Functional organization of the glnB-glnA cluster of Azospirillum brasilense

Abstract

The functional organization of the glnB-A cluster ofAzospiriUum brasilense, which codes for the PI, 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 a7o and cr-4 types, respectively, upstream of glnB and a third unidentified promoter upstream of ginA. Data obtained with lacZ fusions in the wild-type strain confirmed that cotranscription of glnBA and transcription ofglnA alone were oppositely regulated by the cell N status. Quantification of promoter activities showed a high level of transcription from ginBpp2 and a low level from ginAp under conditions of nitrogen limitation. The opposite situation prevails under conditions of nitrogen excess. As a consequence, PI, polypeptide synthesis is increased under conditions of nitrogen fixation, which strongly suggests that PI, plays an important role under these conditions. Null mutant strains of ginB, ntrB-ntrC, nifA, and point mutant strains in ginA were analyzed. NtrB and NtrC are not involved in the regulation of ginBA expression, in contrast to PII and glutamine synthetase. Glutamine synthetase probably acts by modulating the intracellular N status, and PI, 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 nWfA from A. brasilense. This mutant strain is not impaired in NifA synthesis, which is relatively independent of the growth conditions inA. brasilense. It is therefore most likely that PI, is required for NiWA 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 cr factors.The nitrogen fixation (nif) genes are largely conserved among diazotrophs, and the requirement of a nifA product for their activation seems to be a general rule in proteobacteria (for recent reviews, see references 10 and 25). However, different regulatory pathways for the overall regulation of nitrogen metabolism have evolved in different groups of nitrogen-fixing bacteria.In the model system of Kiebsiella pneumoniae, ntrBC and ginA, which code for the two-component regulatory proteins NtrB and NtrC and glutamine synthetase (GS), respectively, are clustered. The PI1 protein, encoded by glnB, that maps elsewhere on the chromosome plays a central role by coordinating the response of the Ntr system to the intracellular nitrogen status (3,14,28,33

Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum

“…A similar regulation of NifA activity by GlnB has been seen in Azospirillum brasilense, Rhodobacter capsulatus, and Herbaspirillum seropedicae (15)(16)(17). However, the mechanism for the activation of NifA activity (whether it is direct or indirect) is unknown in all cases.…”

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

“…In A. brasilense, transcription of glnB is elevated five-to sixfold by nitrogen limitation, although this expression is not NRI dependent (de Zamaroczy et al, 1993). As in A. brasilense, the glnB gene in both Bradyrhizobium japonicum (Martin et al, 1989) and in Rhodobacter capsulatus (Foster-Hartnett and Kranz, 1994) is transcribed from tandem promoters.…”

Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

“…(i) In Azospirillum brasilense, H. seropedicae and Rs. rubrum, GlnB is essential for NifA activity (Benelli et al, 1997;de Zamaroczy et al, 1993;Liang et al, 1992;Zhang et al, 2000), most likely involving a GlnB-mediated activation of preformed NifA under N-limiting conditions. (ii) In K. pneumoniae and Azotobacter vinelandii, GlnK functions as a signal transduction protein modulating the activity of NifL, which inactivates NifA under nitrogen-sufficient conditions (Arcondéguy et al, 1999;He et al, 1998;Holtel & Merrick, 1989;Jack et al, 1999;Little et al, 2000Little et al, , 2002Reyes-Ramirez et al, 2001;Rudnick et al, 2002).…”

“…Both GlnD and GlnB are involved in sensing the nitrogen status of the cells by direct interaction with glutamine and 2-oxoglutarate, respectively. In E. coli, GlnB controls the activity of glutamine synthetase and the sensor kinase NtrB (Ninfa & Atkinson, 2000), whereas the GlnB proteins of Azospirillum brasilense, Herbaspirillum seropedicae and Rhodospirillum rubrum also play essential roles in nitrogen fixation, since glnB mutations lead to synthesis of inactive NifA in the latter three organisms (Benelli et al, 1997;de Zamaroczy et al, 1993;Liang et al, 1992;Souza et al, 1999;Zhang et al, 2000). In addition, many bacteria contain a second glnB-like gene designated glnK (for a review, see Thomas et al, 2000;Arcondéguy et al, 2001).…”