Skip to content
1887

Microbiology Society logo Microbiology

Volume 140, Issue 10

Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Free

Abstract

An mutant of was constructed by replacing the chromosomal gene by an internally deleted derivative containing a cassette. Southern and Western blotting confirmed that the gene had been replaced by the disrupted gene and that the aconitase A protein was no longer expressed. However, the mutant failed to exhibit the anticipated glutamate auxotrophy and it retained a residual aconitase activity. This activity was due to an analogous unstable enzyme(s) designated aconitase B. Studies on the regulation of aconitase A synthesis using an translational fusion showed that the gene resembles other citric acid cycle genes in being subject to CRP-mediated catabolite repression and ArcA-mediated anaerobic repression. In addition to being activated by the SoxRS oxidative stress regulatory system, the gene appeared to be activated by the ferric uptake regulator (Fur). It was concluded that the gene belongs to at least four global regulatory networks, and . In contrast, the aconitase B activity decreased after exposure to oxidative stress and was less affected by anaerobiosis. Comparable studies with the fumarase genes (and ) indicated that (encoding the unstable aerobic iron-sulphur-containing fumarase) is by the ferric uptake regulator (Fur) and (encoding the stable fumarase) is activated by the SoxRS oxidative stress regulatory system.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): aconitase , citric acid cycle , Escherichia coli , fumarase and Fur-mediated transcriptional regulation
© Society for General Microbiology 1994
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-140-10-2531
1994-10-01
2025-05-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/140/10/mic-140-10-2531.html?itemId=/content/journal/micro/10.1099/00221287-140-10-2531&mimeType=html&fmt=ahah

