Adaptation of the yeast URA3 selection system to gram-negative bacteria and generation of a {delta}betCDE Pseudomonas putida strain.
A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5'-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.
Galvão
Teca Calcagno
TC
Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain. vdlorenzo@cnb.uam.es.
de Lorenzo
Víctor
V
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D017353:Gene Deletion; D005819:Genetic Markers; D005821:Genetic Techniques; D005822:Genetic Vectors; D009876:Operon; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D009997:Osmotic Pressure; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012641:Selection, Genetic
D001426:Bacterial Proteins; D005819:Genetic Markers; D009964:Orotidine-5'-Phosphate Decarboxylase; D002794:Choline