Screening of the interacting proteins with NifA in Azospirillum brasilense Sp7 by the yeast two-hybrid system

Abstract

NifA in Azospirillum brasilense plays a key role in regulating the synthesis and activity of nitrogenase in response to ammonia and oxygen available. In this work we used the yeast two-hybrid system to identify the proteins that interact with NifA. The nifA gene was fused to the yeast two-hybrid vector pGBD-C2, and three A. brasilense Sp7 genomic libraries for use in yeast two-hybrid studies were constructed. Screening of the libraries identified four clones encoding proteins that interact with NifA. The confirmation of the interactions of each gene product of the four clones and NifA were carried out by exchanging the vectors for nifA and the four clones and by mutageneses of the four clones with shift reading frame experiments in yeast two-hybrid studies. DNA sequence analyses showed that two clones encode proteins containing PAS domains that play an important role in signal transduction. One clone has high similarity with the fhuE gene of Escherichia coli, whose gene product is involved in iron uptake and transportation, and the other clone encodes an unknown protein.