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. 1990 May;172(5):2413–2420. doi: 10.1128/jb.172.5.2413-2420.1990

Rhizobium meliloti glutamate synthase: cloning and initial characterization of the glt locus.

T A Lewis 1, R Gonzalez 1, J L Botsford 1
PMCID: PMC208877  PMID: 2185218

Abstract

The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berberich M. A. A glutamate-dependent phenotype in E. coli K12: the result of two mutations. Biochem Biophys Res Commun. 1972 Jun 28;47(6):1498–1503. doi: 10.1016/0006-291x(72)90242-2. [DOI] [PubMed] [Google Scholar]
  3. Botsford J. L., Lewis T. A. Osmoregulation in Rhizobium meliloti: Production of Glutamic Acid in Response to Osmotic Stress. Appl Environ Microbiol. 1990 Feb;56(2):488–494. doi: 10.1128/aem.56.2.488-494.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  6. Castaño I., Bastarrachea F., Covarrubias A. A. gltBDF operon of Escherichia coli. J Bacteriol. 1988 Feb;170(2):821–827. doi: 10.1128/jb.170.2.821-827.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  10. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hirsch P. R., Beringer J. E. A physical map of pPH1JI and pJB4JI. Plasmid. 1984 Sep;12(2):139–141. doi: 10.1016/0147-619x(84)90059-3. [DOI] [PubMed] [Google Scholar]
  13. Hua S. S., Tsai V. Y., Lichens G. M., Noma A. T. Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity. Appl Environ Microbiol. 1982 Jul;44(1):135–140. doi: 10.1128/aem.44.1.135-140.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lozoya E., Sanchez-Pescador R., Covarrubias A., Vichido I., Bolivar F. Tight linkage of genes that encode the two glutamate synthase subunits of Escherichia coli K-12. J Bacteriol. 1980 Nov;144(2):616–621. doi: 10.1128/jb.144.2.616-621.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin G. B., Chapman K. A., Chelm B. K. Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII). J Bacteriol. 1988 Dec;170(12):5452–5459. doi: 10.1128/jb.170.12.5452-5459.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Martin G. B., Thomashow M. F., Chelm B. K. Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters. J Bacteriol. 1989 Oct;171(10):5638–5645. doi: 10.1128/jb.171.10.5638-5645.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Measures J. C. Role of amino acids in osmoregulation of non-halophilic bacteria. Nature. 1975 Oct 2;257(5525):398–400. doi: 10.1038/257398a0. [DOI] [PubMed] [Google Scholar]
  18. Miller R. E., Stadtman E. R. Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein. J Biol Chem. 1972 Nov 25;247(22):7407–7419. [PubMed] [Google Scholar]
  19. Oliver G., Gosset G., Sanchez-Pescador R., Lozoya E., Ku L. M., Flores N., Becerril B., Valle F., Bolivar F. Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12. Gene. 1987;60(1):1–11. doi: 10.1016/0378-1119(87)90207-1. [DOI] [PubMed] [Google Scholar]
  20. Osburne M. S., Signer E. R. Ammonium assimilation in Rhizobium meliloti. J Bacteriol. 1980 Sep;143(3):1234–1240. doi: 10.1128/jb.143.3.1234-1240.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ronson C. W., Nixon B. T., Albright L. M., Ausubel F. M. Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol. 1987 Jun;169(6):2424–2431. doi: 10.1128/jb.169.6.2424-2431.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  23. Somerville J. E., Kahn M. L. Cloning of the glutamine synthetase I gene from Rhizobium meliloti. J Bacteriol. 1983 Oct;156(1):168–176. doi: 10.1128/jb.156.1.168-176.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Szeto W. W., Nixon B. T., Ronson C. W., Ausubel F. M. Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol. 1987 Apr;169(4):1423–1432. doi: 10.1128/jb.169.4.1423-1432.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tempest D. W., Meers J. L., Brown C. M. Influence of environment on the content and composition of microbial free amino acid pools. J Gen Microbiol. 1970 Dec;64(2):171–185. doi: 10.1099/00221287-64-2-171. [DOI] [PubMed] [Google Scholar]
  26. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  27. de Bruijn F. J., Rossbach S., Schneider M., Ratet P., Messmer S., Szeto W. W., Ausubel F. M., Schell J. Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation. J Bacteriol. 1989 Mar;171(3):1673–1682. doi: 10.1128/jb.171.3.1673-1682.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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