[en] Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.
Ahmer, B. M., M. G. Thomas, R. A. Larsen, and K. Postle. 1995. Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability. J. Bacteriol. 177:4742-4747.
Almiron, M., M. Martinez, N. Sanjuan, and R. A. Ugalde. 2001. Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence. Infect. Immun. 69:6225-6230.
Al-Tawfiq, J. A., K. R. Fortney, B. P. Katz, A. F. Hood, C. Elkins, and S. M. Spinola. 2000. An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection. J. Infect. Dis. 181:1049-1054.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
Andrews, S. C., A. K. Robinson, and F. Rodriguez-Quinones. 2003. Bacterial iron homeostasis. FEMS Microbiol. Rev. 27:215-237.
a. Ausubel, F. M., R. Brent, R. E. Kingston, D. E. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1991. Current protocols in molecular biology. Green Publishing Associates, New York, N.Y.
Bellaire, B. H., P. H. Elzer, C. L. Baldwin, and R M. Roop II. 2003. Production of the siderophore 2,3-dihydroxybenzoic acid is required for wild-type growth of Brucella abortus in the presence of erythritol under low-iron conditions in vitro. Infect. Immun. 71:2927-2932.
Bellaire, B. H., P. H. Elzer, C. L. Baldwin, and R. M. Roop II. 1999. The siderophore 2,3-dihydroxybenzoic acid is not required for virulence of Brucella abortus in BALB/c mice. Infect. Immun. 67:2615-2618.
Bellaire, B. H., P. H. Elzer, S. Hagius, J. Walker, C. L. Baldwin, and R. M. Roop II. 2003. Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. Infect. Immun. 71:4-1803.
Bellefontaine, A. F., C. E. Pierreux, P. Mertens, J. Vandenhaute, J. J. Letesson, and X. De Bolle. 2002. Plasticity of a transcriptional regulation network among alpha-proteobacteria is supported by the identification of CtrA targets in Brucella abortus. Mol. Microbiol. 43:945-960.
Braun, V. 1995. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol. Rev. 16:295-307.
Braun, V. 1998. Pumping iron through cell membranes. Science 282:2202-2203.
Braun, V. 1989. The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity. J. Bacteriol. 171:6387-6390.
Braun, V., S. Gaisser, C. Herrmann, K. Kampfenkel, H. Killmann, and I. Traub. 1996. Energy-coupled transport across the outer membrane of Escherichia coli: ExbB binds ExbD and TonB in vitro, and leucine 132 in the periplasmic region and aspartate 25 in the transmembrane region are important for ExbD activity. J. Bacteriol. 178:2836-2845.
Braun, V., and C. Herrmann. 1993. Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins. Mol. Microbiol. 8:261-268.
Caldon, C. E., and P. E. March. 2003. Function of the universally conserved bacterial GTPases. Curr. Opin. Microbiol. 6:135-139.
Caldon, C. E., P. Yoong, and P. E. March. 2001. Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function. Mol. Microbiol. 41:289-297.
Cruz-Vera, L. R., J. M. Galindo, and G. Guarneros. 2002. Transcriptional analysis of the gene encoding peptidyl-tRNA hydrolase in Escherichia coli. Microbiology 148:3457-3466.
Danese, I., A. Tibor, P. A. Denoel, V. Weynants, F. Godfroid, and J.-J. Letesson. 1996. Transposition mutagenesis of Brucella melitensis 16M with a mini-Tn5-Kmcat and evaluation of the reporter gene expression. Arch. Physiol. Biochem. 104:45.
de Lorenzo, V., M. Herrero, U. Jakubzik, and K. N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172:6568-6572.
Delrue, R. M., M. Martinez-Lorenzo, P. Lestrate, I. Danese, V. Bielarz, P. Mertens, X. De Bolle, A. Tibor, J. P. Gorvel, and J. J. Letesson. 2001. Identification of Brucella spp. genes involved in intracellular trafficking. Cell Microbiol. 3:487-497.
DelVecchio, V. G., V. Kapatral, P. Elzer, G. Patra, and C. V. Mujer. 2002. The genome of Brucella melitensis. Vet. Microbiol. 90:587-592.
De Voss, J. J., K. Rutter, B. G. Schroeder, H. Su, Y. Zhu, and C. E. Barry III. 2000. The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc. Natl. Acad. Sci. USA 97:1252-1257.
