Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 May;65(5):1659–1668. doi: 10.1128/iai.65.5.1659-1668.1997

Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis.

S W Bearden 1, J D Fetherston 1, R D Perry 1
PMCID: PMC175193  PMID: 9125544

Abstract

We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.

Full Text

The Full Text of this article is available as a PDF (999.2 KB).

Selected References

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

  1. 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]
  2. BURROWS T. W., JACKSON S. The pigmentation of Pasteurella pestis on a defined medium containing haemin. Br J Exp Pathol. 1956 Dec;37(6):570–576. [PMC free article] [PubMed] [Google Scholar]
  3. BURROWS T. W., JACKSON S. The virulence-enhancing effect of iron on nonpigmented mutants of virulent strains of Pasteurella pestis. Br J Exp Pathol. 1956 Dec;37(6):577–583. [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brubaker R. R. Factors promoting acute and chronic diseases caused by yersiniae. Clin Microbiol Rev. 1991 Jul;4(3):309–324. doi: 10.1128/cmr.4.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brubaker R. R. Mutation rate to nonpigmentation in Pasteurella pestis. J Bacteriol. 1969 Jun;98(3):1404–1406. doi: 10.1128/jb.98.3.1404-1406.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carniel E., Guiyoule A., Guilvout I., Mercereau-Puijalon O. Molecular cloning, iron-regulation and mutagenesis of the irp2 gene encoding HMWP2, a protein specific for the highly pathogenic Yersinia. Mol Microbiol. 1992 Feb;6(3):379–388. doi: 10.1111/j.1365-2958.1992.tb01481.x. [DOI] [PubMed] [Google Scholar]
  8. Carniel E., Mazigh D., Mollaret H. H. Expression of iron-regulated proteins in Yersinia species and their relation to virulence. Infect Immun. 1987 Jan;55(1):277–280. doi: 10.1128/iai.55.1.277-280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carniel E., Mercereau-Puijalon O., Bonnefoy S. The gene coding for the 190,000-dalton iron-regulated protein of Yersinia species is present only in the highly pathogenic strains. Infect Immun. 1989 Apr;57(4):1211–1217. doi: 10.1128/iai.57.4.1211-1217.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chambers C. E., McIntyre D. D., Mouck M., Sokol P. A. Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica. Biometals. 1996 Apr;9(2):157–167. doi: 10.1007/BF00144621. [DOI] [PubMed] [Google Scholar]
  11. Chambers C. E., Sokol P. A. Comparison of siderophore production and utilization in pathogenic and environmental isolates of Yersinia enterocolitica. J Clin Microbiol. 1994 Jan;32(1):32–39. doi: 10.1128/jcm.32.1.32-39.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cho H., Cronan J. E., Jr Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme. J Biol Chem. 1993 May 5;268(13):9238–9245. [PubMed] [Google Scholar]
  13. Cosmina P., Rodriguez F., de Ferra F., Grandi G., Perego M., Venema G., van Sinderen D. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol Microbiol. 1993 May;8(5):821–831. doi: 10.1111/j.1365-2958.1993.tb01629.x. [DOI] [PubMed] [Google Scholar]
  14. Crichton R. R., Charloteaux-Wauters M. Iron transport and storage. Eur J Biochem. 1987 May 4;164(3):485–506. doi: 10.1111/j.1432-1033.1987.tb11155.x. [DOI] [PubMed] [Google Scholar]
  15. Donnenberg M. S., Kaper J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310–4317. doi: 10.1128/iai.59.12.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Farrell D. H., Mikesell P., Actis L. A., Crosa J. H. A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron. Gene. 1990 Jan 31;86(1):45–51. doi: 10.1016/0378-1119(90)90112-5. [DOI] [PubMed] [Google Scholar]
  17. Fetherston J. D., Bearden S. W., Perry R. D. YbtA, an AraC-type regulator of the Yersinia pestis pesticin/yersiniabactin receptor. Mol Microbiol. 1996 Oct;22(2):315–325. doi: 10.1046/j.1365-2958.1996.00118.x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Fetherston J. D., Perry R. D. The pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2. Mol Microbiol. 1994 Aug;13(4):697–708. doi: 10.1111/j.1365-2958.1994.tb00463.x. [DOI] [PubMed] [Google Scholar]
