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. 1997 May;179(9):2907–2914. doi: 10.1128/jb.179.9.2907-2914.1997

Regulation of heme biosynthesis in Salmonella typhimurium: activity of glutamyl-tRNA reductase (HemA) is greatly elevated during heme limitation by a mechanism which increases abundance of the protein.

L Y Wang 1, L Brown 1, M Elliott 1, T Elliott 1
PMCID: PMC179053  PMID: 9139907

Abstract

In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in heme biosynthesis. We report that when heme limitation is imposed on cultures of S. typhimurium, glutamyl-tRNA reductase (HemA) enzyme activity is increased 10- to 25-fold. Heme limitation was achieved by a complete starvation for heme in hemB, hemE, and hemH mutants or during exponential growth of a hemL mutant in the absence of heme supplementation. Equivalent results were obtained by both methods. To determine the basis for this induction, we developed a panel of monoclonal antibodies reactive with HemA, which can detect the small amount of protein present in a wild-type strain. Western blot (immunoblot) analysis with these antibodies reveals that the increase in HemA enzyme activity during heme limitation is mediated by an increase in the abundance of the HemA protein. Increased HemA protein levels were also observed in heme-limited cells of a hemL mutant in two different E. coli backgrounds, suggesting that the observed regulation is conserved between E. coli and S. typhimurium. In S. typhimurium, the increase in HemA enzyme and protein levels was accompanied by a minimal (less than twofold) increase in the expression of hemA-lac operon fusions; thus HemA regulation is mediated either at a posttranscriptional step or through modulation of protein stability.

