein-producing bacteria and function as “suicide vector”)

the mob region from the broad-host-range plasmid RP4 (mobilizable by conjugation into a wide range of Gram-negative bacteria)

a multiple cloning sites (EcoRⅠXbaⅠApaⅠSfiⅠSacⅠNotⅠSpeⅠNdeⅠSacⅡBglⅡ)

a kanamycin resistance marker for orthoselection

and a sacB gene for inverse selection. After the construction

the vector was used to carry out a gene deletion experiment by deleting yeeZ gene from Citrobacter freundii.     Results    The designed vector plasmid was constructed successfully and named as pYG4. It did work in the gene deletion experiment.     Conclusion    Our constructed vector plasmid is useful and convenient for generating gene deletion in gram-negative bacteria.