1887

Abstract

Summary: Mutants of were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to and which are part of a gene cluster which codes for malate dehydrogenase (), succinyl-CoA synthetase () and components of the 2-oxoglutarate dehydrogenase complex (). Mutation of either or prevents expression of 2-oxoglutarate dehydrogenase (). Conversely, mutation of or results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes may constitute an operon. mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of or the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of was strongly inhibited in the mutants, even though the transcription level of the operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (). Mutants of succinyl-CoA synthetase () or 2-oxoglutarate dehydrogenase () form ineffective nodules. However, mutants of which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and recluctant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-143-7-2209
1997-07-01
2021-08-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/143/7/mic-143-7-2209.html?itemId=/content/journal/micro/10.1099/00221287-143-7-2209&mimeType=html&fmt=ahah

References

  1. Appels M. A., Haaker H. 1991; Glutamate oxaloacetate transaminase in pea root nodules – participation in a malate/aspartate shuttle between plant and bacteroid. Plant Physiol 95:740–747
    [Google Scholar]
  2. Arwas R., McKay I. A., Rowney F. R. P., Dilworth M. J., Glenn A. R. 1985; Properties of organic acid utilization mutants of Rhizobium leguminosarum strain 300. J Gen Microbiol 131:2059–2066
    [Google Scholar]
  3. Bergersen F. J., Turner G. L. 1990; Bacteroids from soybean root nodules: accumulation of poly-β-hydroxybutyrate during supply of malate and succinate in relation to N2 fixation in flow-chamber reactions. Proc R Soc Lond Ser B Biol Sci 240:39–59
    [Google Scholar]
  4. Bergmeyer H. U., Bernt E., Mollering H., Pfleiderer G. 1974 l-Aspartate and l-asparagine. . In Methods of Enzymatic Analysis , 2nd edn, pp. 1696–1700 . Edited by Bergmeyer H. U. New York: Academic Press;
    [Google Scholar]
  5. Beringer J. E. 1974; R factor transfer in Rhizobium leguminosarum . J Gen Microbiol 84:188–198
    [Google Scholar]
  6. Bernt E., Bergmeyer H. U. 1974 l-Glutamate UV-assay with glutamate dehydrogenase and NAD. . In Methods of Enzymatic Analysis , 2nd edn, pp 1704–1708 . Edited by Bergmeyer H. U. New York: Academic Press;
    [Google Scholar]
  7. Bolton E., Higgisson B., Harrington A., O’Gara F. 1986; Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes. Arch Microbiol 144:142–146
    [Google Scholar]
  8. Buchanan-Wollaston V. 1979; Generalized transduction in Rhizobium leguminosarum . J Gen Microbiol 112:135–142
    [Google Scholar]
  9. Buck D., Spencer M. E., Guest J. R. 1985; Primary structure of the succinyl-CoA synthetase of Escherichia coli . Biochemistry 24:6245–6252
    [Google Scholar]
  10. Cevallos M. A., Encarnacion S., Leija A., Mora Y., Mora J. 1996; Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. J Bacteriol 178:1646–1654
    [Google Scholar]
  11. Darlison M. G., Spencer M. E., Guest J. R. 1984; Nucleotide-sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12. Eur J Biochem 141:351–359
    [Google Scholar]
  12. Day D. A., Udvardi M. K. 1993; Metabolite exchange across symbiosome membranes. Symbiosis 14:175–189
    [Google Scholar]
  13. Dilworth M. J., Arwas R., McKay I. A., Saroso S., Glenn A. R. 1986; Pentose metabolism in Rhizobium leguminosarum MNF300 and in cowpea Rhizobium NGR234. J Gen Microbiol 132:2733–2742
    [Google Scholar]
  14. Duncan M. J., Fraenkel D. G. 1979; α-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti . J Bacteriol 37:415–419
    [Google Scholar]
  15. Encarnacion S., Dunn M., Willms K., Mora J. 1995; Fermentative and aerobic metabolism in Rhizobium etli . J Bacteriol 177:3058–3066
    [Google Scholar]
  16. Engelke T., Jagadish M. N., Pühler A. 1987; Biochemical and genetical analysis of Rhizobium meliloti mutants defective in C4-dicarboxylate transport. J Gen Microbiol 133:3019–3029
    [Google Scholar]
  17. Finan T. M., Wood J. M., Jordan D. C. 1983; Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum . J Bacteriol 154:1403–1413
    [Google Scholar]
  18. Finan T. M., McWhinnie E., Driscoll B., Watson R. J. 1991; Complex symbiotic phenotypes result from gluconeogenic mutations in Rhizobium meliloti . Mol Plant–Microbe Interact 4:386–392
    [Google Scholar]
