Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Jul;171(7):3948–3960. doi: 10.1128/jb.171.7.3948-3960.1989

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

T Elliott 1
PMCID: PMC210147  PMID: 2544564

Abstract

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.

Full text

PDF
3948

Images in this article

Selected References

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

  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. Aota S., Gojobori T., Ishibashi F., Maruyama T., Ikemura T. Codon usage tabulated from the GenBank Genetic Sequence Data. Nucleic Acids Res. 1988;16 (Suppl):r315–r402. doi: 10.1093/nar/16.suppl.r315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BURNHAM B. F., PIERCE W. S., WILLIAMS K. R., BOYER M. H., KIRBY C. K. delta-aminolaevulate dehydratase from Rhodopseudomonas spheroides. Biochem J. 1963 Jun;87:462–472. doi: 10.1042/bj0870462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beaudet A. L., Caskey C. T. Release factor translation of RNA phage terminator codons. Nature. 1970 Jul 4;227(5253):38–40. doi: 10.1038/227038a0. [DOI] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  7. Borthwick I. A., Srivastava G., Day A. R., Pirola B. A., Snoswell M. A., May B. K., Elliott W. H. Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor. Eur J Biochem. 1985 Aug 1;150(3):481–484. doi: 10.1111/j.1432-1033.1985.tb09047.x. [DOI] [PubMed] [Google Scholar]
  8. Bossi L. Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. J Mol Biol. 1983 Feb 15;164(1):73–87. doi: 10.1016/0022-2836(83)90088-8. [DOI] [PubMed] [Google Scholar]
  9. Bower S. G., Hove-Jensen B., Switzer R. L. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium. J Bacteriol. 1988 Jul;170(7):3243–3248. doi: 10.1128/jb.170.7.3243-3248.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  11. Brendel V., Hamm G. H., Trifonov E. N. Terminators of transcription with RNA polymerase from Escherichia coli: what they look like and how to find them. J Biomol Struct Dyn. 1986 Feb;3(4):705–723. doi: 10.1080/07391102.1986.10508457. [DOI] [PubMed] [Google Scholar]
  12. Cauthen S. E., Foster M. A., Woods D. D. Methionine synthesis by extracts of Salmonella typhimurium. Biochem J. 1966 Feb;98(2):630–635. doi: 10.1042/bj0980630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chang G. W., Chang J. T. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature. 1975 Mar 13;254(5496):150–151. doi: 10.1038/254150a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Cox R., Charles H. P. Porphyrin-accumulating mutants of Escherichia coli. J Bacteriol. 1973 Jan;113(1):122–132. doi: 10.1128/jb.113.1.122-132.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Craigen W. J., Cook R. G., Tate W. P., Caskey C. T. Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3616–3620. doi: 10.1073/pnas.82.11.3616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elliott T., Roth J. R. Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance. Mol Gen Genet. 1988 Aug;213(2-3):332–338. doi: 10.1007/BF00339599. [DOI] [PubMed] [Google Scholar]
  18. Ganoza M. C., Tomkins J. K. Polypeptide chain termination in vitro: competition for nonsense codons between a purified release factor and suppressor tRNA. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1455–1463. doi: 10.1016/0006-291x(70)90031-8. [DOI] [PubMed] [Google Scholar]
  19. Hahn D. R., Myers R. S., Kent C. R., Maloy S. R. Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region. Mol Gen Genet. 1988 Jul;213(1):125–133. doi: 10.1007/BF00333408. [DOI] [PubMed] [Google Scholar]
  20. Hall M. N., Hereford L., Herskowitz I. Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell. 1984 Apr;36(4):1057–1065. doi: 10.1016/0092-8674(84)90055-2. [DOI] [PubMed] [Google Scholar]
