Bradyrhizobium japonicum induces the formation of nitrogen-fixing symbiotic root nodules on soybean plants. The B. japonicum genome encodes two isoforms of glutamine synthetase (GS). One form, GSI, encoded by the gene glnA, is similar in structure and activity to the enzyme found in all other bacteria. The second form, GSII, encoded by glnII, is structurally related to the eucaryotic enzyme. Genetic analyses indicate that glnA or glnII alone is sufficient to provide glutamine prototrophy, whereas the double mutation glnA glnII produces glutamine auxotrophy. The glnA gene is transcribed from a single promoter that has a structure most similar to that of the bacterial consensus promoter. The level of transcription of glnA is not specifically affected by nitrogen limitation of growth. The glnII gene is also transcribed from a single promoter; however, this promoter has structural features characteristic of promoters controlled by the nitrogen regulation system. In contrast to glnA, physiological studies indicate that glnII transcription is regulated in response to nitrogen source availability. Under aerobic growth conditions, expression of glnII is induced when growth is limited by nitrogen source depletion as expected for regulation by the nitrogen regulation system.

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We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial ginA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 2 base pairs upstream from the initiation codon. This ginA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nf promoters of the same B. japonwum strain.