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. 1997 Feb;65(2):571–578. doi: 10.1128/iai.65.2.571-578.1997

Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

C R Roy 1, R R Isberg 1
PMCID: PMC176098  PMID: 9009315

Abstract

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 and 73 amino acids and a carboxyl-terminal cytoplasmic domain of 122 amino acids. Protein fractionation studies were consistent with DotA residing in the inner membrane. An alkaline phosphatase fusion located 9 amino acids upstream from the C terminus of DotA still retained function and was able to restore intracellular growth when harbored by two L. pneumophila dotA mutants. A hybrid protein from which the carboxyl-terminal 48 amino acids of DotA were deleted was unable to complement the intracellular growth defect in the dotA mutants, indicating that this cytoplasmic region is required for function.

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Selected References

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  1. Albano M. A., Arroyo J., Eisenstein B. I., Engleberg N. C. PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA. Mol Microbiol. 1992 Jul;6(13):1829–1839. doi: 10.1111/j.1365-2958.1992.tb01355.x. [DOI] [PubMed] [Google Scholar]
  2. Berger K. H., Isberg R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993 Jan;7(1):7–19. doi: 10.1111/j.1365-2958.1993.tb01092.x. [DOI] [PubMed] [Google Scholar]
  3. Berger K. H., Merriam J. J., Isberg R. R. Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol. 1994 Nov;14(4):809–822. doi: 10.1111/j.1365-2958.1994.tb01317.x. [DOI] [PubMed] [Google Scholar]
  4. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brand B. C., Sadosky A. B., Shuman H. A. The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol Microbiol. 1994 Nov;14(4):797–808. doi: 10.1111/j.1365-2958.1994.tb01316.x. [DOI] [PubMed] [Google Scholar]
  6. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  7. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  8. Claros M. G., von Heijne G. TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci. 1994 Dec;10(6):685–686. doi: 10.1093/bioinformatics/10.6.685. [DOI] [PubMed] [Google Scholar]
  9. Dassa E. Sequence-function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli. Mol Microbiol. 1993 Jan;7(1):39–47. doi: 10.1111/j.1365-2958.1993.tb01095.x. [DOI] [PubMed] [Google Scholar]
  10. Davidson A. L., Shuman H. A., Nikaido H. Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2360–2364. doi: 10.1073/pnas.89.6.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol. 1986 Jul;167(1):66–72. doi: 10.1128/jb.167.1.66-72.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fraser D. W., Tsai T. R., Orenstein W., Parkin W. E., Beecham H. J., Sharrar R. G., Harris J., Mallison G. F., Martin S. M., McDade J. E. Legionnaires' disease: description of an epidemic of pneumonia. N Engl J Med. 1977 Dec 1;297(22):1189–1197. doi: 10.1056/NEJM197712012972201. [DOI] [PubMed] [Google Scholar]
  14. Froshauer S., Beckwith J. The nucleotide sequence of the gene for malF protein, an inner membrane component of the maltose transport system of Escherichia coli. Repeated DNA sequences are found in the malE-malF intercistronic region. J Biol Chem. 1984 Sep 10;259(17):10896–10903. [PubMed] [Google Scholar]
  15. 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]
  16. Higgins C. F. The ABC of channel regulation. Cell. 1995 Sep 8;82(5):693–696. doi: 10.1016/0092-8674(95)90465-4. [DOI] [PubMed] [Google Scholar]
  17. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hor L. I., Shuman H. A. Genetic analysis of periplasmic binding protein dependent transport in Escherichia coli. Each lobe of maltose-binding protein interacts with a different subunit of the MalFGK2 membrane transport complex. J Mol Biol. 1993 Oct 20;233(4):659–670. doi: 10.1006/jmbi.1993.1543. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Ishidate K., Creeger E. S., Zrike J., Deb S., Glauner B., MacAlister T. J., Rothfield L. I. Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope. J Biol Chem. 1986 Jan 5;261(1):428–443. [PubMed] [Google Scholar]
  25. Ito K. Identification of the secY (prlA) gene product involved in protein export in Escherichia coli. Mol Gen Genet. 1984;197(2):204–208. doi: 10.1007/BF00330964. [DOI] [PubMed] [Google Scholar]
  26. Kaufmann A. F., McDade J. E., Patton C. M., Bennett J. V., Skaliy P., Feeley J. C., Anderson D. C., Potter M. E., Newhouse V. F., Gregg M. B. Pontiac fever: isolation of the etiologic agent (Legionella pneumophilia) and demonstration of its mode of transmission. Am J Epidemiol. 1981 Sep;114(3):337–347. doi: 10.1093/oxfordjournals.aje.a113200. [DOI] [PubMed] [Google Scholar]
  27. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Manoil C. Analysis of protein localization by use of gene fusions with complementary properties. J Bacteriol. 1990 Feb;172(2):1035–1042. doi: 10.1128/jb.172.2.1035-1042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  31. Marra A., Blander S. J., Horwitz M. A., Shuman H. A. Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9607–9611. doi: 10.1073/pnas.89.20.9607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikaido H. Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS Lett. 1994 Jun 6;346(1):55–58. doi: 10.1016/0014-5793(94)00315-7. [DOI] [PubMed] [Google Scholar]
  33. Pearlman E., Jiwa A. H., Engleberg N. C., Eisenstein B. I. Growth of Legionella pneumophila in a human macrophage-like (U937) cell line. Microb Pathog. 1988 Aug;5(2):87–95. doi: 10.1016/0882-4010(88)90011-3. [DOI] [PubMed] [Google Scholar]
  34. Plano G. V., Barve S. S., Straley S. C. LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol. 1991 Nov;173(22):7293–7303. doi: 10.1128/jb.173.22.7293-7303.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Swanson M. S., Isberg R. R. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun. 1995 Sep;63(9):3609–3620. doi: 10.1128/iai.63.9.3609-3620.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]

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