pTn5cat: a Tn5-derived genetic element to facilitate insertion mutagenesis, promoter probing, physical mapping, cloning, and marker exchange in phytopathogenic and other gram-negative bacteria.

摘要

A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence in Pseudomonas syringae pv. phaseolicola. To enhance the rate of mutation this Tn5 derivative was constructed carrying a mutant transposase which was placed in cis to the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterless cat (chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters being expressed under specific conditions. To facilitate cloning and manipulations in Escherichia coli, a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using an E. coli strain such as S17-1 to provide the tra functions. Sites for the rare cutters PacI and PmeI have also been included to facilitate locating the insertions on a PacI and/or PmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat. It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested in P.s. phaseolicola and Xanthomonas campestris pv. campestris. Copyright 1998 Academic Press.