Abstract
Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.
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- 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]
- 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]
- Blander S. J., Breiman R. F., Horwitz M. A. A live avirulent mutant Legionella pneumophila vaccine induces protective immunity against lethal aerosol challenge. J Clin Invest. 1989 Mar;83(3):810–815. doi: 10.1172/JCI113962. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Carpenter P. B., Ordal G. W. Bacillus subtilis FlhA: a flagellar protein related to a new family of signal-transducing receptors. Mol Microbiol. 1993 Mar;7(5):735–743. doi: 10.1111/j.1365-2958.1993.tb01164.x. [DOI] [PubMed] [Google Scholar]
- Clemens D. L., Horwitz M. A. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med. 1995 Jan 1;181(1):257–270. doi: 10.1084/jem.181.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clemens D. L., Horwitz M. A. Hypoexpression of major histocompatibility complex molecules on Legionella pneumophila phagosomes and phagolysosomes. Infect Immun. 1993 Jul;61(7):2803–2812. doi: 10.1128/iai.61.7.2803-2812.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dreyfus G., Williams A. W., Kawagishi I., Macnab R. M. Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J Bacteriol. 1993 May;175(10):3131–3138. doi: 10.1128/jb.175.10.3131-3138.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eichelberg K., Ginocchio C. C., Galán J. E. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol. 1994 Aug;176(15):4501–4510. doi: 10.1128/jb.176.15.4501-4510.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elliott J. A., Johnson W. Immunological and biochemical relationships among flagella isolated from Legionella pneumophila serogroups 1, 2, and 3. Infect Immun. 1981 Aug;33(2):602–610. doi: 10.1128/iai.33.2.602-610.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Fields B. S., Nerad T. A., Sawyer T. K., King C. H., Barbaree J. M., Martin W. T., Morrill W. E., Sanden G. N. Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis. J Protozool. 1990 Nov-Dec;37(6):581–583. doi: 10.1111/j.1550-7408.1990.tb01269.x. [DOI] [PubMed] [Google Scholar]
- Hermant D., Ménard R., Arricau N., Parsot C., Popoff M. Y. Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells. Mol Microbiol. 1995 Aug;17(4):781–789. doi: 10.1111/j.1365-2958.1995.mmi_17040781.x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Horwitz M. A., Silverstein S. C. Interaction of the legionnaires' disease bacterium (Legionella pneumophila) with human phagocytes. II. Antibody promotes binding of L. pneumophila to monocytes but does not inhibit intracellular multiplication. J Exp Med. 1981 Feb 1;153(2):398–406. doi: 10.1084/jem.153.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Leong J. M., Fournier R. S., Isberg R. R. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein. EMBO J. 1990 Jun;9(6):1979–1989. doi: 10.1002/j.1460-2075.1990.tb08326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ménard R., Sansonetti P. J., Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol. 1993 Sep;175(18):5899–5906. doi: 10.1128/jb.175.18.5899-5906.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Müller A., Hacker J., Brand B. C. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. Infect Immun. 1996 Dec;64(12):4900–4906. doi: 10.1128/iai.64.12.4900-4906.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Persson C., Nordfelth R., Holmström A., Håkansson S., Rosqvist R., Wolf-Watz H. Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol Microbiol. 1995 Oct;18(1):135–150. doi: 10.1111/j.1365-2958.1995.mmi_18010135.x. [DOI] [PubMed] [Google Scholar]
- Pruckler J. M., Benson R. F., Moyenuddin M., Martin W. T., Fields B. S. Association of flagellum expression and intracellular growth of Legionella pneumophila. Infect Immun. 1995 Dec;63(12):4928–4932. doi: 10.1128/iai.63.12.4928-4932.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosqvist R., Håkansson S., Forsberg A., Wolf-Watz H. Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae. EMBO J. 1995 Sep 1;14(17):4187–4195. doi: 10.1002/j.1460-2075.1995.tb00092.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rowbotham T. J. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol. 1980 Dec;33(12):1179–1183. doi: 10.1136/jcp.33.12.1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Salmond G. P., Reeves P. J. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci. 1993 Jan;18(1):7–12. doi: 10.1016/0968-0004(93)90080-7. [DOI] [PubMed] [Google Scholar]
- Simon R., O'Connell M., Labes M., Pühler A. Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria. Methods Enzymol. 1986;118:640–659. doi: 10.1016/0076-6879(86)18106-7. [DOI] [PubMed] [Google Scholar]
- Sory M. P., Boland A., Lambermont I., Cornelis G. R. Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):11998–12002. doi: 10.1073/pnas.92.26.11998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Swanson M. S., Isberg R. R. Identification of Legionella pneumophila mutants that have aberrant intracellular fates. Infect Immun. 1996 Jul;64(7):2585–2594. doi: 10.1128/iai.64.7.2585-2594.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Venkatesan M. M., Buysse J. M., Oaks E. V. Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus. J Bacteriol. 1992 Mar;174(6):1990–2001. doi: 10.1128/jb.174.6.1990-2001.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vogler A. P., Homma M., Irikura V. M., Macnab R. M. Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits. J Bacteriol. 1991 Jun;173(11):3564–3572. doi: 10.1128/jb.173.11.3564-3572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woestyn S., Allaoui A., Wattiau P., Cornelis G. R. YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol. 1994 Mar;176(6):1561–1569. doi: 10.1128/jb.176.6.1561-1569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ying C. W., Scoffone F., Albertini A. M., Galizzi A., Ordal G. W. Properties of the Bacillus subtilis chemotaxis protein CheF, a homolog of the Salmonella typhimurium flagellar protein FliJ. J Bacteriol. 1991 Jun;173(11):3584–3586. doi: 10.1128/jb.173.11.3584-3586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zierler M. K., Galán J. E. Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ. Infect Immun. 1995 Oct;63(10):4024–4028. doi: 10.1128/iai.63.10.4024-4028.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- d'Enfert C., Reyss I., Wandersman C., Pugsley A. P. Protein secretion by gram-negative bacteria. Characterization of two membrane proteins required for pullulanase secretion by Escherichia coli K-12. J Biol Chem. 1989 Oct 15;264(29):17462–17468. [PubMed] [Google Scholar]