mini-Tn7 insertion in bacteria with secondary, non-glmS-linked attTn7 sites: example Proteus mirabilis HI4320.

We previously constructed a series of mini-Tn7 chromosome integration vectors that, when provided only with the site-specific transposition machinery, generally transpose to a naturally evolved, neutral attTn7 site that is located 25-bp downstream of the glmS gene. Here we provide a protocol for application of the mini-Tn7 system in Proteus mirabilis as an example of a bacterium with a secondary attTn7 site that is not linked to glmS but, in this case, located in the carAB operon. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into P. mirabilis by transformation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions; and last, optional Flp-mediated excision of the antibiotic-resistance selection marker present on the chromosomally integrated mini-Tn7 element. When transposon-containing cells are selected on rich medium, insertions occur at both attTn7 sites with equal efficiency and frequency. Because carA mutants are arginine and pyrimidine auxotrophs, single-site insertions at the glmS attTn7 sites can be obtained by selection on minimal medium. From start to verification of the insertion events, the whole procedure takes 5 d. This chromosome integration system in P. mirabilis provides an important tool for animal and biofilm studies based on this bacterium. Vectors are available for gene complementation and expression, gene fusion analyses and tagging with a green fluorescent protein (GFP)-encoding reporter gene.