Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Mar;177(6):1435–1443. doi: 10.1128/jb.177.6.1435-1443.1995

Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa.

C Hungerer 1, B Troup 1, U Römling 1, D Jahn 1
PMCID: PMC176757  PMID: 7883699

Abstract

The general tetrapyrrole precursor 5-aminolevulinic acid is formed in bacteria via two different biosynthetic pathways. Members of the alpha group of the proteobacteria use 5-aminolevulinic acid synthase for the condensation of succinyl-coenzyme A and glycine, while other bacteria utilize a two-step pathway from aminoacylated tRNA(Glu). The tRNA-dependent pathway, involving the enzymes glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde-2,1-aminomutase (encoded by hemL), was demonstrated to be used by Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Comamonas testosteroni, Azotobacter vinelandii, and Acinetobacter calcoaceticus. To study the regulation of the pathway, the glutamyl-tRNA reductase gene (hemA) from P. aeruginosa was cloned by complementation of an Escherichia coli hemA mutant. The hemA gene was mapped to the SpeI A fragment and the DpnIL fragment of the P. aeruginosa chromosome corresponding to min 24.1 to 26.8. The cloned hemA gene, coding for a protein of 423 amino acids with a calculated molecular mass of 46,234 Da, forms an operon with the gene for protein release factor 1 (prf1). This translational factor mediates the termination of the protein chain at the ribosome at amber and ochre codons. Since the cloned hemA gene did not possess one of the appropriate stop codons, an autoregulatory mechanism such as that postulated for the enterobacterial system was ruled out. Three open reading frames of unknown function transcribed in the opposite direction to the hemA gene were found. hemM/orf1 and orf2 were found to be homologous to open reading frames located in the 5' region of enterobacterial hemA genes. Utilization of both transcription start sites was changed in a P. aeruginosa mutant missing the oxygen regulator Anr (Fnr analog), indicating the involvement of the transcription factor in hemA expression. DNA sequences homologous to one half of an Anr binding site were detected at one of the determined transcription start sites.

Full Text

The Full Text of this article is available as a PDF (413.1 KB).

