Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jun;176(12):3790–3799. doi: 10.1128/jb.176.12.3790-3799.1994

The iron superoxide dismutase of Legionella pneumophila is essential for viability.

A B Sadosky 1, J W Wilson 1, H M Steinman 1, H A Shuman 1
PMCID: PMC205569  PMID: 8206858

Abstract

Legionella pneumophila, the causative agent of Legionnaires' disease, contains two superoxide dismutases (SODs), a cytoplasmic iron enzyme (FeSOD) and a periplasmic copper-zinc SOD. To study the role of the FeSOD in L. pneumophila, the cloned FeSOD gene (sodB) was inactivated with Tn903dIIlacZ, forming a sodB::lacZ gene fusion. By using this fusion, expression of sodB was shown to be unaffected by a variety of conditions, including several that influence sod expression in Escherichia coli: aeration, oxidants, the redox cycling compound paraquat, manipulation of iron levels in the medium, and the stage of growth. A reproducible twofold decrease in sodB expression was found during growth on agar medium containing charcoal, a potential scavenger of oxyradicals, in comparison with growth on the same medium without charcoal. No induction was seen during growth in human macrophages. Additional copies of sodB+ in trans increased resistance to paraquat. Construction of a sodB mutant was attempted by allelic exchange of the sodB::lacZ fusion with the chromosomal copy of sodB. The mutant could not be isolated, and the allelic exchange was possible only if wild-type sodB was present in trans. These results indicate that the periplasmic copper-zinc SOD cannot replace the FeSOD. The data strongly suggest that sodB is an essential gene and that FeSOD is required for the viability of L. pneumophila. In contrast, Sod- mutants of E. coli and Streptococcus mutans grow aerobically and SOD is not required for viability in these species.

