Dept of Microbiology, China Agricultural University, Beijing 100094, China
Pn is specifically required for nitrogen fixation in A. brasilense. Pn activates NifA, preventing an inhibitory effect on the N-terminal domain (Arsene et al. 1996). However, the mechanism of Pn interacting with NifA directly or indirectly is unknown. Here we investigate the interaction between Pn and NifA in A. brasilense Sp7 by using yeast two-hybrid system.
The glnB gene encoding Pn was PCR amplified and fused to the DNA-binding domain in the pGBD-C2 plasmid vector to generate a recombinant pGBD-GlnB plasmid. Four DNA fragments corresponding respectively to the N-terminal domain, the central domain, the C-terminal domain of NifA and the complete NifA protein were PCR amplified and fused respectively to the DNA-activation domain in the pGAD-C2 plasmid vector to generate the four recombinant pGAD-NifAN, pGAD-NifAM, pGAD-NifAC and pGAD-NifA plasmids. After introducing appropriate plasmid combinations in yeast cells, the existence of direct interaction between NifA and Pn was analyzed with the yeast two-hybrid system by testing for the expression of lacZ, trp, leu, his and ade genes. The experimental results showed that Pn directly interacts with the N-terminal domain of NifA and that Pn did not have any interactions with the central domain and the C-terminal domain of NifA. No interaction occurred if glnB was frame-shift mutated by cloning into pGBD-C3.
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