Characterization of Azorhizobium caulinodans glnB and glnA genes: involvement of the P(II) protein in symbiotic nitrogen fixation

Abstract

The nucleotide sequence and transcriptional organization of Azorhizobium caulinodans ORS571 glnA, the structural gene for glutamine synthetase (GS), and glnB, the structural gene for the P II protein, have been determined. glnB and glnA are organized as a single operon transcribed from the same start site, under conditions of both nitrogen limitation and nitrogen excess. This start site may be used by two different promoters since the expression of a glnB-lacZ fusion was high in the presence of ammonia and enhanced under conditions of nitrogen limitation in the wild-type strain. The increase was not observed in rpoN or ntrC mutants. In addition, this fusion was overexpressed under both growth conditions, in the glnB mutant strain, suggesting that P II negatively regulates its own expression. A DNA motif, similar to a 54 -dependent promoter consensus, was found in the 5 nontranscribed region. Thus, the glnBA operon seems to be transcribed from a 54 -dependent promoter that operates under conditions of nitrogen limitation and from another uncharacterized promoter in the presence of ammonia. Both glnB and glnBA mutant strains derepress their nitrogenase in the free-living state, but only the glnBA mutant, auxotrophic for glutamine, does not utilize molecular nitrogen for growth. The level of GS adenylylation is not affected in the glnB mutant as compared to that in the wild type. Under symbiotic conditions, the glnB and glnBA mutant strains induced Fix ؊ nodules on Sesbania rostrata roots. P II is the first example in A. caulinodans of a protein required for symbiotic nitrogen fixation but dispensable in bacteria growing in the free-living state.Azorhizobium caulinodans ORS571, isolated from stem nodules of its host plant, the tropical legume Sesbania rostrata, fixes nitrogen both during symbiosis and in the free-living state (15). In the free-living state, this strain assimilates fixed ammonium for growth, via the glutamine synthetase (GS)/glutamate synthetase pathway (14). During symbiosis, ammonium produced by nitrogen fixation is exported from the bacteroid to the vegetal cell, where it is assimilated by the plant GS. As in enteric bacteria, only one GS (GSI) has been characterized in A. caulinodans (14), whereas two forms (GSI and GSII) have been identified in most rhizobia (9). In enteric bacteria and rhizobia, GS activity is modulated by reversible adenylylation in response to changes in the intracellular glutamine/2-ketoglutarate ratio, reflecting the level of cellular nitrogen (20). In Escherichia coli, under conditions of nitrogen limitation, the P II protein, encoded by glnB, is uridylylated by the glnD gene product. Under conditions of nitrogen excess, P II is deuridylylated and activates an adenylyltransferase (28). This enzyme transfers an AMP group to a tyrosine residue in each of the 12 subunits of the GS; the fully adenylylated form of the enzyme is less active (33). The structural gene for GS (glnA) is part of the glnALG operon, where glnL and glnG (also designated ntrB and ntrC) are the structura...

PIISignal Transduction Proteins, Pivotal Players in Microbial Nitrogen Control

“…Hence, both GlnB and GlnK may be involved in the regulation of A. caulinaudans nitrogenase activity (159). As discussed earlier, A. caulinodans glnB and glnBA mutants induce Fix Ϫ nodules on Sesbania rostrata roots despite expressing a pnifHlacZ fusion at a similar level to the wild type, a situation which again suggests control of nitrogenase activity by GlnB (158).…”

“…Although Northern blotting analysis revealed the presence of two transcripts, no glnA promoter was detected, suggesting that the glnA mRNA is produced by processing (34,113). As in Rhodospirillum rubrum and Rhodobacter capsulatus, Azorhizobium caulinaudans glnB and glnA appear to be cotranscribed (158). In both nitrogen excess and nitrogen limitation, the glnBA operon is transcribed from two overlapping promoters having the same start site, one of them 54 and NtrC dependent and the other uncharacterized.…”

“…In both nitrogen excess and nitrogen limitation, the glnBA operon is transcribed from two overlapping promoters having the same start site, one of them 54 and NtrC dependent and the other uncharacterized. Again, no promoter was detected upstream of glnA, and an mRNA processing event was proposed (158).…”

“…One gene encodes a P II protein that is very similar to E. coli GlnB and is located upstream of and cotranscribed with glnA (the structural gene for glutamine synthetase I) (7,27,41,94,113,127,151,158,262). The second gene encodes a protein similar to GlnK and is linked to an amtB homologue (125,157,182,185,222,254) (Y. Zhang and G. Roberts, personal communication).…”

Distinct Roles of P II -Like Signal Transmitter Proteins and amtB in Regulation of nif Gene Expression, Nitrogenase Activity, and Posttranslational Modification of NifH in Azoarcus sp. Strain BH72

“…amtB genes encode integral membrane proteins which were identified as high-affinity ammonium transporters in Saccharamyces cerevisiae (38) and Arabidopsis thaliana (45). An involvement in ammonium transport is also assumed for bacteria (9,43,54). It has been proposed that the conservation of physical linkage of glnK and amtB reflects a functional relationship and a physical interaction of these proteins (57).…”