Full text

PDF
3790

Images in this article

3796Image
on p.3796

Selected References

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

  1. Beaman L., Beaman B. L. The role of oxygen and its derivatives in microbial pathogenesis and host defense. Annu Rev Microbiol. 1984;38:27–48. doi: 10.1146/annurev.mi.38.100184.000331. [DOI] [PubMed] [Google Scholar]
  2. Beck B. L., Tabatabai L. B., Mayfield J. E. A protein isolated from Brucella abortus is a Cu-Zn superoxide dismutase. Biochemistry. 1990 Jan 16;29(2):372–376. doi: 10.1021/bi00454a010. [DOI] [PubMed] [Google Scholar]
  3. Bellinger-Kawahara C., Horwitz M. A. Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes. J Exp Med. 1990 Oct 1;172(4):1201–1210. doi: 10.1084/jem.172.4.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bey R. F., Johnson R. C. Protein-free and low-protein media for the cultivation of Leptospira. Infect Immun. 1978 Feb;19(2):562–569. doi: 10.1128/iai.19.2.562-569.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beyer W., Imlay J., Fridovich I. Superoxide dismutases. Prog Nucleic Acid Res Mol Biol. 1991;40:221–253. doi: 10.1016/s0079-6603(08)60843-0. [DOI] [PubMed] [Google Scholar]
  6. Carlioz A., Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986 Mar;5(3):623–630. doi: 10.1002/j.1460-2075.1986.tb04256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A. 1978 May;75(5):2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farr S. B., D'Ari R., Touati D. Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8268–8272. doi: 10.1073/pnas.83.21.8268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fee J. A. Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function. Mol Microbiol. 1991 Nov;5(11):2599–2610. doi: 10.1111/j.1365-2958.1991.tb01968.x. [DOI] [PubMed] [Google Scholar]
  10. Feeley J. C., Gibson R. J., Gorman G. W., Langford N. C., Rasheed J. K., Mackel D. C., Baine W. B. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol. 1979 Oct;10(4):437–441. doi: 10.1128/jcm.10.4.437-441.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Franzon V. L., Arondel J., Sansonetti P. J. Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis. Infect Immun. 1990 Feb;58(2):529–535. doi: 10.1128/iai.58.2.529-535.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fridovich I. Superoxide radical: an endogenous toxicant. Annu Rev Pharmacol Toxicol. 1983;23:239–257. doi: 10.1146/annurev.pa.23.040183.001323. [DOI] [PubMed] [Google Scholar]
  14. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  15. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  16. Gardner P. R., Fridovich I. Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical. J Biol Chem. 1992 May 5;267(13):8757–8763. [PubMed] [Google Scholar]
  17. Gardner P. R., Fridovich I. Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem. 1991 Oct 15;266(29):19328–19333. [PubMed] [Google Scholar]
  18. Halliwell B., Gutteridge J. M. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 1984 Apr 1;219(1):1–14. doi: 10.1042/bj2190001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hassett D. J., Cohen M. S. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 1989 Dec;3(14):2574–2582. doi: 10.1096/fasebj.3.14.2556311. [DOI] [PubMed] [Google Scholar]
  20. Hoffman P. S., Pine L., Bell S. Production of superoxide and hydrogen peroxide in medium used to culture Legionella pneumophila: catalytic decomposition by charcoal. Appl Environ Microbiol. 1983 Mar;45(3):784–791. doi: 10.1128/aem.45.3.784-791.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horwitz M. A., Silverstein S. C. Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin. J Clin Invest. 1983 Jan;71(1):15–26. doi: 10.1172/JCI110744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keele B. B., Jr, McCord J. M., Fridovich I. Superoxide dismutase from escherichia coli B. A new manganese-containing enzyme. J Biol Chem. 1970 Nov 25;245(22):6176–6181. [PubMed] [Google Scholar]
  23. Kroll J. S., Langford P. R., Loynds B. M. Copper-zinc superoxide dismutase of Haemophilus influenzae and H. parainfluenzae. J Bacteriol. 1991 Dec;173(23):7449–7457. doi: 10.1128/jb.173.23.7449-7457.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MUELLER J. H., MILLER P. A. Variable factors influencing the production of tetanus toxin. J Bacteriol. 1954 Mar;67(3):271–277. doi: 10.1128/jb.67.3.271-277.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marra A., Shuman H. A. Isolation of a Legionella pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J Bacteriol. 1989 Apr;171(4):2238–2240. doi: 10.1128/jb.171.4.2238-2240.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mengaud J. M., Horwitz M. A. The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein. J Bacteriol. 1993 Sep;175(17):5666–5676. doi: 10.1128/jb.175.17.5666-5676.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moody C. S., Hassan H. M. Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli. J Biol Chem. 1984 Oct 25;259(20):12821–12825. [PubMed] [Google Scholar]
  28. Morales V. M., Bäckman A., Bagdasarian M. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene. 1991 Jan 2;97(1):39–47. doi: 10.1016/0378-1119(91)90007-x. [DOI] [PubMed] [Google Scholar]
  29. Nakayama K. Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants. J Bacteriol. 1992 Aug;174(15):4928–4934. doi: 10.1128/jb.174.15.4928-4934.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Niederhoffer E. C., 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 Apr;172(4):1930–1938. doi: 10.1128/jb.172.4.1930-1938.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Payne N. R., Horwitz M. A. Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med. 1987 Nov 1;166(5):1377–1389. doi: 10.1084/jem.166.5.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pine L., George J. R., Reeves M. W., Harrell W. K. Development of a chemically defined liquid medium for growth of Legionella pneumophila. J Clin Microbiol. 1979 May;9(5):615–626. doi: 10.1128/jcm.9.5.615-626.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pine L., Hoffman P. S., Malcolm G. B., Benson R. F., Keen M. G. Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella. J Clin Microbiol. 1984 Sep;20(3):421–429. doi: 10.1128/jcm.20.3.421-429.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prohaska J. R. Changes in tissue growth, concentrations of copper, iron, cytochrome oxidase and superoxide dismutase subsequent to dietary or genetic copper deficiency in mice. J Nutr. 1983 Oct;113(10):2048–2058. doi: 10.1093/jn/113.10.2048. [DOI] [PubMed] [Google Scholar]
  35. Puget K., Michelson A. M. Isolation of a new copper-containing superoxide dismutase bacteriocuprein. Biochem Biophys Res Commun. 1974 Jun 4;58(3):830–838. doi: 10.1016/s0006-291x(74)80492-4. [DOI] [PubMed] [Google Scholar]
  36. Sadosky A. B., Wiater L. A., Shuman H. A. Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun. 1993 Dec;61(12):5361–5373. doi: 10.1128/iai.61.12.5361-5373.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Salin M. L., Bridges S. M. Isolation and characterization of an iron-containing superoxide dismutase from a eucaryote, Brassica campestris. Arch Biochem Biophys. 1980 May;201(2):369–374. doi: 10.1016/0003-9861(80)90524-x. [DOI] [PubMed] [Google Scholar]
  38. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  40. Steinman H. M. Bacteriocuprein superoxide dismutases in pseudomonads. J Bacteriol. 1985 Jun;162(3):1255–1260. doi: 10.1128/jb.162.3.1255-1260.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Steinman H. M. Construction of an Escherichia coli K-12 strain deleted for manganese and iron superoxide dismutase genes and its use in cloning the iron superoxide dismutase gene of Legionella pneumophila. Mol Gen Genet. 1992 Apr;232(3):427–430. doi: 10.1007/BF00266247. [DOI] [PubMed] [Google Scholar]
  42. Steinman H. M. Copper-zinc superoxide dismutase from Caulobacter crescentus CB15. A novel bacteriocuprein form of the enzyme. J Biol Chem. 1982 Sep 10;257(17):10283–10293. [PubMed] [Google Scholar]
  43. Tardat B., Touati D. Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA. Mol Microbiol. 1993 Jul;9(1):53–63. doi: 10.1111/j.1365-2958.1993.tb01668.x. [DOI] [PubMed] [Google Scholar]
  44. Tardat B., Touati D. Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli::Fur (ferric uptake regulation) and Arc (aerobic respiration control). Mol Microbiol. 1991 Feb;5(2):455–465. doi: 10.1111/j.1365-2958.1991.tb02129.x. [DOI] [PubMed] [Google Scholar]
  45. Touati D. The molecular genetics of superoxide dismutase in E. coli. An approach to understanding the biological role and regulation of SODS in relation to other elements of the defence system against oxygen toxicity. Free Radic Res Commun. 1989;8(1):1–9. doi: 10.3109/10715768909087967. [DOI] [PubMed] [Google Scholar]
  46. Touati D. Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions. J Bacteriol. 1988 Jun;170(6):2511–2520. doi: 10.1128/jb.170.6.2511-2520.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wiater L. A., Sadosky A. B., Shuman H. A. Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene. Mol Microbiol. 1994 Feb;11(4):641–653. doi: 10.1111/j.1365-2958.1994.tb00343.x. [DOI] [PubMed] [Google Scholar]
  48. Winn W. C., Jr Legionnaires disease: historical perspective. Clin Microbiol Rev. 1988 Jan;1(1):60–81. doi: 10.1128/cmr.1.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989 May 11;17(9):3469–3478. doi: 10.1093/nar/17.9.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yost F. J., Jr, Fridovich I. An iron-containing superoxide dismutase from Escherichia coli. J Biol Chem. 1973 Jul 25;248(14):4905–4908. [PubMed] [Google Scholar]

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

RESOURCES