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 chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. 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 intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter PA1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 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 Tn5 transposon. Tn5 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 Tn7-gfp marker system was combined with a Tn5-delivered 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.
Koch
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B
Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 C, Frederiksberg, Denmark. bik@kvl.dk
Jensen
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LE
Nybroe
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O
D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004352:Drug Resistance, Microbial / Q000235:genetics; D005822:Genetic Vectors; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011551:Pseudomonas fluorescens / Q000235:genetics
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)