Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium.
A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI--BglII fragment between the kanamycin resistance (Kmr) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Kmr transfer frequency of 10(-6) per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8 x 10(6) cpm for a cell density of 10(3) colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.
Boivin
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Département de Phytologie, Faculté des Sciences, Université Laval, Québec, Canada.
Chalifour
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Dion
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D004251:DNA Transposable Elements; D008163:Luminescent Measurements; D010641:Phenotype; D010944:Plants / Q000235:genetics; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D012231:Rhizobium / Q000235:genetics*; D013045:Species Specificity
D004251:DNA Transposable Elements