FUNCTIONAL-ORGANIZATION OF THE GLNB-GLNA CLUSTER OF AZOSPIRILLUM-BRASILENSE

The functional organization of the glnB-A cluster of Azospirillum brasilense, which codes for the P(II) protein and glutamine synthetase, respectively, was studied with the aid of lacZ fusions, deletion mapping, site-directed mutagenesis, and complementation. It was shown previously by mRNA mapping that the cluster contains two tandemly organized promoters, glnBp1 and glnBp2, of the sigma70 and sigma54 types, respectively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription of glnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, P(II) polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that P(II) plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, nifA, and point mutant strains in glnA were analyzed. NtrB and NtrC are not involved in the regulation of glnBA expression, in contrast to P(II) and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and P(II) acts by modifying the properties of an unidentified regulator which might be a functional homolog of NtrC. In addition, a Nif-null mutant strain of glnB was characterized further. A Nif+ phenotype was restored to the strain by nifA from Klebsiella pneumoniae but not by nifA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions in A. brasilense. It is therefore most likely that P(II) is required for NifA activation under conditions of nitrogen fixation. Deletion mapping and site-directed mutagenesis showed that glnAp was located within a 45-bp DNA fragment upstream of the mRNA start site, dissimilar to previously described consensus sites for sigma factors.