A panel of Tn 7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site

Abstract

The use of Tn 7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn 7 delivery vectors. In one group of vectors, the miniTn 7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter P A1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn 7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn 5 transposon. Tn 5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker/reporter system, the Tn 7- gfp marker system was combined with a Tn 5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn 5-tagged locus could be determined by measuring bioluminescence.