Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.
In bacteria, the first reaction of the tryptophan biosynthetic pathway involves the conversion of chorismate and glutamine to anthranilate by the action of anthranilate synthase, which is composed of the alpha (trpE gene product) and beta (trpG gene product) subunits. In this study, the tryptophan biosynthetic gene trpE of the spirochaete Leptospira meyeri was interrupted by a kanamycin-resistance cassette by homologous recombination. The trpE double cross-over mutant was not able to grow on solid or in liquid EMJH medium. In contrast, the trpE mutant showed a wild-type phenotype when tryptophan or anthranilate was added to the media, therefore showing that disruption of the L. meyeri trpE gene resulted in tryptophan auxotrophy. The authors have also characterized a second selectable marker that allows the construction of a spectinomycin-resistant L. meyeri-E. coli shuttle vector and the functional complementation of the L. meyeri trpE mutant.
Bauby
Hélène
H
Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France.
Saint Girons
Isabelle
I
Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France.
Picardeau
Mathieu
M
Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur, 28 rue du docteur Roux, 75724 Paris Cedex 15, France.
D000878:Anthranilate Synthase / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007612:Kanamycin / Q000494:pharmacology; D007919:Leptospira / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019884:Nitrogenous Group Transferases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D014364:Tryptophan / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000378:metabolism
D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D007612:Kanamycin; D014364:Tryptophan; D019884:Nitrogenous Group Transferases; D000878:Anthranilate Synthase; C020655:anthranilate synthase, glutamine amidotransferase subunit