TnMax — a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis

A series of Tn7721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site ( res), a suicide replication origin ( ori fd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (Tn Max2). Other versions of Tn Max (Tn Max3and Tn Max4) carry a promoterless alkaline phosphatase-encoding gene ( phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10 −2 events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn 1721, Tn Max variants prefer random insertion into plasmids rather than into the E. colichromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The Tn Max-borne ori fd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of Tn Max plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. colihost permissive for ori fd -directed replication.