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A system for stable tagging of gram-negative bacteria with the firefly luciferase gene,luc , is described. A previously constructed fusion constitutively expressingluc from the λp R promoter was used. Stable integration into the bacterial genome was achieved by use of mini-Tn5 delivery vectors. The procedure developed was applied for tagging of representative gram-negative bacteria, such asEscherichia coli, Rhizobium meliloti, Pseudomonas putida , andAgrobacterium tumefaciens . The system permitted the detection of taggedR. meliloti in the presence of more than 105 CFU per plate without the use of any selective markers (such as antibiotic resistance genes). No significant differences in growth rates or soil survival were found between the marked strain and the wild-type strain. Studies of bioluminescentR. meliloti also revealed a good correlation between cell biomass and bioluminescence. The firefly luciferase tagging system is an easy, safe, and sensitive method for the detection and enumeration of bacteria in the environment.

Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring