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.
Bibliographical noteFunding Information:
This work is supported by grants (RC-0007-12) from the Initiative for Renewable Energy & the Environment (Institute on the Environment); the MnDRIVE transdisciplinary research initiative through the University of Minnesota based on funding from the state of Minnesota to B.M.B; the National Institute of Food and Agriculture (Project Numbers MIN-12-070 and MIN-12-081); and the Biotechnology Institute at the University of Minnesota for fellowship funding to L.J.E. We thank Matt Posewitz for helpful suggestions related to this project. We also thank the reviewers of this manuscript for helpful suggestions and pointing out previous work done by others using sacB in A. vinelandii.
- Azotobacter vinelandii