Developing a Two-Hybrid System in Azotobacter vinelandii

MORENO D; MARTÍNEZ-NOEL G; RUBIO L

Ginebra, Suiza

Conferencia; 9th European Nitrogen Fixation Conference; 2010

Abstract Book

The Two-Hybrid System is a powerful method that allows determining in vivo protein-protein interactions (1). We are developing an adapted Two-Hybrid System in the nitrogen-fixing bacterium Azotobacter vinelandii. This organism is an obligate aerobe specialized to support diverse anaerobic metabolic processes (2), an essential condition for any oxygen-sensitive protein. Furthermore, it has a high-frequency of transformation and is highly amenable to genetic manipulation. This system will be the experimental basis to study the interactions of proteins involved in the biosynthesis of iron-molybdenum cofactor (FeMo-co) of nitrogenase in their normal anaerobic environment. A new feature of our bacterial Two-Hybrid System is the mechanism to introduce the bait and target proteins. While others include the bait and target sequences in vectors, we will insert both of them in the chromosome by recombination, solving the problem to maintain the plasmids in the reporter strain. In conclusion, our new two-hybrid system would be a useful tool to study in vivo interactions between oxygen-sensitive proteins.