Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Jun;177(11):3326–3331. doi: 10.1128/jb.177.11.3326-3331.1995

Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase.

B Troup 1, C Hungerer 1, D Jahn 1
PMCID: PMC177029  PMID: 7768836

Abstract

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression.

Full Text

The Full Text of this article is available as a PDF (366.0 KB).

Selected References

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

  1. Angov E., Brusilow W. S. Use of lac fusions to measure in vivo regulation of expression of Escherichia coli proton-translocating ATPase (unc) genes. J Bacteriol. 1988 Jan;170(1):459–462. doi: 10.1128/jb.170.1.459-462.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Coomber S. A., Jones R. M., Jordan P. M., Hunter C. N. A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene. Mol Microbiol. 1992 Nov;6(21):3159–3169. doi: 10.1111/j.1365-2958.1992.tb01772.x. [DOI] [PubMed] [Google Scholar]
  6. Cotter P. A., Darie S., Gunsalus R. P. The effect of iron limitation on expression of the aerobic and anaerobic electron transport pathway genes in Escherichia coli. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):227–232. doi: 10.1111/j.1574-6968.1992.tb14045.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Hungerer C., Troup B., Römling U., Jahn D. Regulation of the hemA gene during 5-aminolevulinic acid formation in Pseudomonas aeruginosa. J Bacteriol. 1995 Mar;177(6):1435–1443. doi: 10.1128/jb.177.6.1435-1443.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Keng T. HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Jun;12(6):2616–2623. doi: 10.1128/mcb.12.6.2616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  18. Madsen O., Sandal L., Sandal N. N., Marcker K. A. A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules. Plant Mol Biol. 1993 Oct;23(1):35–43. doi: 10.1007/BF00021417. [DOI] [PubMed] [Google Scholar]
  19. Martasek P., Camadro J. M., Delfau-Larue M. H., Dumas J. B., Montagne J. J., de Verneuil H., Labbe P., Grandchamp B. Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3024–3028. doi: 10.1073/pnas.91.8.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Ostrow K. S., Silhavy T. J., Garrett S. cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12. J Bacteriol. 1986 Dec;168(3):1165–1171. doi: 10.1128/jb.168.3.1165-1171.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Plunkett G., 3rd, Burland V., Daniels D. L., Blattner F. R. Analysis of the Escherichia coli genome. III. DNA sequence of the region from 87.2 to 89.2 minutes. Nucleic Acids Res. 1993 Jul 25;21(15):3391–3398. doi: 10.1093/nar/21.15.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Poulson R., Polglase W. J. Aerobic and anaerobic coproporphyrinogenase activities in extracts from Saccharomyces cerevisiae. J Biol Chem. 1974 Oct 25;249(20):6367–6371. [PubMed] [Google Scholar]
  25. Tait G. H. Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D. Biochem J. 1972 Aug;128(5):1159–1169. doi: 10.1042/bj1281159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tait G. H. Coproporphyrinogenase activity in extracts from Rhodopseudomonas spheroides. Biochem Biophys Res Commun. 1969 Sep 24;37(1):116–122. doi: 10.1016/0006-291x(69)90888-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Verdière J., Gaisne M., Labbe-Bois R. CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcribed genes in yeast [corrected]. Mol Gen Genet. 1991 Aug;228(1-2):300–306. doi: 10.1007/BF00282480. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Xu K., Elliott T. An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium. J Bacteriol. 1993 Aug;175(16):4990–4999. doi: 10.1128/jb.175.16.4990-4999.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Xu K., Elliott T. Cloning, DNA sequence, and complementation analysis of the Salmonella typhimurium hemN gene encoding a putative oxygen-independent coproporphyrinogen III oxidase. J Bacteriol. 1994 Jun;176(11):3196–3203. doi: 10.1128/jb.176.11.3196-3203.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zagorec M., Buhler J. M., Treich I., Keng T., Guarente L., Labbe-Bois R. Isolation, sequence, and regulation by oxygen of the yeast HEM13 gene coding for coproporphyrinogen oxidase. J Biol Chem. 1988 Jul 15;263(20):9718–9724. [PubMed] [Google Scholar]

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

RESOURCES