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Azorhizobium caulinodans reduces atmospheric nitrogen both in the free-living state and in symbiosis with its host plant, the tropical legume Sesbania rostrata (11). In pure culture, this bacterium grows using molecular nitrogen, whereas during symbiosis, fixed nitrogen is exported from the bacteroid to the plant cell and assimilated by the host. Thus, the coupling between nitrogen fixation and ammonia assimilation that exists in the free-living state must be abolished during symbiosis.

Ammonia assimilation proceeds through the glutamine synthetase (GS)-glutamine oxoglutarate aminotransferase pathway. A. caulinodans has a single GS (encoded by glnA), the activity of which is regulated by adenylylation (10). Two genes with products similar to P_II have been characterized in A. caulinodans: glnB, which is cotranscribed with glnA (17); and glnK, which is cotranscribed with amtB, a gene encoding a protein similar to a known ammonium transporter (18). As in Escherichia coli, glnB is constitutively transcribed whereas glnK expression is regulated by ammonia (17, 22). Neither P_II nor GlnK is required for nitrogen fixation in the free-living state. glnB mutants are impaired in symbiotic nitrogen fixation (Fix), whereas glnK mutants are not (Fix⁺). P_II and GlnK have a minor effect on GS adenylylation (17, 18).

Two proteins similar to P_II (P_II and GlnK) have been identified in several gram-negative bacteria, including Herbaspirillum seropedicae, Azospirillum brasilense, and E. coli (3, 8, 22). These two proteins are not equivalent in H. seropedicae and A. brasilense because glnB single mutants have impaired nitrogen fixation (3, 9). In contrast, E. coli P_II and GlnK seem to control GS deadenylylation in the absence of ammonia (2).

We report herein the properties of an A. caulinodans glnB glnK double mutant. In contrast to the glnB and glnK single mutants, GS deadenylylation was strongly impaired during nitrogenase derepression in the double mutant. We also found that the glnB glnK double mutant, but not the wild type, derepressed nitrogenase activity in the presence of ammonia, indicating that P_II and GlnK are also involved in the regulation of nitrogen fixation.

Characterization of the growth properties of the glnB glnK double mutant. A glnB glnK double mutant (strain 57625) was constructed by transferring the glnK interposon mutation (18) into the glnB mutant strain (57620) by conjugation, in order to study the effect of the absence of both proteins.

P_II or GlnK was required for GS deadenylylation. Unadenylylated and total GS activities were measured by the -glutamyltransferase assay in the presence and the absence, respectively, of 60 mM Mg²⁺ (Table 1), on whole cells cultured under nitrogenase-derepressing conditions (17) with and without shock by addition of 0.2% NH₄⁺. As reported for the glnB mutant (57620) (17), total GS activity, which depends on the total amount of enzyme, was higher in the glnB glnK mutant (57625) than in the wild type. This may be due to there being more glnA transcription under the control of the promoter of the aphII gene (which confers kanamycin resistance) inserted in the glnB coding sequence. For all strains tested, there was less or equal amount of unadenylylated (or active) GS after ammonia shock than under nitrogenase-derepressing conditions, suggesting that GS adenylylation does not require P_II or GlnK.

The glnB glnK double mutant excreted ammonia. A. caulinodans, unlike Bradyrhizobium species, can grow by consuming molecular nitrogen in pure culture. In the free-living state, only 10% of fixed nitrogen (NH₃) is exported from the cell, the remaining 90% being used for growth (13), whereas Bradyrhizobium cultures export all their fixed nitrogen to the medium (4). The absence or inhibition of GS activity blocks ammonium transport in Klebsiella pneumoniae (16). We tested whether the low level of unadenylylated GS in the glnB glnK double mutant (57625) or the absence of GS in the glnBA mutant (57619) affected ammonium excretion during nitrogen fixation in the free-living state by the indophenol method (6). No NH₄⁺ was detected in the supernatants of cultures of glnB or glnK single mutant strains (57620 and 57621), as was also previously reported for the wild-type strain (13). A large amount of NH₄⁺ was present in the supernatants of cultures of the glnBA mutant and glnB glnK double mutant (310 and 362 µM extracellular NH₄⁺/optical density unit, respectively). Excretion of NH₄⁺ was completely abolished in the glnB glnK mutant by expression from plasmids of either glnB (57625/pRS1045) or glnK (57625/pRS1046). Thus, the absence or inactivation of GS may lead to the accumulation of fixed nitrogen in A. caulinodans cells and ultimately to its excretion into the medium.

The glnB glnK mutant strain expressed nitrogen fixation genes in the presence of ammonia. The P_II and GlnK proteins control ammonium metabolism, in response to ammonia availability, by regulating GS activity. Ammonia negatively regulates nitrogen fixation genes in A. caulinodans. In particular it affects nifA transcription (15, 21). Thus, we investigated whether P_II and GlnK were also involved in the regulation of nifA expression. The A. caulinodans NifA protein has an estimated molecular mass of 66.8 kDa. It was detected by Western blot analysis in whole-cell extracts from wild-type and mutant strains, using Bradyrhizobium japonicum anti-NifA antibodies (19) (Fig. 1). NifA was detected in the wild-type strain and in glnB and glnK single mutants (57620 and 57621) cultivated under nitrogenase-derepressing conditions (Fig. 1, lanes 1, 4, and 6) but not in the presence of NH₄⁺ (lanes 2, 5, and 7) or in the nifA mutant (lane 3) (20). NifA was detected in the presence and absence of ammonia in the glnB glnK double mutant (lanes 8 and 9) and in the nifA mutant containing either the A. caulinodans nifA gene (lanes 10 and 11) or the B. japonicum nifA gene expressed constitutively in the presence of ammonia (lanes 12 and 13).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Immunodetection of NifA from A. caulinodans cells incubated under microaerobic conditions either in nitrogen-free medium (lanes 1, 3, 4, 6, 8, 10, and 12) or in the presence of 15 mM ammonia (lanes 2, 5, 7, 9, 11, and 13). Lanes 1 and 2, ORS571 (wild type); lane 3, ORS571A5 (nifA mutant); lanes 4 and 5, 57620 (glnB mutant); lanes 6 and 7, 57621 (glnK mutant); lanes 8 and 9, 57625 (glnB glnK mutant); lanes 10 and 11, ORS571A5/pRS1022 (containing A. caulinodans constitutive nifA [15]); and lanes 12 and 13, ORS571A5/pRJ7556 (containing B. japonicum constitutive nifA [12]).

View this table: [in this window] [in a new window]   TABLE 2.   -Galactosidase activities of the translational nifH-lacZY fusion recombined into the chromosomes of the wild-type and mutant strains of A. caulinodans carrying or not carrying the constitutively expressed nifA from A. caulinodans or B. japonicum

Nitrogenase was active in the presence of ammonia in the glnB glnK mutant strain. As nifH was expressed in the presence of ammonia in the glnB glnK mutant strain, we investigated whether the nitrogenase was active (17). Nitrogenase activities were similar in the wild-type strain and the glnB glnK mutant strain under nitrogenase-derepressing conditions (Fig. 2). No nitrogenase activity was detected in the wild-type strain in the presence of 10 mM NH₄⁺, whereas nitrogenase activity was detected in the glnB glnK mutant strain (Fig. 2), suggesting that P_II and GlnK may also be required for the regulation of nitrogenase activity.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Kinetics of nitrogenase activities in ORS571 (wild type) (open squares) or the 57625 strain (glnB glnK mutant) (open circles) under microaerobic conditions in nitrogen-free medium or of the same strains in medium with 10 mM ammonia added to the medium at time 0 (closed squares and closed circles, respectively).