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

The functional organization of the glnB-A cluster of Azospirillum bras ilense, which codes for the P(II) protein and glutamine synthetase, re spectively, was studied with the aid of lacZ fusions, deletion mapping , site-directed mutagenesis, and complementation. It was shown previou sly by mRNA mapping that the cluster contains two tandemly organized p romoters, glnBp1 and glnBp2, of the sigma70 and sigma54 types, respect ively, upstream of glnB and a third unidentified promoter upstream of glnA. Data obtained with lacZ fusions in the wild-type strain confirme d that cotranscription of glnBA and transcription of glnA alone were o ppositely regulated by the cell N status. Quantification of promoter a ctivities showed a high level of transcription from glnBp1p2 and a low level from glnAp under conditions of nitrogen limitation. The opposit e situation prevails under conditions of nitrogen excess. As a consequ ence, P(II) polypeptide synthesis is increased under conditions of nit rogen fixation, which strongly suggests that P(II) plays an important role under these conditions. Null mutant strains of glnB, ntrB-ntrC, n ifA, 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 mo dulating the intracellular N status, and P(II) acts by modifying the p roperties of an unidentified regulator which might be a functional hom olog of NtrC. In addition, a Nif-null mutant strain of glnB was charac terized further. A Nif+ phenotype was restored to the strain by nifA f rom Klebsiella pneumoniae but not by nifA from A. brasilense. This mut ant strain is not impaired in NifA synthesis, which is relatively inde pendent of the growth conditions in A. brasilense. It is therefore mos t likely that P(II) is required for NifA activation under conditions o f nitrogen fixation. Deletion mapping and site-directed mutagenesis sh owed that glnAp was located within a 45-bp DNA fragment upstream of th e mRNA start site, dissimilar to previously described consensus sites for sigma factors.