References

  1. Amabile-Cuevas C.F., Demple B. Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon.. Nucleic Acids Res 1991; 19:4479–4484
     [Google Scholar]
  2. Andrews S.C., Harrison P.M., Guest J.R. Cloning, sequencing and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12. J Bacteriol 1989; 171:3940–3947
     [Google Scholar]
  3. Bagg A., Neilands J.B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 1987; 26:5471–5477
     [Google Scholar]
  4. Bell P.J., Andrews S.C., Sivak M.N., Guest J.R. Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12. J Bacteriol 1989; 171:3494–3503
     [Google Scholar]
  5. Chambers S.P., Prior S.E., Barstow D.A., Minton N.P. The pMTL nic−cloning vectors. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 1988; 68:139–149
     [Google Scholar]
  6. Cole S.T., Guest J.R. Genetic and physical characterization of lambda transducing phages (λfrdA) containing the fumarate reductase gene of Escherichia coli K12. Mol & Gen Genet 1980; 178:409–418
     [Google Scholar]
  7. Compan I., Touati D. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K12. J Bacteriol 1993; 175:1687–1696
     [Google Scholar]
  8. Compan I., Touati D. Anaerobic activation of arc A transcription in Escherichia coli: roles for Fnr and ArcA. Mol Microbiol 1994; 11:955–964
     [Google Scholar]
  9. Constable A., Quick S., Gray N.K., Hentze M.W. Modulation of the RNA-binding activity of a regulatory protein by iron in vitro, switching between enzymatic and genetic function. Proc Natl Acad Sci USA 1992; 89:4554–4558
     [Google Scholar]
  10. Emery-Goodman A., Hirling H., Scarpellino L., Henderson B., Kuhn L. Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase. Nucleic Acids Res 1993; 21:1457–1461
     [Google Scholar]
  11. Flint D.H., Emptage M.H., Guest J.R. Fumarase A from Escherichia coli: purification and characterization as an iron-sulfur containing enzyme. Biochemistry 1992; 31:10331–10337
     [Google Scholar]
  12. Gardner P.R., Fridovich I. Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem 1991; 266:19328–19333
     [Google Scholar]
  13. Gardner P.R., Fridovich I. Inactivation-reactivation of aconitase in Escherichia coli - a sensitive measure of superoxide radical. J Biol Chem 1992; 267:8757–8763
     [Google Scholar]
  14. Gray C.T., Wimpenny J.W.T., Mossman M.R. Regulation of metabolism in facultative bacteria. II. Effects of aerobiosis and nutrition on the formation of Krebs cycle enzymes in Escherichia coli. Biochim Biophys Acta 1966; 117:33–41
     [Google Scholar]
  15. Greenberg J.T., Monach P., Chou J.H., Josephy D., Demple B. Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci USA 1990; 87:6181–6185
     [Google Scholar]
  16. Guest J.R. Partial replacement of succinate dehydrogenase function by phage- and plasmid-specified fumarate reductase in Escherichia coli. J Gen Microbiol 1981; 122:171–179
     [Google Scholar]
  17. Guest J.R., Russell G.C. Complexes and complexities of the citric acid cycle in Escherichia coli. Curr Top Cell Regulation 1992; 33:231–247
     [Google Scholar]
  18. Guest J.R., Angier S.J., Russell G.C. Structure, expression, and protein engineering of the pyruvate dehydrogenase complex of Escherichia coli. Ann NY Acad Sci 1989; 573:76–99
     [Google Scholar]
  19. Haile D.J., Rouault T.A., Harford J.B., Kennedy M.C., Blondin G.A., Beinert H., Klausner R.D. Cellular regulation of the iron—responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Natl Acad Sci USA 1992a; 89:11735–11739
     [Google Scholar]
  20. Haile D.J., Rouault T.A., Tang C.K., Chin J., Harford J.B., Klausner R.D. Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein: role of the iron—sulfur cluster. Proc Natl Acad Sci USA 1992b; 89:7536–7540
     [Google Scholar]
  21. Hamilton C.M., Aldea M., Washburn B.K., Babitzke P., Kushner S.R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 1989; 171:4617–4622
     [Google Scholar]
  22. Hidalgo E., Demple B. An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein. EMBOJ 1994; 13:138–146
     [Google Scholar]
  23. Iuchi S., Lin E.C.C. arc A {dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci USA 1988; 85:1888–1892
     [Google Scholar]
  24. Jin S.F., Sonenshein A.L. Identification of two distinct Bacillus subtilis citrate synthase genes. J Bacteriol 1994; 176:4669–4679
     [Google Scholar]
  25. Kaptain S., Downey W.E., Tang C., Philpott C., Haile D., Orloff D.G., Harford J.B., Rouault T.A., Klausner R.D. A regulated RNA binding protein also possesses aconitase activity. Proc Natl Acad Sci USA 1991; 88:10109–10113
     [Google Scholar]
  26. Kennedy M.C., Emptage M.H., Dreyer J.-L., Bienert H. The role of iron in the activation-inactivation of aconitase. J Biol Chem 1983; 258:11098–11105
     [Google Scholar]
  27. Kennedy M.C., Mende-Mueller L., Blondin G.A., Beinert H. Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci USA 1992; 89:11730–11734
     [Google Scholar]
  28. Klausner R.D., Rouault T.A. A double life: cytosolic aconitase as a regulatory RNA binding protein. Mol Biol Cell 1993; 4:1–5
     [Google Scholar]
  29. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 277:680–685
     [Google Scholar]
  30. Lakshmi T.M., Helling R.B. Selection for citrate synthase deficiency in icd mutants of Escherichia coli. J Bacteriol 1976; 127:76–83
     [Google Scholar]
  31. Liochev S.I., Fridovich I. Fumarase C, the stable fumarase of Fsscherichia coli, is controlled by the toxRT regulon. Proc Natl Acad Sci USA 1992; 89:5892–5896
     [Google Scholar]
  32. Miles J.M., Guest J.R. Complete nucleotide sequence of the fumarase gene fumA of Escherichia coli. Nucleic Acids Res 1984; 12:3631–3642
     [Google Scholar]
  33. Miller J.H. Experiments in Molecular Genetics 1972 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  34. Minton N. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 1984; 31:269–273
     [Google Scholar]
  35. Niederhoffer E.G., Naranjo C.M., Bradley K.L., Fee J.A. Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus. J Bacteriol 1990; 172:1930–1938
     [Google Scholar]
  36. Patton A.J., Hough D.W., Towner P., Danson M.J. Does Escherichia coli possess a second citrate synthase gene?. Eur Biochem 1993; 214:75–81
     [Google Scholar]
  37. Perham R.N. Genes, proteins and metabolic functions o enzymes. In Genetics and Human Nutrition 1990 Edited by Randle P.J., Bell J.I., Scott J. London: John Libbey; pp 1–14
     [Google Scholar]
  38. Prodromou G., Haynes M.J., Guest J.R. The aconitase of Escherichia coli: purification of the enzyme and molecular cloninj and map location of the gene (acn). J Gen Microbiol 1991; 137:2505–2515
     [Google Scholar]
  39. Prodromou G., Artymiuk P.J., Guest J.R. The aconitasi of Escherichia coli: nucleotide sequence of the aconitase gene anc amino acid sequence similarity with mitochondrial aconitases, thi iron-responsive-element-binding protein and isopropylmalati isomerases. Eur J Biochem 1992; 204:599–609
     [Google Scholar]
  40. Reaney S.K., Bungard S.J., Guest J.R. Molecular anc enzymological evidence for two classes of fumarase in Bacillu stearothermophilus (var non-diastaticus). J Gen Microbiol 1993; 139:403–416
     [Google Scholar]
  41. Richarme G. Purification of a new dihydrolipoamidi dehydrogenase from Escherichia coli. J Bacteriol 1989; 171:6580–6585
     [Google Scholar]
  42. Robbins A.H., Stout C.D. Structure of activatec aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc Nat Acad Sci USA 1989; 86:3639–3643
     [Google Scholar]
  43. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning a Eaboratory Manual, 1989, 2nd edn. Cold Spring Harbor, NY: Cole Spring Harbor Laboratory;
     [Google Scholar]
  44. Silhavy T.J., Barman M.L., Enquist L.W. Experiment with Gene Fusions 1984 Cold Spring Harbor, NY: Cold Spring Harbo: Laboratory;
     [Google Scholar]
  45. Spiro S., Guest J.R. Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol 1987; 133:3279–3288
     [Google Scholar]
  46. Spiro S., Roberts R.E., Guest J.R. FNR-dependen repression of the ndh gene of Escherichia coli and metal ior requirement for FNR-regulated gene expression. Mol Microbiol 1989; 3:601–608
     [Google Scholar]
  47. Woods S.A., Guest J.R. Differential roles of the Escherichia coli fumarase and fnr-dependent expression of fumarase B and aspartase. FEMS Microbiol Lett 1987; 48:219–224
     [Google Scholar]
  48. Woods S.A., Miles J.S., Roberts R.E., Guest J.R. Structural and functional relationships between fumarase anc aspartase: nucleotide sequences of the fumarase (fumC) anc aspartase (aspA) genes of Escherichia coli K12. Biochem J 1986; 237:547–557
     [Google Scholar]
  49. Woods S.A., Schartzbach S.D., Guest J.R. Two biochemically distinct classes of fumarases in Escherichia coli. Biochim Biophys Acta 1988; 954:14–26
     [Google Scholar]
/content/journal/micro/10.1099/00221287-140-10-2531
Loading
Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli
Microbiology 140, 2531 (1994); https://doi.org/10.1099/00221287-140-10-2531
/content/journal/micro/10.1099/00221287-140-10-2531
/content/journal/micro/10.1099/00221287-140-10-2531
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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