title: A methodology for markerless genetic modifications in Azotobacter vinelandii.
abstract: OBJECTIVE
  Efficient manipulation of multiple regions within a genome can be improved by counter-selection approaches. In this work, we sought to develop a method to manipulate Azotobacter vinelandii using a counter-selection approach based on the presence of the pyrF gene.
  
  
  RESULTS
  A background uracil auxotroph of A. vinelandii was first constructed by deleting the pyrF gene coding orotidine-5'-phosphate decarboxylase. The pyrF gene and promoter were also incorporated together with an antibiotic marker to create a selection and counter-selection cassette to shuttle into various plasmids. The constructed cassette could then be removed using a plasmid lacking the pyrF gene via counter-selection resulting from the production of 5-fluorouracil. The process could be repeated multiple times using the same procedure for selection and counter-selection. Following completion, the pyrF gene may be reintroduced to the genome in its original location, leaving a completed strain devoid of any antibiotic markers.
  
  
  CONCLUSIONS
  Utilization of the pyrF gene for counter-selection is a powerful tool that can be used effectively to make multiple gene deletions in A. vinelandii.
  
  
  CONCLUSIONS
  This study demonstrates the successful application of a counter-selection approach to yield markerless genetic modifications to A. vinelandii, which should be of interest for a range of applications in this important model bacterium.
authors: Department of Bioproducts and Biosystems Engineering, Biotechnology Institute, University of Minnesota, St. Paul, MN, USA.
authors: L J
authors: LJ
authors: Eberhart
authors: Department of Bioproducts and Biosystems Engineering, Biotechnology Institute, University of Minnesota, St. Paul, MN, USA.
authors: C M
authors: CM
authors: Knutson
authors: Department of Bioproducts and Biosystems Engineering, Biotechnology Institute, University of Minnesota, St. Paul, MN, USA.
authors: B M
authors: BM
authors: Barney
mesh_terms: D016948:Azotobacter vinelandii / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005821:Genetic Techniques; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: Azotobacter vinelandii; counter-selection; markerless; nitrogen; nitrogenase; polyhydroxyalkanoate; pyrF