  20. Fetherston J. D., Schuetze P., Perry R. D. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol Microbiol. 1992 Sep;6(18):2693–2704. doi: 10.1111/j.1365-2958.1992.tb01446.x. [DOI] [PubMed] [Google Scholar]
  21. Gensberg K., Hughes K., Smith A. W. Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa. J Gen Microbiol. 1992 Nov;138(11):2381–2387. doi: 10.1099/00221287-138-11-2381. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Guilvout I., Mercereau-Puijalon O., Bonnefoy S., Pugsley A. P., Carniel E. High-molecular-weight protein 2 of Yersinia enterocolitica is homologous to AngR of Vibrio anguillarum and belongs to a family of proteins involved in nonribosomal peptide synthesis. J Bacteriol. 1993 Sep;175(17):5488–5504. doi: 10.1128/jb.175.17.5488-5504.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. HIGUCHI K., SMITH J. L. Studies on the nutrition and physiology of Pasteurella pestis. VI. A differential plating medium for the estimation of the mutation rate to avirulence. J Bacteriol. 1961 Apr;81:605–608. doi: 10.1128/jb.81.4.605-608.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Haag H., Hantke K., Drechsel H., Stojiljkovic I., Jung G., Zähner H. Purification of yersiniabactin: a siderophore and possible virulence factor of Yersinia enterocolitica. J Gen Microbiol. 1993 Sep;139(9):2159–2165. doi: 10.1099/00221287-139-9-2159. [DOI] [PubMed] [Google Scholar]
  26. Heinrichs D. E., Poole K. Cloning and sequence analysis of a gene (pchR) encoding an AraC family activator of pyochelin and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. J Bacteriol. 1993 Sep;175(18):5882–5889. doi: 10.1128/jb.175.18.5882-5889.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Humphreys G. O., Willshaw G. A., Anderson E. S. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. doi: 10.1016/0005-2787(75)90318-4. [DOI] [PubMed] [Google Scholar]
  29. Krätzschmar J., Krause M., Marahiel M. A. Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J Bacteriol. 1989 Oct;171(10):5422–5429. doi: 10.1128/jb.171.10.5422-5429.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kutyrev V. V., Filippov A. A., Oparina O. S., Protsenko O. A. Analysis of Yersinia pestis chromosomal determinants Pgm+ and Psts associated with virulence. Microb Pathog. 1992 Mar;12(3):177–186. doi: 10.1016/0882-4010(92)90051-o. [DOI] [PubMed] [Google Scholar]
  31. Lindler L. E., Klempner M. S., Straley S. C. Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague. Infect Immun. 1990 Aug;58(8):2569–2577. doi: 10.1128/iai.58.8.2569-2577.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lindler L. E., Tall B. D. Yersinia pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages. Mol Microbiol. 1993 Apr;8(2):311–324. doi: 10.1111/j.1365-2958.1993.tb01575.x. [DOI] [PubMed] [Google Scholar]
  33. Litwin C. M., Calderwood S. B. Role of iron in regulation of virulence genes. Clin Microbiol Rev. 1993 Apr;6(2):137–149. doi: 10.1128/cmr.6.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Merriman T. R., Merriman M. E., Lamont I. L. Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases. J Bacteriol. 1995 Jan;177(1):252–258. doi: 10.1128/jb.177.1.252-258.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mietzner T. A., Morse S. A. The role of iron-binding proteins in the survival of pathogenic bacteria. Annu Rev Nutr. 1994;14:471–493. doi: 10.1146/annurev.nu.14.070194.002351. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Ochs M., Veitinger S., Kim I., Welz D., Angerer A., Braun V. Regulation of citrate-dependent iron transport of Escherichia coli: fecR is required for transcription activation by FecI. Mol Microbiol. 1995 Jan;15(1):119–132. doi: 10.1111/j.1365-2958.1995.tb02226.x. [DOI] [PubMed] [Google Scholar]