  21. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  22. Hashimoto-Gotoh T., Franklin F. C., Nordheim A., Timmis K. N. Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors. Gene. 1981 Dec;16(1-3):227–235. doi: 10.1016/0378-1119(81)90079-2. [DOI] [PubMed] [Google Scholar]
  23. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Hoppe I., Roth J. Specialized transducing phages derived from salmonella phage P22. Genetics. 1974 Apr;76(4):633–654. doi: 10.1093/genetics/76.4.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Housley P. R., Leavitt A. D., Whitfield H. J. Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity. J Bacteriol. 1981 Jul;147(1):13–24. doi: 10.1128/jb.147.1.13-24.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hughes K. T., Roth J. R. Conditionally transposition-defective derivative of Mu d1(Amp Lac). J Bacteriol. 1984 Jul;159(1):130–137. doi: 10.1128/jb.159.1.130-137.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Imlay J. A., Linn S. DNA damage and oxygen radical toxicity. Science. 1988 Jun 3;240(4857):1302–1309. doi: 10.1126/science.3287616. [DOI] [PubMed] [Google Scholar]
  29. Imlay J. A., Linn S. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J Bacteriol. 1987 Jul;169(7):2967–2976. doi: 10.1128/jb.169.7.2967-2976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kannangara C. G., Gough S. P., Bruyant P., Hoober J. K., Kahn A., von Wettstein D. tRNA(Glu) as a cofactor in delta-aminolevulinate biosynthesis: steps that regulate chlorophyll synthesis. Trends Biochem Sci. 1988 Apr;13(4):139–143. doi: 10.1016/0968-0004(88)90071-0. [DOI] [PubMed] [Google Scholar]
  31. Klein H., Capecchi M. R. Polypetide chain termination. Purification of the release factors, R1 and R2, from Escherichia coli. J Biol Chem. 1971 Feb 25;246(4):1055–1061. [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lathe R., Kieny M. P., Skory S., Lecocq J. P. Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA. 1984;3(2):173–182. doi: 10.1089/dna.1984.3.173. [DOI] [PubMed] [Google Scholar]
  34. Lee C. C., Kohara Y., Akiyama K., Smith C. L., Craigen W. J., Caskey C. T. Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches. J Bacteriol. 1988 Oct;170(10):4537–4541. doi: 10.1128/jb.170.10.4537-4541.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee J. H., Nishitani J., Wilcox G. Genetic characterization of Salmonella typhimurium LT2 ara mutations. J Bacteriol. 1984 Apr;158(1):344–346. doi: 10.1128/jb.158.1.344-346.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lee N., Gielow W., Martin R., Hamilton E., Fowler A. The organization of the araBAD operon of Escherichia coli. Gene. 1986;47(2-3):231–244. doi: 10.1016/0378-1119(86)90067-3. [DOI] [PubMed] [Google Scholar]
  37. Leong S. A., Williams P. H., Ditta G. S. Analysis of the 5' regulatory region of the gene for delta-aminolevulinic acid synthetase of Rhizobium meliloti. Nucleic Acids Res. 1985 Aug 26;13(16):5965–5976. doi: 10.1093/nar/13.16.5965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  39. Matsuyama S., Mizushima S. Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for OmpF synthesis in Escherichia coli. J Bacteriol. 1985 Jun;162(3):1196–1202. doi: 10.1128/jb.162.3.1196-1202.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. McClung C. R., Somerville J. E., Guerinot M. L., Chelm B. K. Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase. Gene. 1987;54(1):133–139. doi: 10.1016/0378-1119(87)90355-6. [DOI] [PubMed] [Google Scholar]