  19. Fitzmaurice A. M., O’Gara F. 1993; A Rhizobium meliloti mutant, lacking a functional gamma-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation. FEMS Microbiol Lett 109:195–202
    [Google Scholar]
  20. Glenn A. R., Dilworth M. J. 1981; Oxidation of substrates by isolated bacteroids and free-living cells of Rhizobium leguminosarum 3841. J Gen Microbiol 126:243–247
    [Google Scholar]
  21. Glenn A. R., Poole P. S., Hudman J. F. 1980; Succinate uptake by free-living and bacteroid forms of Rhizobium leguminosarum . J Gen Microbiol 119:267–271
    [Google Scholar]
  22. Glenn A. R., McKay I. A., Arwas R., Dilworth M. J. 1984; Sugar metabolism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum . J Gen Microbiol 130:239–245
    [Google Scholar]
  23. Glenn A. R., McKay I. A., Arwas R., Dilworth M. J. 1984; Sugar metabolism and the symbiotic properties of carbohydrate mutants of Rhizobium leguminosarum . J Gen Microbiol 130:239–245
    [Google Scholar]
  24. Guest J. R. 1992; Oxygen-regulated gene expression in Escherichia coli . J Gen Microbiol 138:2253–2263
    [Google Scholar]
  25. Guest J. R., Russell G. C. 1992; Complexes and complexities of the citric acid cycle in Escherichia coli . Curr Top Cell Regul 33:231–247
    [Google Scholar]
  26. Hirsch P. R., Beringer J. E. 1984; A physical map of pPH1JI and pJB4JI. Plasmid 12:139–141
    [Google Scholar]
  27. Jin H. N., Dilworth M. J., Glenn A. R. 1990; 4-Aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules. Arch Microbiol 153:455–462
    [Google Scholar]
  28. Johnston A. W. B., Beringer J. E. 1975; Identification of the Rhizobium strains in pea root nodules using genetic markers. J Gen Microbiol 87:343–350
    [Google Scholar]
  29. Kahn M. L., Kraus J., Sommerville J. E. 1985 A model of nutrient exchange in the Rhizobium–legume symbiosis. . In Nitrogen Fixation Research Progress , pp. 193–199 . Edited by Evans H. J., Bottomley P. J., Newton W. E. Dordrecht: Martinus Nijhoff;
    [Google Scholar]
  30. Kouchi H., Fukai K., Kihara A. 1991; Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules. J Gen Microbiol 137:2901–2910
    [Google Scholar]
  31. Kramer R. 1994; Secretion of amino acids by bacteria–physiology and mechanism. FEMS Microbiol Rev 13:75–93
    [Google Scholar]
  32. Law J. H., Slepecky R. A. 1961; Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–42
    [Google Scholar]
  33. McDermott T. R., Griffith S. M., Vance C. P., Graham P. H. 1989; Carbon metabolism in Bradyrhizobium japonicum bacteroids. FEMS Microbiol Lett 63:327–340
    [Google Scholar]
  34. McKay I. A., Glenn A. R., Dilworth M. J. 1985; Gluconeogenesis in Rhizobium leguminosarum MNF3841. J Gen Microbiol 131:2067–2073
    [Google Scholar]
  35. Meissner P. S., Sisk W. P., Berman M. L. 1987; Bacteriophage cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci USA 84:4171–4175
    [Google Scholar]
  36. Miles J. S., Guest J. R. 1987; Molecular genetic aspects of the citric acid cycle of Escherichia coli . Biochem Soc Symp 54:45–65
    [Google Scholar]
  37. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  38. Miller R. W., McRae D. G., Jot K. 1991; Glutamate and gamma-aminobutyrate metabolism in isolated Rhizobium meliloti bacteroids. Mol Plant–Microbe Interact 4:37–45
    [Google Scholar]
  39. Nicholls D. J., Sundaram T., ढ., Atkinson T., Minton N. P. 1990; Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B. FEMS Microbiol Lett 70:7–14
    [Google Scholar]
  40. Nishiyama M., Horinouchi S., Beppu T. 1991; Characterization of an operon encoding succinyl-CoA synthetase and malate dehydrogenase from Thermus flavus AT-62 and its expression in Escherichia coli . Mol Gen Genet 226:1–9
    [Google Scholar]
  41. Osteras M., Finan T. M., Stanley J. 1991; Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase-gluconeogenesis and host-dependent symbiotic phenotype. Mol Gen Genet 230:257–269
    [Google Scholar]
  42. Patriarca E. J., Chiurazzi M., Manco G., Riccio A., Lamberti A., Depaolis A., Rossi M., Defez R., Iaccarino M. 1992; Activation of the Rhizobium leguminosarum glnII gene by NtrC is dependent on upstream DNA sequences. Mol Gen Genet 234:337–345
    [Google Scholar]
  43. Poole P. S., Dilworth M. J., Glenn A. R. 1984; Acquisition of aspartase activity in Rhizobium leguminosarum WU235. J Gen Microbiol 130:881–886
    [Google Scholar]
  44. Poole P. S., Franklin M., Glenn A. R., Dilworth M. J. 1985; The transport of l-glutamate by Rhizobium leguminosarum involves a common amino acid carrier. J Gen Microbiol 131:1441–1448
    [Google Scholar]
  45. Poole P. S., Blyth A., Reid C. J., Walters K. 1994a; myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae. Microbiology 140:2787–2795
    [Google Scholar]
  46. Poole P. S., Schofield N. A., Reid C. J., Drew E. M., Walshaw D. L. 1994b; Identification of chromosomal genes located downstream of dctD that affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum . Microbiology 140:2797–2809
    [Google Scholar]
  47. Povolo S., Tombolini R., Morea A., Anderson A. J., Casella S., Nuti M. P. 1994; Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-beta-hydroxybutyrate. Can J Microbiol 40:823–829
    [Google Scholar]
  48. Quandt J., Hynes M. F. 1993; Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127:15–21
    [Google Scholar]
  49. Reeves H. C., Rabin R., Wegener W. S., Ajl S. J. 1971; Assays of enzymes of the tricarboxylic acid and glyoxylate cycles. Methods Micro 6A:425–462
    [Google Scholar]
  50. Reid C. J., Walshaw D. L., Poole P. S. 1996; Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons. Microbiology 142:2603–2612
    [Google Scholar]
  51. Ronson C. W., Primrose S. B. 1979; Carbohydrate metabolism in Rhizobium trifolii: identification and symbiotic properties of mutants. J Gen Microbiol 112:77–88
    [Google Scholar]
  52. Ronson C. W., Lyttleton P., Robertson J. G. 1981; C4-Dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens . Proc Natl Acad Sci USA 78:4284–4288
    [Google Scholar]
  53. Rosendahl L., Dilworth M. J., Glenn A. R. 1992; Exchange of metabolites across the peribacteroid membrane in pea root nodules. J Plant Physiol 139:635–638
    [Google Scholar]
  54. Ruvkun G. B., Ausubel F. M. 1981; A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88
    [Google Scholar]
  55. Salminen S. O., Streeter J. G. 1987; Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids. J Bacteriol 169:495–499
    [Google Scholar]
  56. Salminen S. O., Streeter J. G. 1990; Factors contributing to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions. J Gen Microbiol 136:2119–2126
    [Google Scholar]
  57. Salminen S. O., Streeter J. G. 1992; Labeling of carbon pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae bacteroids following incubation of intact nodules with 14CO2 . Plant Physiol 100:597–604
    [Google Scholar]
  58. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor; NY: Cold Spring Harbor Laboratory:
    [Google Scholar]
  59. Saroso S., Dilworth M. J., Glenn A. R. 1986; The use of activities of carbon catabolic enzymes as a probe for the carbon nutrition of snakebean nodule bacteroids. J Gen Microbiol 132:243–249
    [Google Scholar]
  60. Simon R., Priefer U., Pühler A. 1983; A broad host-range mobilization system for in vivo genetic engineering: transposon mutagenesis of Gram-negative bacteria. Biotechnology 1:784–791
    [Google Scholar]
  61. Simon R., Quandt J., Klipp W. 1989; New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80:161–169
    [Google Scholar]
  62. Spaink H. P., Okker R. J. H., Wijffelman C. A., Pees E., Lugtenberg B. J. J. 1987; Promoters in the nodulation region of the Rhizobium leguminosarum SYM plasmid PRL1JI. Plant Mol Biol 9:27–39
    [Google Scholar]
  63. Spencer M. E., Darlison M. G., Stephens P. E., Duckenfield I. K., Guest J. R. 1984; Nucleotide-sequence of the sucB gene encoding the dihydrolipoamide succinyltransferase of Escherichia coli K12 and homology with the corresponding acetyltransferase. Eur J Biochem 141:361–374
    [Google Scholar]
  64. Streeter J. G. 1987; Carbohydrate, organic acid, and amino acid composition of bacteroids and cytosol from soybean nodules. Plant Physiol 85:768–773
    [Google Scholar]
  65. Tempest D. W., Neijssel O. M. 1992; Physiological and energetic aspects of bacterial metabolite overproduction. FEMS Microbiol Lett 100:169–176
    [Google Scholar]
  66. Udvardi M., ढ., Ou Yang L.-J., Young S., Day D. A. 1990; Sugar and amino acid transport across symbiotic membranes from soybean nodules. Mol Plant–Microbe Interact 3:334–340
    [Google Scholar]
  67. Walshaw D. L., Poole P. S. 1996; The general l-amino acid permease of Rhizobium leguminosarum is an ABC uptake system that influences efflux of solutes. Mol Microbiol 21:1239–1252
    [Google Scholar]
  68. Wood W. B. 1966; Host specificity of DNA produced by Escherichia coli; bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16:118–133
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-143-7-2209
Loading
/content/journal/micro/10.1099/00221287-143-7-2209
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error