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

A series of Tn1721-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 (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) 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 Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd 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 TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.