Genomic Manipulations of the Diazotroph Azotobacter vinelandii

The biological reduction of nitrogen gas to ammonia is limited to a select group of nitrogen-fixing prokaryotes. While nitrogenase is the catalyst of nitrogen fixation in these biological systems, a consortium of additional gene products is required for the synthesis, activation, and catalytic competency of this oxygen-sensitive metalloenzyme. Thus, the biochemical complexity of this process often requires functional studies and isolation of gene products from the native nitrogen-fixing organisms. The strict aerobe Azotobacter vinelandii is the best-studied model bacterium among diazotrophs. This chapter provides a description of procedures for targeted genomic manipulation and isolation of A. vinelandii strains. These methods have enabled identification and characterization of gene products with roles in nitrogen fixation and other related aspects of metabolism. The ability to modify and control expression levels of targeted sequences provides a biotechnological tool to uncover molecular details associated with nitrogen fixation, as well as to exploit this model system as a host for expression of oxygen-sensitive proteins.