Elkins, C., P. A. Totten, B. Olsen, and C. E. Thomas. 1998. Role of the Haemophilus ducreyi Ton system in internalization of heme from hemoglobin. Infect. Immun. 66:151-160.
Elzer, P. H., R. W. Phillips, M. E. Kovach, K. M. Peterson, and R. M. Roop II. 1994. Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant. Infect. Immun. 62:4135-4139.
Enright, F. M., L. N. Araya, P. H. Elzer, G. E. Rowe, and A. J. Winter. 1990. Comparative histopathology in BALB/c mice infected with virulent and attenuated strains of Brucella abortus. Vet. Immunol. Immunopathol. 26:171-182.
Eskra, L., A. Canavessi, M. Carey, and G. Splitter. 2001. Brucella abortus genes identified following constitutive growth and macrophage infection. Infect. Immun. 69:7736-7742.
Fuqua, W. C. 1992. An improved chloramphenicol resistance gene cassette for site-directed marker replacement mutagenesis. BioTechniques 12:223-225.
Furrer, J. L., D. N. Sanders, I. G. Hook-Barnard, and M. A. McIntosh. 2002. Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter. Mol. Microbiol. 44:1225-1234.
Genco, C. A., and D. W. Dixon. 2001. Emerging strategies in microbial haem capture. Mol. Microbiol. 39:1-11.
Godfroid, F., B. Taminiau, I. Danese, P. Denoel, A. Tiber, V. Weynants, A. Cloeckaert, J. Godfroid, and J. J. Letesson. 1998. Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect. Immun. 66:5485-5493.
González Carreró, M. I., F. J. Sangari, J. Aguero, and J. M. Garcia Lobo. 2002. Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore. Microbiology 148:353-360.
Hancock, R. E., K. Hantke, and V. Braun. 1977. Iron transport in Escherichia coli K-12. 2,3-Dihydroxybenzoate-promoted iron uptake. Arch. Microbiol. 114:231-239.
Hantke, K. 1987. Ferrous iron transport mutants in Escherichia coli K12. FEM Microbiol. Lett. 44:53-57.
Hantke, K. 2003. Is the bacterial ferrous iron transporter FeoB a living fossil? Trends Microbiol. 11:192-195.
Higgs, P. I., P. S. Myers, and K. Postle. 1998. Interactions in the TonB-dependent energy transduction complex: ExbB and ExbD form homomultimers. J. Bacteriol. 180:6031-6038.
Janakiraman, A., and J. M. Slauch. 2000. The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium. Mol. Microbiol. 35:1146-1155.
Jiang, X., and C. L. Baldwin. 1993. Effects of cytokines on intracellular growth of Brucella abortus. Infect. Immun. 61:124-134.
Jiang, X., and C. L. Baldwin. 1993. Iron augments macrophage-mediated killing of Brucella abortus alone and in conjunction with interferon-gamma. Cell. Immunol. 148:397-407.
Karlsson, M., K. Hannavy, and C. F. Higgins. 1993. ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm. Mol. Microbiol. 8:389-396.
Kim, S., M. Watarai, Y. Kondo, J. Erdenebaatar, S. Makino, and T. Shirahata. 2003. Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells. Infect. Immun. 71:3020-3027.
Kovach, M. E., R. W. Phillips, P. H. Elzer, R. M. Roop I I, and K. M. Peterson. 1994. pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16:800-802.
Lambrecht, R. S., and M. T. Collins. 1993. Inability to detect mycobactin in mycobacteria-infected tissues suggests an alternative iron acquisition mechanism by mycobacteria in vivo. Microb. Pathog. 14:229-238.
Lawlor, K. M., P. A. Daskaleros, R. E. Robinson, and S. M. Payne. 1987. Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds. Infect. Immun. 55:594-599.
Leonard, B. A., I. Lopez-Goni, and C. L. Baldwin. 1997. Brucella abortus siderophore 2,3-dihydroxybenzoic acid protects brucellae from killing by macrophages. Vet. Res. 28:87-92.
Lopez-Goni, I., I. Moriyon, and J. B. Neilands. 1992. Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore. Infect. Immun. 60:4496-4503.
Marchler-Bauer, A., A. R. Panchenko, B. A. Shoemaker, P. A. Thiessen, L. Y. Geer, and S. H. Bryant. 2002. CDD: a database of conserved domain alignments with links to domain three-dimensional structure. Nucleic Acids Res. 30:281-283.