Selected References

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

  1. Adamski F. M., McCaughan K. K., Jørgensen F., Kurland C. G., Tate W. P. The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli. J Mol Biol. 1994 May 6;238(3):302–308. doi: 10.1006/jmbi.1994.1293. [DOI] [PubMed] [Google Scholar]
  2. Allefs J. J., Salentijn E. M., Krens F. A., Rouwendal G. J. Optimization of non-radioactive Southern blot hybridization: single copy detection and reuse of blots. Nucleic Acids Res. 1990 May 25;18(10):3099–3100. doi: 10.1093/nar/18.10.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asahara N., Murakami K., Korbrisate S., Hashimoto Y., Murooka Y. Cloning and characterization of the hemA gene for synthesis of delta-aminolevulinic acid in Xanthomonas campestris pv. phaseoli. Appl Microbiol Biotechnol. 1994 Feb;40(6):846–850. doi: 10.1007/BF00173986. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Boorstein W. R., Craig E. A. Primer extension analysis of RNA. Methods Enzymol. 1989;180:347–369. doi: 10.1016/0076-6879(89)80111-9. [DOI] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  9. Chen M. W., Jahn D., O'Neill G. P., Söll D. Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in delta-aminolevulinic acid formation during chlorophyll biosynthesis. J Biol Chem. 1990 Mar 5;265(7):4058–4063. [PubMed] [Google Scholar]
  10. Chen M. W., Jahn D., Schön A., O'Neill G. P., Söll D. Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme. J Biol Chem. 1990 Mar 5;265(7):4054–4057. [PubMed] [Google Scholar]
  11. 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]
  12. Diver J. M., Bryan L. E., Sokol P. A. Transformation of Pseudomonas aeruginosa by electroporation. Anal Biochem. 1990 Aug 15;189(1):75–79. doi: 10.1016/0003-2697(90)90046-c. [DOI] [PubMed] [Google Scholar]
  13. Doss M., Philipp-Dormston W. K. Regulatory link between lactate dehydrogenase and biosynthesis of porphyrin and heme in microorganisms. Enzyme. 1973;16(1):28–41. doi: 10.1159/000459359. [DOI] [PubMed] [Google Scholar]
  14. Drolet M., Péloquin L., Echelard Y., Cousineau L., Sasarman A. Isolation and nucleotide sequence of the hemA gene of Escherichia coli K12. Mol Gen Genet. 1989 Apr;216(2-3):347–352. doi: 10.1007/BF00334375. [DOI] [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., 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]
  19. Grimm B., Bull A., Breu V. Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli. Mol Gen Genet. 1991 Jan;225(1):1–10. doi: 10.1007/BF00282635. [DOI] [PubMed] [Google Scholar]
  20. Hansson M., Rutberg L., Schröder I., Hederstedt L. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J Bacteriol. 1991 Apr;173(8):2590–2599. doi: 10.1128/jb.173.8.2590-2599.1991. [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. Ikemi M., Murakami K., Hashimoto M., Murooka Y. Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli. Gene. 1992 Nov 2;121(1):127–132. doi: 10.1016/0378-1119(92)90170-t. [DOI] [PubMed] [Google Scholar]
  23. Ilag L. L., Jahn D. Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R. Biochemistry. 1992 Aug 11;31(31):7143–7151. doi: 10.1021/bi00146a016. [DOI] [PubMed] [Google Scholar]
  24. Ilag L. L., Jahn D., Eggertsson G., Söll D. The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase. J Bacteriol. 1991 Jun;173(11):3408–3413. doi: 10.1128/jb.173.11.3408-3413.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ilag L. L., Kumar A. M., Söll D. Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis. Plant Cell. 1994 Feb;6(2):265–275. doi: 10.1105/tpc.6.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jacobs N. J., Jacobs J. M., Mills B. A. Role of oxygen in the late steps of heme synthesis in pseudomonads and Escherichia coli. Enzyme. 1973;16(1):50–56. doi: 10.1159/000459361. [DOI] [PubMed] [Google Scholar]
  27. Jacobs N. J., Jacobs J. M., Morgan H. E., Jr Comparative effect of oxygen and nitrate on protoporphyrin and heme synthesis from delta-amino levulinic acid in bacterial cultures. J Bacteriol. 1972 Dec;112(3):1444–1445. doi: 10.1128/jb.112.3.1444-1445.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jahn D., Kim Y. C., Ishino Y., Chen M. W., Söll D. Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. J Biol Chem. 1990 May 15;265(14):8059–8064. [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Jin S., Ishimoto K., Lory S. Nucleotide sequence of the rpoN gene and characterization of two downstream open reading frames in Pseudomonas aeruginosa. J Bacteriol. 1994 Mar;176(5):1316–1322. doi: 10.1128/jb.176.5.1316-1322.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li J. M., Russell C. S., Cosloy S. D. Cloning and structure of the hem A gene of Escherichia coli K-12. Gene. 1989 Oct 30;82(2):209–217. doi: 10.1016/0378-1119(89)90046-2. [DOI] [PubMed] [Google Scholar]
  33. MacGregor C. H., Wolff J. A., Arora S. K., Phibbs P. V., Jr Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa. J Bacteriol. 1991 Nov;173(22):7204–7212. doi: 10.1128/jb.173.22.7204-7212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Majumdar D., Avissar Y. J., Wyche J. H., Beale S. I. Structure and expression of the Chlorobium vibrioforme hemA gene. Arch Microbiol. 1991;156(4):281–289. doi: 10.1007/BF00262999. [DOI] [PubMed] [Google Scholar]
