A panel of Tn7-based vectors for insertion of the gfp marker gene or for delivery of cloned DNA into Gram-negative bacteria at a neutral chromosomal site

The use of Tn7-based systems for site-specific insertion of DNA into the ch romosome of Gram-negative bacteria has been limited due to the lack of appr opriate vectors. We therefore developed a flexible panel of Tn7 delivery ve ctors. In one group of vectors, the miniTn7 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 int ended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter P-A1/04/03, several transcrip tional 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 fluor escent Pseudomonas strains. The gfp-tagging vector containing a gentamicin- resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are fre quently used to generate bacterial mutants and to deliver reporter construc tions in gene expression studies. To demonstrate the utility of a dual mark er / reporter system, the Tn7-gfp marker system was combined with a Tn5-del ivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence. ( C) 2001 Elsevier Science B.V. All nights reserved.