Marlovits, T. C., W. Haase, C. Herrmann, S. G. Aller, and V. M. Unger. 2002. The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria. Proc. Natl. Acad. Sci. USA 99:16243-16248.
Moeck, G. S., and J. W. Coulton. 1998. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol. Microbiol. 28:675-681.
Nakai, K., and P. Horton. 1999. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24:34-35.
Nakai, K., and M. Kanehisa. 1991. Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 11:95-110.
Nassif, X., M. C. Mazert, J. Mounier, and P. J. Sansonetti. 1987. Evaluation with an iuc::Tn10 mutant of the role of aerobactin production in the virulence of Shigella flexneri. Infect. Immun. 55:1963-1969.
Occhino, D. A., E. E. Wyckoff, D. P. Henderson, T. J. Wrona, and S. M. Payne. 1998. Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes. Mol. Microbiol. 29:1493-1507.
Pardon, P., and J. Marly. 1976. Resistance of Brucella abortus infected mice to intravenous or intraperitoneal Brucella reinfection. Ann. Immunol. 127C: 57-70.
Pizarro-Cerda, J., S. Meresse, R. G. Parton, G. van der Goot, A. Sola-Landa, I. Lopez-Goni, E. Moreno, and J. P. Gorvel. 1998. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect. Immun. 66:5711-5724.
Pope, C. D., W. O'Connell, and N. P. Cianciotto. 1996. Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection. Infect. Immun. 64:629-636.
Pradel, E., N. Guiso, F. D. Menozzi, and C. Locht. 2000. Bordetella pertussis TonB, a Bvg-independent virulence determinant. Infect. Immun. 68:1919-1927.
Reeves, S. A., A. G. Torres, and S. M. Payne. 2000. TonB is required for intracellular growth and virulence of Shigella dysenteriae. Infect. Immun. 68:6329-6336.
Rost, B., and C. Sander. 1993. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232:584-599.
Rost, B., C. Sander, and R. Schneider. 1994. PHD - an automatic mail server for protein secondary structure prediction. Comput. Appl. Biosci. 10:53-60.
Salgado, H., G. Moreno-Hagelsieb, T. F. Smith, and J. Collado-Vides. 2000. Operons in Escherichia coli: genomic analyses and predictions. Proc. Natl. Acad. Sci. USA 97:6652-6657.
Screen, J., E. Moya, I. S. Blagbrough, and A. W. Smith. 1995. Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid. FEMS Microbiol. Lett. 127:145-149.
Sigel, S. P., J. A. Stoebner, and S. M. Payne. 1985. Iron-vibriobactin transport system is not required for virulence of Vibrio cholerae. Infect. Immun. 47:360-362.
Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 10:783-791.
Skare, J. T., and K. Postle. 1991. Evidence for a TonB-dependent energy transduction complex in Escherichia coli. Mol. Microbiol. 5:2883-2890.
Stabel, J. R., and T. J. Stabel. 1995. Immortalization and characterization of bovine peritoneal macrophages transfected with SV40 plasmid DNA. Vet. Immunol. Immunopathol. 45:211-220.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.
Tibor-de Walque, A. 1999. Caracterisation de trois protéines de la membrane externe de Brucella abortus. Ph.D. thesis. University of Namur, Namur, Belgium.
Traub, I., S. Gaisser, and V. Braun. 1993. Activity domains of the TonB protein. Mol. Microbiol. 8:409-423.
Tsolis, R. M., A. J. Baumler, F. Heffron, and I. Stojiljkovic. 1996. Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella typhimurium in the mouse. Infect. Immun. 64:4549-4556.
Turner, P. C., C. E. Thomas, C. Elkins, S. Clary, and P. F. Sparling. 1998. Neisseria gonorrhoeae heme biosynthetic mutants utilize heme and hemoglobin as a heme source but fail to grow within epithelial cells. Infect. Immun. 66:5215-5223.
Verger, J. M., M. Grayon, E. Chaslus-Dancla, M. Meurisse, and J. P. Lafont. 1993. Conjugative transfer and in vitro/in vivo stability of the broad-host-range IncP R751 plasmid in Brucella spp. Plasmid 29:142-146.
Wandersman, C., and I. Stojiljkovic. 2000. Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores. Curr. Opin. Microbiol. 3:215-220.
Zhao, Q., and K. Poole. 2000. A second tonB gene in Pseudomonas aeruginosa is linked to the exbB and exbD genes. FEMS Microbiol. Lett. 184:127-132.