  38. Pendrak M. L., Perry R. D. Proteins essential for expression of the Hms+ phenotype of Yersinia pestis. Mol Microbiol. 1993 May;8(5):857–864. doi: 10.1111/j.1365-2958.1993.tb01632.x. [DOI] [PubMed] [Google Scholar]
  39. Perry R. D. Acquisition and storage of inorganic iron and hemin by the yersiniae. Trends Microbiol. 1993 Jul;1(4):142–147. doi: 10.1016/0966-842x(93)90129-f. [DOI] [PubMed] [Google Scholar]
  40. Perry R. D., Brubaker R. R. Accumulation of iron by yersiniae. J Bacteriol. 1979 Mar;137(3):1290–1298. doi: 10.1128/jb.137.3.1290-1298.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Perry R. D., Pendrak M. L., Schuetze P. Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis. J Bacteriol. 1990 Oct;172(10):5929–5937. doi: 10.1128/jb.172.10.5929-5937.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pospiech A., Cluzel B., Bietenhader J., Schupp T. A new Myxococcus xanthus gene cluster for the biosynthesis of the antibiotic saframycin Mx1 encoding a peptide synthetase. Microbiology. 1995 Aug;141(Pt 8):1793–1803. doi: 10.1099/13500872-141-8-1793. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Rusnak F., Sakaitani M., Drueckhammer D., Reichert J., Walsh C. T. Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine. Biochemistry. 1991 Mar 19;30(11):2916–2927. doi: 10.1021/bi00225a027. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schlumbohm W., Stein T., Ullrich C., Vater J., Krause M., Marahiel M. A., Kruft V., Wittmann-Liebold B. An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase. J Biol Chem. 1991 Dec 5;266(34):23135–23141. [PubMed] [Google Scholar]
  47. Sikkema D. J., Brubaker R. R. Outer membrane peptides of Yersinia pestis mediating siderophore-independent assimilation of iron. Biol Met. 1989;2(3):174–184. doi: 10.1007/BF01142557. [DOI] [PubMed] [Google Scholar]
  48. Sikkema D. J., Brubaker R. R. Resistance to pesticin, storage of iron, and invasion of HeLa cells by Yersiniae. Infect Immun. 1987 Mar;55(3):572–578. doi: 10.1128/iai.55.3.572-578.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Staab J. F., Elkins M. F., Earhart C. F. Nucleotide sequence of the Escherichia coli entE gene. FEMS Microbiol Lett. 1989 May;50(1-2):15–19. doi: 10.1016/0378-1097(89)90450-3. [DOI] [PubMed] [Google Scholar]
  51. Staggs T. M., Perry R. D. Fur regulation in Yersinia species. Mol Microbiol. 1992 Sep;6(17):2507–2516. doi: 10.1111/j.1365-2958.1992.tb01427.x. [DOI] [PubMed] [Google Scholar]
  52. Staggs T. M., Perry R. D. Identification and cloning of a fur regulatory gene in Yersinia pestis. J Bacteriol. 1991 Jan;173(2):417–425. doi: 10.1128/jb.173.2.417-425.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Straley S. C., Bowmer W. S. Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun. 1986 Feb;51(2):445–454. doi: 10.1128/iai.51.2.445-454.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Surgalla M. J., Beesley E. D. Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl Microbiol. 1969 Nov;18(5):834–837. doi: 10.1128/am.18.5.834-837.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tognoni A., Franchi E., Magistrelli C., Colombo E., Cosmina P., Grandi G. A putative new peptide synthase operon in Bacillus subtilis: partial characterization. Microbiology. 1995 Mar;141(Pt 3):645–648. doi: 10.1099/13500872-141-3-645. [DOI] [PubMed] [Google Scholar]
  56. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  57. Wake A., Misawa M., Matsui A. Siderochrome production by Yersinia pestis and its relation to virulence. Infect Immun. 1975 Nov;12(5):1211–1213. doi: 10.1128/iai.12.5.1211-1213.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. de Lorenzo V., Giovannini F., Herrero M., Neilands J. B. Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30. J Mol Biol. 1988 Oct 20;203(4):875–884. doi: 10.1016/0022-2836(88)90113-1. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES