PAS domain of the deduced Org35 protein mediates the interaction with NifA

NifA in Azospirillum brasilense plays a key role in regulating the synthesis of nitrogenase in response to ammonia and oxygen available. Recently, our laboratory has identified four clones, whose gene prodcuts interact with NifA, from A. brasilense Sp7 genomic libraries by using the yeast two-hybrid sys- tem with NifA as bait. We are interested in clone S35, one of the four clones, because it contains a PAS-domain coding region. The entire open reading frame (ORF) for the PAS domain-containing protein was isolated and designated as org35 here. org35 gene is 2211-bp long and encodes a protein of 736 aa with a predicted molecular weight of about 78.4 kD. The predicted amino acid sequence of org35 has similarity to some two-component sensor kinase/response regulator hybrids of bacteria. Struc- tural analyses showed that Org35 comprises at least three discrete conserved domains: the N-terminal PAS, the central histidine protein kinase (HPK) and the C-terminal response regulator (RR). The PAS domain of the deduced Org35 protein was found to interact directly with NifA, but the central HPK and the C-terminal RR domains of Org35 were not. These results indicated that interaction between NifA and Org35 was mediated by PAS domain.