[Cloning and functional analysis of glnB from Azospirillum brasilense Yu62]

The glnB gene of A. brasilense Yu62 was determined in a 3.7 kb EcoRI + PstI fragment. The glnA is located downstream of glnB and an ORF for hypothetical protein is on upstream of glnB. The deduced amino acid sequence of PII encoded by glnB is 71%, 77%, 79% and 69% identical to that of K. pneumoniae, Bradyrhizobium japonicum, Rhizobium leguninosarum and E. coli, respectively. A Km-casette was inserted into BglII site of glnB coding region and GlnB- mutant was obtained by homologous recombination. The GlnB- mutant has lost the nitrogenase activity, i.e.: Nif-. For the functional confirmation of glnB gene, a complementary test was carried out and it was shown that C-glnB(glnB::Km/glnB) can restore the nitrogenase activity. When the recombinant plasmid pVK-II which containined the coding region of glnB was introduced into A. brasilense Yu62 and A. brasilense Yu62 DraT-, respectively, the Yu62-II (containing pVK-II) and draT-II(containing pVK-II) showed higher nitrogenase activity than wild type. These results confirmed that glnB plays an important role in the regulation of nitrogen in A. brasilense.