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Selected References

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  1. Ailion M., Bobik T. A., Roth J. R. Two global regulatory systems (Crp and Arc) control the cobalamin/propanediol regulon of Salmonella typhimurium. J Bacteriol. 1993 Nov;175(22):7200–7208. doi: 10.1128/jb.175.22.7200-7208.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Archer C. D., Wang X., Elliott T. Mutants defective in the energy-conserving NADH dehydrogenase of Salmonella typhimurium identified by a decrease in energy-dependent proteolysis after carbon starvation. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9877–9881. doi: 10.1073/pnas.90.21.9877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Avissar Y. J., Beale S. I. Cloning and expression of a structural gene from Chlorobium vibrioforme that complements the hemA mutation in Escherichia coli. J Bacteriol. 1990 Mar;172(3):1656–1659. doi: 10.1128/jb.172.3.1656-1659.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Avissar Y. J., Beale S. I. Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli. J Bacteriol. 1989 Jun;171(6):2919–2924. doi: 10.1128/jb.171.6.2919-2924.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Avissar Y. J., Ormerod J. G., Beale S. I. Distribution of delta-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups. Arch Microbiol. 1989;151(6):513–519. doi: 10.1007/BF00454867. [DOI] [PubMed] [Google Scholar]
  6. Berkowitz D., Hushon J. M., Whitfield H. J., Jr, Roth J., Ames B. N. Procedure for identifying nonsense mutations. J Bacteriol. 1968 Jul;96(1):215–220. doi: 10.1128/jb.96.1.215-220.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bochner B. R., Ames B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed] [Google Scholar]
  8. Breton R., Sanfaçon H., Papayannopoulos I., Biemann K., Lapointe J. Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases. J Biol Chem. 1986 Aug 15;261(23):10610–10617. [PubMed] [Google Scholar]
  9. Brown L., Elliott T. Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene. J Bacteriol. 1996 Jul;178(13):3763–3770. doi: 10.1128/jb.178.13.3763-3770.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen W., Russell C. S., Murooka Y., Cosloy S. D. 5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA. J Bacteriol. 1994 May;176(9):2743–2746. doi: 10.1128/jb.176.9.2743-2746.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Choi P., Wang L., Archer C. D., Elliott T. Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product. J Bacteriol. 1996 Feb;178(3):638–646. doi: 10.1128/jb.178.3.638-646.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Darie S., Gunsalus R. P. Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products. J Bacteriol. 1994 Sep;176(17):5270–5276. doi: 10.1128/jb.176.17.5270-5276.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Desrochers M., Peloquin L., Săsărman A. Mapping of the hemE locus in Salmonella typhimurium. J Bacteriol. 1978 Sep;135(3):1151–1153. doi: 10.1128/jb.135.3.1151-1153.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elliott T. A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon. J Bacteriol. 1992 Jan;174(1):245–253. doi: 10.1128/jb.174.1.245-253.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Elliott T., Avissar Y. J., Rhie G. E., Beale S. I. Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase. J Bacteriol. 1990 Dec;172(12):7071–7084. doi: 10.1128/jb.172.12.7071-7084.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elliott T. Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium. J Bacteriol. 1989 Jul;171(7):3948–3960. doi: 10.1128/jb.171.7.3948-3960.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elliott T., Roth J. R. Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis. Mol Gen Genet. 1989 Apr;216(2-3):303–314. doi: 10.1007/BF00334369. [DOI] [PubMed] [Google Scholar]
  18. Elliott T. Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium. J Bacteriol. 1993 Jan;175(2):325–331. doi: 10.1128/jb.175.2.325-331.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Elliott T., Wang X. Salmonella typhimurium prfA mutants defective in release factor 1. J Bacteriol. 1991 Jul;173(13):4144–4154. doi: 10.1128/jb.173.13.4144-4154.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hart R. A., Kallio P. T., Bailey J. E. Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli. Appl Environ Microbiol. 1994 Jul;60(7):2431–2437. doi: 10.1128/aem.60.7.2431-2437.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hino S., Ishida A. Effect of oxygen on heme and cytochrome content in some facultative bacteria. Enzyme. 1973;16(1):42–49. doi: 10.1159/000459360. [DOI] [PubMed] [Google Scholar]
  22. Hoober J. K., Kahn A., Ash D. E., Gough S., Kannangara C. G. Biosynthesis of delta-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase. Carlsberg Res Commun. 1988;53(1):11–25. doi: 10.1007/BF02908411. [DOI] [PubMed] [Google Scholar]
  23. Jahn D., Michelsen U., Söll D. Two glutamyl-tRNA reductase activities in Escherichia coli. J Biol Chem. 1991 Feb 5;266(4):2542–2548. [PubMed] [Google Scholar]
  24. Jahn D., Verkamp E., Söll D. Glutamyl-transfer RNA: a precursor of heme and chlorophyll biosynthesis. Trends Biochem Sci. 1992 Jun;17(6):215–218. doi: 10.1016/0968-0004(92)90380-r. [DOI] [PubMed] [Google Scholar]
  25. Javor G. T., Febre E. F. Enzymatic basis of thiol-stimulated secretion of porphyrins by Escherichia coli. J Bacteriol. 1992 Feb;174(3):1072–1075. doi: 10.1128/jb.174.3.1072-1075.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kaderbhai M. A., Sligar S. G., Barnfield T., Reames T., Gallagher J., He M., Mercer E. I., Kaderbhai N. A novel series of pEX-PINK expression vectors for screening high-level production of (un)fused foreign proteins by rapid visual detection of PINK Escherichia coli clones. Nucleic Acids Res. 1990 Aug 11;18(15):4629–4630. doi: 10.1093/nar/18.15.4629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kearney J. F., Radbruch A., Liesegang B., Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed] [Google Scholar]
  28. Li J. M., Brathwaite O., Cosloy S. D., Russell C. S. 5-Aminolevulinic acid synthesis in Escherichia coli. J Bacteriol. 1989 May;171(5):2547–2552. doi: 10.1128/jb.171.5.2547-2552.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mogi T., Saiki K., Anraku Y. Biosynthesis and functional role of haem O and haem A. Mol Microbiol. 1994 Nov;14(3):391–398. doi: 10.1111/j.1365-2958.1994.tb02174.x. [DOI] [PubMed] [Google Scholar]
  30. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Neill G. P., Chen M. W., Söll D. delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA. FEMS Microbiol Lett. 1989 Aug;51(3):255–259. doi: 10.1016/0378-1097(89)90406-0. [DOI] [PubMed] [Google Scholar]
  32. RICHMOND M. H., MALLOE O. The rate of growth of Salmonella typhimurium with individual carbon sources related to glucose metabolism or to the Krebs cycle. J Gen Microbiol. 1962 Feb;27:285–297. doi: 10.1099/00221287-27-2-285. [DOI] [PubMed] [Google Scholar]
  33. Ron D., Dressler H. pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins. Biotechniques. 1992 Dec;13(6):866–869. [PubMed] [Google Scholar]
  34. Schmieger H. Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet. 1972;119(1):75–88. doi: 10.1007/BF00270447. [DOI] [PubMed] [Google Scholar]
  35. Schneegurt M. A., Rieble S., Beale S. I. The tRNA Required for in Vitro delta-Aminolevulinic Acid Formation from Glutamate in Synechocystis Extracts : Determination of Activity in a Synechocystis in Vitro Protein Synthesizing System. Plant Physiol. 1988 Dec;88(4):1358–1366. doi: 10.1104/pp.88.4.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schröder I., Hederstedt L., Kannangara C. G., Gough P. Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product. Biochem J. 1992 Feb 1;281(Pt 3):843–850. doi: 10.1042/bj2810843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  38. Verkamp E., Backman V. M., Björnsson J. M., Söll D., Eggertsson G. The periplasmic dipeptide permease system transports 5-aminolevulinic acid in Escherichia coli. J Bacteriol. 1993 Mar;175(5):1452–1456. doi: 10.1128/jb.175.5.1452-1456.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Verkamp E., Jahn M., Jahn D., Kumar A. M., Söll D. Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression. J Biol Chem. 1992 Apr 25;267(12):8275–8280. [PubMed] [Google Scholar]
  40. Warren M. J., Bolt E. L., Roessner C. A., Scott A. I., Spencer J. B., Woodcock S. C. Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein. Biochem J. 1994 Sep 15;302(Pt 3):837–844. doi: 10.1042/bj3020837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Woodard S. I., Dailey H. A. Regulation of heme biosynthesis in Escherichia coli. Arch Biochem Biophys. 1995 Jan 10;316(1):110–115. doi: 10.1006/abbi.1995.1016. [DOI] [PubMed] [Google Scholar]
  42. Xu K., Delling J., Elliott T. The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation. J Bacteriol. 1992 Jun;174(12):3953–3963. doi: 10.1128/jb.174.12.3953-3963.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zeilstra-Ryalls J. H., Kaplan S. Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state. J Bacteriol. 1996 Feb;178(4):985–993. doi: 10.1128/jb.178.4.985-993.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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