The Azospirillum brasilense PII protein, encoded by the glnB gene, is a signal transducer that senses the intracellular nitrogen status. A glnB mutant strain of A. brasilense (7628) is not able to fix nitrogen. The nif gene specific transcriptional activator, the NifA protein, is normally expressed in this mutant, but it cannot activate nif transcription suggesting that GlnB is required for NifA activity, possibly by direct interaction. To identify the specific aminoacid residues of GlnB involved in its interaction with NifA, the glnB gene was submitted to a random mutagenesis. The native and mutant glnB genes were cloned into the vector pLAFR3.18 yielding broad-host-range plasmids expressing GlnB and the mutant forms GlnBV100-A and GlnBL13-P under control of the plac promoter. The plasmids were introduced into A. brasilense strains 7628 (glnB mutant) and JI18 (glnZ^-nifH::lacZ). Neither of the GlnB mutant forms were able to restore the nitrogenase activity of strain 7628. GlnBL13-P and GlnB did not affect expression of the nifH::lacZ fusion in strain JI18. In contrast, GlnBV100-A abolished nifH expression. The results suggest that the GlnBV100 residue is essential to promote NifA activation by GlnB whereas the GlnBL13 residue is not.

Supported by: CNPq/ MCT, Fundação Araucária and Paraná Tecnologia