  35. Mercenier A., Simon J. P., Haas D., Stalon V. Catabolism of L-arginine by Pseudomonas aeruginosa. J Gen Microbiol. 1980 Feb;116(2):381–389. doi: 10.1099/00221287-116-2-381. [DOI] [PubMed] [Google Scholar]
  36. Moffat J. G., Donly B. C., McCaughan K. K., Tate W. P. Functional domains in the Escherichia coli release factors. Activities of hybrids between RF-1 and RF-2. Eur J Biochem. 1993 Apr 15;213(2):749–756. doi: 10.1111/j.1432-1033.1993.tb17816.x. [DOI] [PubMed] [Google Scholar]
  37. Murakami K., Hashimoto Y., Murooka Y. Cloning and characterization of the gene encoding glutamate 1-semialdehyde 2,1-aminomutase, which is involved in delta-aminolevulinic acid synthesis in Propionibacterium freudenreichii. Appl Environ Microbiol. 1993 Jan;59(1):347–350. doi: 10.1128/aem.59.1.347-350.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murakami K., Korbsrisate S., Asahara N., Hashimoto Y., Murooka Y. Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli. Appl Microbiol Biotechnol. 1993 Jan;38(4):502–506. doi: 10.1007/BF00242945. [DOI] [PubMed] [Google Scholar]
  39. O'Neill G. P., Jahn D., Söll D. Transfer RNA involvement in chlorophyll biosynthesis. Subcell Biochem. 1991;17:235–264. doi: 10.1007/978-1-4613-9365-8_11. [DOI] [PubMed] [Google Scholar]
  40. O'Neill G. P., Thorbjarnardóttir S., Michelsen U., Pálsson S., Söll D., Eggertsson G. delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli. J Bacteriol. 1991 Jan;173(1):94–100. doi: 10.1128/jb.173.1.94-100.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Olsen G. J., Woese C. R., Overbeek R. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol. 1994 Jan;176(1):1–6. doi: 10.1128/jb.176.1.1-6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ornston L. N., Stanier R. Y. The conversion of catechol and protocatechuate to beta-ketoadipate by Pseudomonas putida. J Biol Chem. 1966 Aug 25;241(16):3776–3786. [PubMed] [Google Scholar]
  43. Petricek M., Rutberg L., Schröder I., Hederstedt L. Cloning and characterization of the hemA region of the Bacillus subtilis chromosome. J Bacteriol. 1990 May;172(5):2250–2258. doi: 10.1128/jb.172.5.2250-2258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Philipp-Dormston W. K., Doss M. Comparison of porphyrin and heme biosynthesis in various heterotrophic bacteria. Enzyme. 1973;16(1):57–64. doi: 10.1159/000459362. [DOI] [PubMed] [Google Scholar]
  45. Römling U., Tümmler B. The impact of two-dimensional pulsed-field gel electrophoresis techniques for the consistent and complete mapping of bacterial genomes: refined physical map of Pseudomonas aeruginosa PAO. Nucleic Acids Res. 1991 Jun 25;19(12):3199–3206. doi: 10.1093/nar/19.12.3199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Savioz A., Zimmermann A., Haas D. Pseudomonas aeruginosa promoters which contain a conserved GG-N10-GC motif but appear to be RpoN-independent. Mol Gen Genet. 1993 Apr;238(1-2):74–80. doi: 10.1007/BF00279533. [DOI] [PubMed] [Google Scholar]
  47. Sawers R. G. Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli. Mol Microbiol. 1991 Jun;5(6):1469–1481. doi: 10.1111/j.1365-2958.1991.tb00793.x. [DOI] [PubMed] [Google Scholar]
  48. Schweizer H. P. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 1991 Jan 2;97(1):109–121. doi: 10.1016/0378-1119(91)90016-5. [DOI] [PubMed] [Google Scholar]
  49. Southern E. M., Anand R., Brown W. R., Fletcher D. S. A model for the separation of large DNA molecules by crossed field gel electrophoresis. Nucleic Acids Res. 1987 Aug 11;15(15):5925–5943. doi: 10.1093/nar/15.15.5925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thauer R. K., Jungermann K., Decker K. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev. 1977 Mar;41(1):100–180. doi: 10.1128/br.41.1.100-180.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thuring R. W., Sanders J. P., Borst P. A freeze-squeeze method for recovering long DNA from agarose gels. Anal Biochem. 1975 May 26;66(1):213–220. doi: 10.1016/0003-2697(75)90739-3. [DOI] [PubMed] [Google Scholar]
  52. Totten P. A., Lara J. C., Lory S. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J Bacteriol. 1990 Jan;172(1):389–396. doi: 10.1128/jb.172.1.389-396.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Troup B., Jahn M., Hungerer C., Jahn D. Isolation of the hemF operon containing the gene for the Escherichia coli aerobic coproporphyrinogen III oxidase by in vivo complementation of a yeast HEM13 mutant. J Bacteriol. 1994 Feb;176(3):673–680. doi: 10.1128/jb.176.3.673-680.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Verkamp E., Chelm B. K. Isolation, nucleotide sequence, and preliminary characterization of the Escherichia coli K-12 hemA gene. J Bacteriol. 1989 Sep;171(9):4728–4735. doi: 10.1128/jb.171.9.4728-4735.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Yamano Y., Nishikawa T., Komatsu Y. Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1. Mol Microbiol. 1993 May;8(5):993–1004. doi: 10.1111/j.1365-2958.1993.tb01643.x. [DOI] [PubMed] [Google Scholar]
  58. Zimmermann A., Reimmann C., Galimand M., Haas D. Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli. Mol Microbiol. 1991 Jun;5(6):1483–1490. doi: 10.1111/j.1365-2958.1991.tb00794.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES