Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster.

Détails

ID Serval
serval:BIB_A8DAA2E4DD0F
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster.
Périodique
Gene
Auteur⸱e⸱s
Rella M., Mercenier A., Haas D.
ISSN
0378-1119 (Print)
ISSN-L
0378-1119
Statut éditorial
Publié
Date de publication
1985
Volume
33
Numéro
3
Pages
293-303
Langue
anglais
Résumé
For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.
Mots-clé
Arginine/metabolism, Chromosome Mapping, Cloning, Molecular, DNA Restriction Enzymes, DNA Transposable Elements, DNA, Recombinant, Deoxyribonuclease EcoRI, Drug Resistance, Microbial, Genes, Bacterial, Genotype, Mutation, Nucleic Acid Hybridization, Plasmids, Pseudomonas aeruginosa/genetics, Pseudomonas aeruginosa/metabolism, Temperature, Threonine/metabolism
Pubmed
Web of science
Création de la notice
25/01/2008 17:01
Dernière modification de la notice
20/08/2019 15:13
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