  41. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  42. Neidhardt F. C., Bloch P. L., Pedersen S., Reeh S. Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli. J Bacteriol. 1977 Jan;129(1):378–387. doi: 10.1128/jb.129.1.378-387.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Roland K. L., Liu C. G., Turnbough C. L., Jr Role of the ribosome in suppressing transcriptional termination at the pyrBI attenuator of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7149–7153. doi: 10.1073/pnas.85.19.7149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Roland K. L., Powell F. E., Turnbough C. L., Jr Role of translation and attenuation in the control of pyrBI operon expression in Escherichia coli K-12. J Bacteriol. 1985 Sep;163(3):991–999. doi: 10.1128/jb.163.3.991-999.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roof D. M., Roth J. R. Ethanolamine utilization in Salmonella typhimurium. J Bacteriol. 1988 Sep;170(9):3855–3863. doi: 10.1128/jb.170.9.3855-3863.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rydén S. M., Isaksson L. A. A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors. Mol Gen Genet. 1984;193(1):38–45. doi: 10.1007/BF00327411. [DOI] [PubMed] [Google Scholar]
  47. Sanderson K. E., Roth J. R. Linkage map of Salmonella typhimurium, edition VII. Microbiol Rev. 1988 Dec;52(4):485–532. doi: 10.1128/mr.52.4.485-532.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Siegel L. M., Murphy M. J., Kamin H. Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. I. The Escherichia coli hemoflavoprotein: molecular parameters and prosthetic groups. J Biol Chem. 1973 Jan 10;248(1):251–264. [PubMed] [Google Scholar]
  49. Smith H. O. Defective phage formation by lysogens of integration deficient phage P22 mutants. Virology. 1968 Feb;34(2):203–223. doi: 10.1016/0042-6822(68)90231-6. [DOI] [PubMed] [Google Scholar]
  50. Susskind M. M., Wright A., Botstein D. Superinfection exclusion by P22 prophage in lysogens of Salmonella typhimurium. II. Genetic evidence for two exclusion systems. Virology. 1971 Sep;45(3):638–652. doi: 10.1016/0042-6822(71)90178-4. [DOI] [PubMed] [Google Scholar]
  51. Săsărman A., Chartrand P., Proschek R., Desrochers M., Tardif D., Lapointe C. Uroporphyrin-accumulating mutant of Escherichia coli K-12. J Bacteriol. 1975 Dec;124(3):1205–1212. doi: 10.1128/jb.124.3.1205-1212.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Săsărman A., Sanderson K. E., Surdeanu M., Sonea S. Hemin-deficient mutants of Salmonella typhimurium. J Bacteriol. 1970 May;102(2):531–536. doi: 10.1128/jb.102.2.531-536.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Săsărman A., Surdeanu M., Horodniceanu T. Locus determining the synthesis of delta-aminolevulinic acid in Escherichia coli K-12. J Bacteriol. 1968 Nov;96(5):1882–1884. doi: 10.1128/jb.96.5.1882-1884.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Ta G. H. General aspects of haem synthesis. Biochem Soc Symp. 1968;28:19–34. [PubMed] [Google Scholar]
  55. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tai T. N., Moore M. D., Kaplan S. Cloning and characterization of the 5-aminolevulinate synthase gene(s) from Rhodobacter sphaeroides. Gene. 1988 Oct 15;70(1):139–151. doi: 10.1016/0378-1119(88)90112-6. [DOI] [PubMed] [Google Scholar]
  57. Tuerk C., Gauss P., Thermes C., Groebe D. R., Gayle M., Guild N., Stormo G., d'Aubenton-Carafa Y., Uhlenbeck O. C., Tinoco I., Jr CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1364–1368. doi: 10.1073/pnas.85.5.1364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Urban-Grimal D., Volland C., Garnier T., Dehoux P., Labbe-Bois R. The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae. Eur J Biochem. 1986 May 2;156(3):511–519. doi: 10.1111/j.1432-1033.1986.tb09610.x. [DOI] [PubMed] [Google Scholar]
  59. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  60. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  61. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Weiss R. B., Murphy J. P., Gallant J. A. Genetic screen for cloned release factor genes. J Bacteriol. 1984 Apr;158(1):362–364. doi: 10.1128/jb.158.1.362-364.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Youderian P., Sugiono P., Brewer K. L., Higgins N. P., Elliott T. Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages. Genetics. 1988 Apr;118(4):581–592. doi: 10.1093/genetics/118.4.581. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES