Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense

Abstract

The coexistence of two different P II proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding P II ) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [ 32 P]phosphate or [ 3 H]uracil labeling or by cross-reaction with an anti-A. brasilense P II -antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second P II protein, named P Z , was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of P II . Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that P II and P Z are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.Azospirillum brasilense is a plant growth-promoting rhizobacterium that associates with the roots of grasses and fixes nitrogen under microaerobic conditions in the free-living state (33). As in all proteobacteria, the transcription of nif (nitrogen fixation) genes proceeds from 54 (NtrA or RpoN)-dependent promoters with upstream activator sequences that are the binding target of the transcriptional activator NifA (reviewed in reference 10). In contrast, the regulation by nitrogen of both the expression and activity of NifA differs considerably from that described for other free-living diazotrophs. In A. brasilense, NifA is synthesized in an inactive form under conditions incompatible with nitrogen fixation (24), and NtrC is not essential for the regulation of nifA expression (23).A. brasilense possesses a glutamine synthetase (GS) encoded by glnA (5). GS activity is modulated by reversible adenylylation as in enteric bacteria (8,19). However, the regulation of glnA expression has some unusual features. The glnA gene is adjacent to glnB, the structural gene for the P II protein. P II plays an essential role in regulation of nitrogen metabolism in Escherichia coli by controlling the level and activity of GS (reviewed in references 27 and 32). The transcription of the glnB-glnA cluster in A. brasilense depends on three different, selectively used and nitrogen-regulated promoters (11) and does not involve the two-component regulatory system NtrBNtrC (12). A glnB null mutant strain does not have impaired ammonia assimilation but is strictly Nif Ϫ (1...

Partial characterization of nif genes from the bacterium Azospirillum amazonense

“…Faint hybridization signals were detected in A. amazonense total DNA and also with PstI-digested A. brasilense total DNA (Figure 1E, lane 2). Genes homologous to glnB have been found in A. brasilense, Herbaspirillum seropedicae and other bacteria, suggesting the presence of two copies of glnBlike genes in these organisms (17,18). Since no PstI restriction site was found within glnB or glnZ genes from A. brasilense or in the glnB gene from A. amazonense, the presence of two hybridization fragments in both strains may represent similarity between the probe and the two glnB-like genes.…”

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

“…This was explained by the presence of two closely related P II proteins found in A. brasilense (18) that are involved in different regulatory steps of nitrogen metabolism. The P II protein, a product of the glnB gene, is required for modulating NifA activity in response to changes in cellular nitrogen levels (18,33).…”

“…To further analyze the binding of NifA protein, mobility gel shift assays were carried out with NifA preparations (total extracts from MC1061/pAAbA and MC1061/ pGEXAbA), wild-type UAS-carrying frag- (18). The modular structure of transcriptional activators suggests that inactive NifA would be able to bind to its target sequences (35).…”

“…Moreover, its regulation differs considerably from that found in other free living diazotrophs. It has been proposed that, in A. brasilense, the Nterminal domain plays an inhibitory role which leads to the inactivation of NifA in the absence of P II protein (17,18). P II protein plays an essential role in the regulation of nitrogen metabolism in E. coli by controlling the activity of GS (19,20) and it appears that glnB is essential for nitrogen fixation in A. brasilense (18).…”

“…It has been proposed that, in A. brasilense, the Nterminal domain plays an inhibitory role which leads to the inactivation of NifA in the absence of P II protein (17,18). P II protein plays an essential role in the regulation of nitrogen metabolism in E. coli by controlling the activity of GS (19,20) and it appears that glnB is essential for nitrogen fixation in A. brasilense (18). Another important feature of A. brasilense is the regulation of nitrogenase activity at the post-translational level by a switch-off mechanism in response to environmental changes in ammonia concentration (21).…”