Insertion mutagenesis in Citrobacter freundii by Mini-Tn5 transposon

Objective To study the technique of transposon mutagenesis involving the use of Mini-Tn5 on a suicide plasmid for Citrobacter freundii (C. freundii). Methods A conjugation method was used in transposon mutagenesis. The stability of exconjugants was confirmed by series passages under nonselective condition. Detection of anti-ampicillin resistance and purification of the plasmid were conducted for excluding the possibility of plasmid-encoded antibiotic resistance. All exconjugants were screened for auxotrophic mutants and swarming motility mutants. Results Analysis of the generated exconjugants indicated that Mini-Tn5 Km was transposed in C. freundii at a high frequency (ca. 9.2×10 -6). The mutants were stable and retained the transposon-encoded antibiotic resistance after incubation for three passages under nonselective condition. The frequency of auxotrophic mutants in the population confirmed that the insertion of the transposon was random and the C. freundii chromosome did not contain significant insertional hot spots of transposition. No extrachromosomal plasmid DNA was found. Conclusion Transposon mutagenesis of Mini-Tn5 is a suitable technique for the study of differentiation in C. freundii.