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. 1988 Jun;56(6):1449–1455. doi: 10.1128/iai.56.6.1449-1455.1988

Isolation and characterization of auxotrophic mutants of Legionella pneumophila that fail to multiply in human monocytes.

C S Mintz 1, J X Chen 1, H A Shuman 1
PMCID: PMC259420  PMID: 3372016

Abstract

Attempts to isolate auxotrophic mutants of Legionella pneumophila have been hampered by the complex nutritional composition of the media used to cultivate this organism. We developed a semidefined medium, designated CAA, to facilitate the isolation and characterization of Legionella auxotrophs. Unlike previously described chemically defined media for this organism, L. pneumophila formed colonies on CAA agar. Using this medium, we isolated several independent tryptophan auxotrophs of strain Philadelphia-1 after ethyl methanesulfonate mutagenesis and penicillin enrichment. Trimethoprim selection was used to isolate several independent thymidine-requiring mutants of the same strain. The thymidine auxotrophs exhibited a marked decrease in viability when they were deprived of thymidine. The results of monocyte infection experiments with both the tryptophan and thymidine auxotrophs indicated that the thymidine auxotrophs were incapable of intracellular survival or multiplication. In contrast, the tryptophan auxotrophs grew well in monocyte cultures. The isolation of additional auxotrophic mutants will facilitate the study of the nutritional requirements of L. pneumophila for growth in human mononuclear phagocytes.

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Selected References

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  1. Alikhanian S. I., Iljina T. S., Kaliaeva E. S., Kameneva S. V., Sukhodolec V. V. A genetical study of thymineless mutants of E. coli K12. Genet Res. 1966 Aug;8(1):83–100. doi: 10.1017/s0016672300009939. [DOI] [PubMed] [Google Scholar]
  2. Alikhanian S. I., Iljina T. S., Kaliaeva E. S., Kameneva S. V., Sukhodolec V. V. Mutants of Escherichia coli K12 lacking thymine. Nature. 1965 May 22;206(4986):848–849. doi: 10.1038/206848a0. [DOI] [PubMed] [Google Scholar]
  3. Dreyfus L. A., Iglewski B. H. Conjugation-mediated genetic exchange in Legionella pneumophila. J Bacteriol. 1985 Jan;161(1):80–84. doi: 10.1128/jb.161.1.80-84.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elliott J. A., Johnson W. Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs. Infect Immun. 1982 Mar;35(3):943–946. doi: 10.1128/iai.35.3.943-946.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elliott J. A., Winn W. C., Jr Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. Infect Immun. 1986 Jan;51(1):31–36. doi: 10.1128/iai.51.1.31-36.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Fields P. I., Swanson R. V., Haidaris C. G., Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5189–5193. doi: 10.1073/pnas.83.14.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gabay J. E., Horwitz M. A. Isolation and characterization of the cytoplasmic and outer membranes of the Legionnaires' disease bacterium (Legionella pneumophila). J Exp Med. 1985 Feb 1;161(2):409–422. doi: 10.1084/jem.161.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harrison A. P., Jr Thymine incorporation and metabolism by various classes of thymine-less bacteria. J Gen Microbiol. 1965 Dec;41(3):321–333. doi: 10.1099/00221287-41-3-321. [DOI] [PubMed] [Google Scholar]
  10. Hoiseth S. K., Stocker B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981 May 21;291(5812):238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  11. Horwitz M. A. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J Exp Med. 1987 Nov 1;166(5):1310–1328. doi: 10.1084/jem.166.5.1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Horwitz M. A. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med. 1983 Oct 1;158(4):1319–1331. doi: 10.1084/jem.158.4.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horwitz M. A., Maxfield F. R. Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol. 1984 Dec;99(6):1936–1943. doi: 10.1083/jcb.99.6.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horwitz M. A., Silverstein S. C. Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J Clin Invest. 1980 Sep;66(3):441–450. doi: 10.1172/JCI109874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horwitz M. A. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 1983 Dec 1;158(6):2108–2126. doi: 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McDade J. E., Shepard C. C. Virulent to avirulent conversion of Legionnaires' disease bacterium (Legionella pneumophila)--its effect on isolation techniques. J Infect Dis. 1979 Jun;139(6):707–711. doi: 10.1093/infdis/139.6.707. [DOI] [PubMed] [Google Scholar]
  17. O'Donovan G. A., Neuhard J. Pyrimidine metabolism in microorganisms. Bacteriol Rev. 1970 Sep;34(3):278–343. doi: 10.1128/br.34.3.278-343.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Ristroph J. D., Hedlund K. W., Gowda S. Chemically defined medium for Legionella pneumophila growth. J Clin Microbiol. 1981 Jan;13(1):115–119. doi: 10.1128/jcm.13.1.115-119.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smith D. W., Hanawalt P. C. Macromolecular synthesis and thymineless death in Mycoplasma laidlawii B. J Bacteriol. 1968 Dec;96(6):2066–2076. doi: 10.1128/jb.96.6.2066-2076.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wachsman J. T., Kemp S., Kogg L. Thymineless death in Bacillus megaterium. J Bacteriol. 1964 May;87(5):1079–1086. doi: 10.1128/jb.87.5.1079-1086.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Warren W. J., Miller R. D. Growth of Legionnaires disease bacterium (Legionella pneumophila) in chemically defined medium. J Clin Microbiol. 1979 Jul;10(1):50–55. doi: 10.1128/jcm.10.1.50-55.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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