Cloning of the glutamine synthetase I gene from Rhizobium meliloti

Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium. Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII. As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14. A gene bank of R. meliloti was constructed by using the bacteriophage P4 cosmid pMK318. Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source. One of the cosmids, pJS36, contains an insert of 11.9 kilobases. ET8051(pJS36) grows slowly on minimal media. When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45). Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable. pJS36 hybridizes with the glnA structural gene from Escherichia coli. Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity. This interrupted version of a glutamine synthetase gene was substituted for the normal R. meliloti sequence by homologous recombination in R. meliloti. Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source. These mutants are unaffected in nodulation and nitrogen fixation.

[1]  F. Ausubel,et al.  Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae , 1983, Nature.

[2]  M. Merrick,et al.  Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae , 1983, Nature.

[3]  D. Macneil,et al.  Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli , 1982, Journal of bacteriology.

[4]  B. Magasanik.  Genetic control of nitrogen assimilation in bacteria. , 1982, Annual review of genetics.

[5]  R. Haselkorn,et al.  A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[6]  C. M. Joyce,et al.  Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. A. Ludwig.  Regulation of Rhizobium nitrogen fixation by the unadenylylated glutamine synthetase I system. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Thomashow,et al.  Integration and organization of Ti plasmid sequences in crown gall tumors , 1980, Cell.

[9]  Christopher M Thomas,et al.  Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance , 1980, Journal of bacteriology.

[10]  A. H. Gibson,et al.  Methods for legumes in glasshouses and controlled environment cabinets. , 1980 .

[11]  R. Darrow.  Role of Glutamine Synthetase in Nitrogen Fixation , 1980 .

[12]  A. Pain.  Symbiotic Properties of Antibiotic-Resistant and Auxotrophic Mutants of Rhizobium leguminosarum , 1979 .

[13]  M. Israel,et al.  A rapid method for detecting and mapping homology between heterologous DNAs. Evaluation of polyomavirus genomes. , 1979, The Journal of biological chemistry.

[14]  R. Calendar,et al.  Maturation of bacteriophage P2 DNA in vitro: A complex, site-specific system for DNA cleavage. , 1979, Journal of molecular biology.

[15]  W. Reznikoff,et al.  Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance , 1979, Journal of bacteriology.

[16]  R. Kolter,et al.  Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. , 1979, Methods in enzymology.

[17]  J. Beynon,et al.  Transfer of the drug-resistance transposon Tn5 to Rhizobium , 1978, Nature.

[18]  R. A. Ludwig.  Control of ammonium assimilation in Rhizobium 32H1 , 1978, Journal of bacteriology.

[19]  R. Darrow,et al.  Two forms of glutamine synthetase in free-living root-nodule bacteria. , 1977, Biochemical and biophysical research communications.

[20]  R. A. Ludwig,et al.  Glutamine synthetase and control of nitrogen fixation in Rhizobium , 1977, Nature.

[21]  B. Magasanik,et al.  Biochemical parameters of glutamine synthetase from Klebsiella aerogenes , 1977, Journal of bacteriology.

[22]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[23]  B. Sauer,et al.  A bacterial mutation blocking P2 phage late gene expression. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[24]  C. Yanofsky,et al.  Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[26]  R. Calendar,et al.  P2 phage amber mutants: characterization by use of a polarity suppressor. , 1971, Virology.

[27]  J. Vincent.  A manual for the practical study of root-nodule bacteria , 1971 .