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Mutations at two sites, glnA and glnB, of the Klebsiella aerogenes chromosome result in the loss of glutamine synthetase. The locations of these sites on the chromosome were established by complementation by episomes of Escherichia coli and by determination of their linkage to other genetic sites by transduction with phage P1. The glnB gene is located at a position corresponding to 48 min on the Taylor map of the E. coli chromosome; it is linked to tryA, nadB, and GUA. The glnA gene is at a position corresponding to 77 min on the Taylor map and is linked to rha and metB; it is also closely linked to rbs, located in E. coli at 74 min, indicating a difference in this chromosomal region between E. coli and K. aerogenes. Mutations in the glnA site can also lead to nonrepressible synthesis of active glutamine synthetase. The examination of the fine genetic structure of glnA revealed that one such mutation is located between two mutations leading to the loss of enzymatic activity. This result, together with evidence that the structural gene for glutamine synthetase is at glnA, suggests that glutamine synthetase controls expression of its own structural gene by repression.

The enzymatic activity of glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from the Gram-positive bacterium Streptomyces cattleya is regulated by covalent modification. In whole cells containing high levels of GS the addition of ammonium chloride leads to a rapid decline in GS activity. Crude extracts prepared from such ammonia-shocked cells had very low levels of GS activity as measured by biosynthetic and gamma-glutamyltransferase assays. Incubation of the crude extracts with snake venom phosphodiesterase restored GS activity. In cell extracts, GS was also inactivated by an ATP- and glutamine-dependent reaction. Radioactive labeling studies demonstrated the incorporation of an AmP moiety into GS protein upon modification. Our results suggest a covalent modification of GS in a Gram-positive bacterium. This modification appears to be adenylylation of the GS subunit similar to that found in the Gram-negative bacteria.

We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.

We have developed two procedures which allow the very rapid purification of glutamine synthetase (GS) from a diverse variety of bacteria. The first procedure, based upon differential sedimentation, depends upon the association of GS with deoxyribonucleic acid in cell extracts. The second procedure, derived from the method of C. Gross et al (J. Bacteriol. 128:382-389, 1976) for purifying ribonucleic acid polymerase by polyethylene glycol (PEG) precipitation, enabled us to obtain high yields of GS from either small or large quantities of cells. We used the PEG procedure to purify GS from Klebsiella aerogenes, K. pneumoniae, Escherichia coli, Salmonella typhimurium, Rhizobium sp. strain 32H1, R. meliloti, Azotobacter vinelandii, Pseudomonas putida, Caulobacter crescentus, and Rhodopseudomonas capsulata. The purity of the GS obtained, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was high, and in many instances only a single protein band was detected.

The glnD mutation of Klebsiella aerogenes is cotransducible by phage P1 with pan (requirement for pantothenate) and leads to a loss of uridylytransferase and uridylyl-removing enzyme, components of the glutamine synthetase adenylylation system. This defect results in an inability to deadenylylate glutamine synthetase rapidly and in a requirement for glutamine for normal growth. Suppression of the glnD mutation are located at the glutamine synthetase structural gene glnA.

In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.