A methodology for markerless genetic modifications in Azotobacter vinelandii

Aims 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. Methods and 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 . Significance and Impact of the Study 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.