Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(24):7265–7275. doi: 10.1128/jb.178.24.7265-7275.1996

The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors.

K LeVier 1, M L Guerinot 1
PMCID: PMC178643  PMID: 8955412

Abstract

Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Actis L. A., Tolmasky M. E., Farrell D. H., Crosa J. H. Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system. J Biol Chem. 1988 Feb 25;263(6):2853–2860. [PubMed] [Google Scholar]
  2. Adams T. H., McClung C. R., Chelm B. K. Physical organization of the Bradyrhizobium japonicum nitrogenase gene region. J Bacteriol. 1984 Sep;159(3):857–862. doi: 10.1128/jb.159.3.857-862.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Ankenbauer R. G., Quan H. N. FptA, the Fe(III)-pyochelin receptor of Pseudomonas aeruginosa: a phenolate siderophore receptor homologous to hydroxamate siderophore receptors. J Bacteriol. 1994 Jan;176(2):307–319. doi: 10.1128/jb.176.2.307-319.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bal A. K., Shantharam S., Verma D. P. Changes in the outer cell wall of Rhizobium during development of root nodule symbiosis in soybean. Can J Microbiol. 1980 Sep;26(9):1096–1103. doi: 10.1139/m80-182. [DOI] [PubMed] [Google Scholar]
  6. Bergman T., Jörnvall H. Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis. Eur J Biochem. 1987 Nov 16;169(1):9–12. doi: 10.1111/j.1432-1033.1987.tb13573.x. [DOI] [PubMed] [Google Scholar]
  7. Biswas G. D., Sparling P. F. Characterization of lbpA, the structural gene for a lactoferrin receptor in Neisseria gonorrhoeae. Infect Immun. 1995 Aug;63(8):2958–2967. doi: 10.1128/iai.63.8.2958-2967.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bitter W., Marugg J. D., de Weger L. A., Tommassen J., Weisbeek P. J. The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein. Mol Microbiol. 1991 Mar;5(3):647–655. doi: 10.1111/j.1365-2958.1991.tb00736.x. [DOI] [PubMed] [Google Scholar]
  9. Braun V. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol Rev. 1995 Jul;16(4):295–307. doi: 10.1111/j.1574-6976.1995.tb00177.x. [DOI] [PubMed] [Google Scholar]
  10. Butterton J. R., Stoebner J. A., Payne S. M., Calderwood S. B. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992 Jun;174(11):3729–3738. doi: 10.1128/jb.174.11.3729-3738.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bäumler A. J., Hantke K. Ferrioxamine uptake in Yersinia enterocolitica: characterization of the receptor protein FoxA. Mol Microbiol. 1992 May;6(10):1309–1321. doi: 10.1111/j.1365-2958.1992.tb00852.x. [DOI] [PubMed] [Google Scholar]
  12. Carson K. C., Holliday S., Glenn A. R., Dilworth M. J. Siderophore and organic acid production in root nodule bacteria. Arch Microbiol. 1992;157(3):264–271. doi: 10.1007/BF00245160. [DOI] [PubMed] [Google Scholar]
  13. Coleman J., Green P. J., Inouye M. The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes. Cell. 1984 Jun;37(2):429–436. doi: 10.1016/0092-8674(84)90373-8. [DOI] [PubMed] [Google Scholar]
  14. Cornelissen C. N., Biswas G. D., Tsai J., Paruchuri D. K., Thompson S. A., Sparling P. F. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors. J Bacteriol. 1992 Sep;174(18):5788–5797. doi: 10.1128/jb.174.18.5788-5797.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Coulton J. W., Mason P., Cameron D. R., Carmel G., Jean R., Rode H. N. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):181–192. doi: 10.1128/jb.165.1.181-192.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Daban M., Medrano A., Querol E. Cloning, sequencing and expression of the transferrin-binding protein 1 gene from Actinobacillus pleuropneumoniae. Biochem J. 1996 Apr 1;315(Pt 1):257–264. doi: 10.1042/bj3150257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dean C. R., Poole K. Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa. J Bacteriol. 1993 Jan;175(2):317–324. doi: 10.1128/jb.175.2.317-324.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Expert D., Enard C., Masclaux C. The role of iron in plant host-pathogen interactions. Trends Microbiol. 1996 Jun;4(6):232–237. doi: 10.1016/0966-842X(96)10038-X. [DOI] [PubMed] [Google Scholar]
  19. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  20. Fetherston J. D., Lillard J. W., Jr, Perry R. D. Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore. J Bacteriol. 1995 Apr;177(7):1824–1833. doi: 10.1128/jb.177.7.1824-1833.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fischer H. M. Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev. 1994 Sep;58(3):352–386. doi: 10.1128/mr.58.3.352-386.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gill P. R., Jr, Neilands J. B. Cloning a genomic region required for a high-affinity iron-uptake system in Rhizobium meliloti 1021. Mol Microbiol. 1989 Sep;3(9):1183–1189. doi: 10.1111/j.1365-2958.1989.tb00268.x. [DOI] [PubMed] [Google Scholar]
  23. Gish W., States D. J. Identification of protein coding regions by database similarity search. Nat Genet. 1993 Mar;3(3):266–272. doi: 10.1038/ng0393-266. [DOI] [PubMed] [Google Scholar]
  24. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  25. Goldberg M. B., Boyko S. A., Butterton J. R., Stoebner J. A., Payne S. M., Calderwood S. B. Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins. Mol Microbiol. 1992 Aug;6(16):2407–2418. doi: 10.1111/j.1365-2958.1992.tb01415.x. [DOI] [PubMed] [Google Scholar]
  26. Gonzalez G. C., Yu R. H., Rosteck P. R., Jr, Schryvers A. B. Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes. Microbiology. 1995 Oct;141(Pt 10):2405–2416. doi: 10.1099/13500872-141-10-2405. [DOI] [PubMed] [Google Scholar]
  27. Guerinot M. L., Chelm B. K. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1837–1841. doi: 10.1073/pnas.83.6.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Guerinot M. L., Meidl E. J., Plessner O. Citrate as a siderophore in Bradyrhizobium japonicum. J Bacteriol. 1990 Jun;172(6):3298–3303. doi: 10.1128/jb.172.6.3298-3303.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Guerinot M. L. Microbial iron transport. Annu Rev Microbiol. 1994;48:743–772. doi: 10.1146/annurev.mi.48.100194.003523. [DOI] [PubMed] [Google Scholar]
  30. Hartz D., McPheeters D. S., Traut R., Gold L. Extension inhibition analysis of translation initiation complexes. Methods Enzymol. 1988;164:419–425. doi: 10.1016/s0076-6879(88)64058-4. [DOI] [PubMed] [Google Scholar]
  31. Heller K. J., Kadner R. J., Günther K. Suppression of the btuB451 mutation by mutations in the tonB gene suggests a direct interaction between TonB and TonB-dependent receptor proteins in the outer membrane of Escherichia coli. Gene. 1988 Apr 15;64(1):147–153. doi: 10.1016/0378-1119(88)90488-x. [DOI] [PubMed] [Google Scholar]
  32. Heller K., Kadner R. J. Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli. J Bacteriol. 1985 Mar;161(3):904–908. doi: 10.1128/jb.161.3.904-908.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Killmann H., Benz R., Braun V. Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli. EMBO J. 1993 Aug;12(8):3007–3016. doi: 10.1002/j.1460-2075.1993.tb05969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Koebnik R., Hantke K., Braun V. The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co-evolution of receptor structure and substrate specificity. Mol Microbiol. 1993 Feb;7(3):383–393. doi: 10.1111/j.1365-2958.1993.tb01130.x. [DOI] [PubMed] [Google Scholar]
  35. Koster M., van de Vossenberg J., Leong J., Weisbeek P. J. Identification and characterization of the pupB gene encoding an inducible ferric-pseudobactin receptor of Pseudomonas putida WCS358. Mol Microbiol. 1993 May;8(3):591–601. doi: 10.1111/j.1365-2958.1993.tb01603.x. [DOI] [PubMed] [Google Scholar]
  36. Kühn S., Braun V., Köster W. Ferric rhizoferrin uptake into Morganella morganii: characterization of genes involved in the uptake of a polyhydroxycarboxylate siderophore. J Bacteriol. 1996 Jan;178(2):496–504. doi: 10.1128/jb.178.2.496-504.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lisser S., Margalit H. Compilation of E. coli mRNA promoter sequences. Nucleic Acids Res. 1993 Apr 11;21(7):1507–1516. doi: 10.1093/nar/21.7.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Loosmore S. M., Yang Y. P., Coleman D. C., Shortreed J. M., England D. M., Harkness R. E., Chong P. S., Klein M. H. Cloning and expression of the Haemophilus influenzae transferrin receptor genes. Mol Microbiol. 1996 Feb;19(3):575–586. doi: 10.1046/j.1365-2958.1996.406943.x. [DOI] [PubMed] [Google Scholar]
  39. Lundrigan M. D., Kadner R. J. Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. J Biol Chem. 1986 Aug 15;261(23):10797–10801. [PubMed] [Google Scholar]
  40. Morris J., Donnelly D. F., O'Neill E., McConnell F., O'Gara F. Nucleotide sequence analysis and potential environmental distribution of a ferric pseudobactin receptor gene of Pseudomonas sp. strain M114. Mol Gen Genet. 1994 Jan;242(1):9–16. doi: 10.1007/BF00277342. [DOI] [PubMed] [Google Scholar]
  41. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  42. Nau C. D., Konisky J. Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene. J Bacteriol. 1989 Feb;171(2):1041–1047. doi: 10.1128/jb.171.2.1041-1047.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Neilands J. B. Microbial envelope proteins related to iron. Annu Rev Microbiol. 1982;36:285–309. doi: 10.1146/annurev.mi.36.100182.001441. [DOI] [PubMed] [Google Scholar]
  44. Neilands J. B. Siderophores: structure and function of microbial iron transport compounds. J Biol Chem. 1995 Nov 10;270(45):26723–26726. doi: 10.1074/jbc.270.45.26723. [DOI] [PubMed] [Google Scholar]
  45. Nikaido H., Saier M. H., Jr Transport proteins in bacteria: common themes in their design. Science. 1992 Nov 6;258(5084):936–942. doi: 10.1126/science.1279804. [DOI] [PubMed] [Google Scholar]
  46. Page K. M., Connolly E. L., Guerinot M. L. Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene. J Bacteriol. 1994 Mar;176(5):1535–1538. doi: 10.1128/jb.176.5.1535-1538.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Plessner O., Klapatch T., Guerinot M. L. Siderophore Utilization by Bradyrhizobium japonicum. Appl Environ Microbiol. 1993 May;59(5):1688–1690. doi: 10.1128/aem.59.5.1688-1690.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Poole K., Neshat S., Krebes K., Heinrichs D. E. Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J Bacteriol. 1993 Aug;175(15):4597–4604. doi: 10.1128/jb.175.15.4597-4604.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pressler U., Staudenmaier H., Zimmermann L., Braun V. Genetics of the iron dicitrate transport system of Escherichia coli. J Bacteriol. 1988 Jun;170(6):2716–2724. doi: 10.1128/jb.170.6.2716-2724.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rakin A., Saken E., Harmsen D., Heesemann J. The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function. Mol Microbiol. 1994 Jul;13(2):253–263. doi: 10.1111/j.1365-2958.1994.tb00420.x. [DOI] [PubMed] [Google Scholar]
  51. Ramseier T. M., Göttfert M. Codon usage and G + C content in Bradyrhizobium japonicum genes are not uniform. Arch Microbiol. 1991;156(4):270–276. doi: 10.1007/BF00262997. [DOI] [PubMed] [Google Scholar]
  52. Reigh G., O'Connell M. Siderophore-mediated iron transport correlates with the presence of specific iron-regulated proteins in the outer membrane of Rhizobium meliloti. J Bacteriol. 1993 Jan;175(1):94–102. doi: 10.1128/jb.175.1.94-102.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rutz J. M., Liu J., Lyons J. A., Goranson J., Armstrong S. K., McIntosh M. A., Feix J. B., Klebba P. E. Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane. Science. 1992 Oct 16;258(5081):471–475. doi: 10.1126/science.1411544. [DOI] [PubMed] [Google Scholar]
  54. Sauer M., Hantke K., Braun V. Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants. Mol Microbiol. 1990 Mar;4(3):427–437. doi: 10.1111/j.1365-2958.1990.tb00609.x. [DOI] [PubMed] [Google Scholar]
  55. Schöffler H., Braun V. Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane. Mol Gen Genet. 1989 Jun;217(2-3):378–383. doi: 10.1007/BF02464907. [DOI] [PubMed] [Google Scholar]
  56. Scott-Craig J. S., Guerinot M. L., Chelm B. K. Isolation of Bradyrhizobium japonicum DNA sequences that are transcribed at high levels in bacteroids. Mol Gen Genet. 1991 Sep;228(3):356–360. doi: 10.1007/BF00260627. [DOI] [PubMed] [Google Scholar]
  57. Sen K., Nikaido H. Lipopolysaccharide structure required for in vitro trimerization of Escherichia coli OmpF porin. J Bacteriol. 1991 Jan;173(2):926–928. doi: 10.1128/jb.173.2.926-928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  59. Struyvé M., Moons M., Tommassen J. Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein. J Mol Biol. 1991 Mar 5;218(1):141–148. doi: 10.1016/0022-2836(91)90880-f. [DOI] [PubMed] [Google Scholar]
  60. Talian J. C., Olmsted J. B., Goldman R. D. A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture. J Cell Biol. 1983 Oct;97(4):1277–1282. doi: 10.1083/jcb.97.4.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Touati D., Jacques M., Tardat B., Bouchard L., Despied S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase. J Bacteriol. 1995 May;177(9):2305–2314. doi: 10.1128/jb.177.9.2305-2314.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. Wei B. Y., Bradbeer C., Kadner R. J. Conserved structural and regulatory regions in the Salmonella typhimurium btuB gene for the outer membrane vitamin B12 transport protein. Res Microbiol. 1992 Jun;143(5):459–466. doi: 10.1016/0923-2508(92)90091-2. [DOI] [PubMed] [Google Scholar]
  64. Weidenhaupt M., Rossi P., Beck C., Fischer H. M., Hennecke H. Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes: fixA, fixB and etfS, etfL. Arch Microbiol. 1996 Mar;165(3):169–178. doi: 10.1007/BF01692858. [DOI] [PubMed] [Google Scholar]
  65. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. 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]
  68. von Heijne G. The signal peptide. J Membr Biol. 1990 May;115(3):195–201. doi: 10.1007/BF01868635. [DOI] [PubMed] [Google Scholar]

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

RESOURCES