Biology of Metals

, Volume 4, Issue 1, pp 41–47 | Cite as

Characterization of a hemin-storage locus ofYersinia pestis

  • Michael L. Pendrak
  • Robert D. Perry
Original Articles


The pigmentation phenotype (Pgm+) ofYersinia pestis refers to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics. Spontaneous mutation to a Pgm phenotype occurs via a large chromosomal deletion event and results in the inability to express the Pgm+ characteristics. In this study, we have used transposon insertion mutants to define two regions of a hemin-storage (hms) locus. A clone (pHMSI) encompassing this locus reinstates expression of hemin storage (Hms+) inY. pestis spontaneous Pgm strains KIM and Kuma but not inEscherichia coli. Complementation analysis using subclones of pHMS1 inY. pestis transposon mutants indicates that both regions (hmsA andhmsB), which are separated by about 4 kb of intervening DNA, are essential for expression of the Hms+ phenotype. The 9.1-kb insert of pHMS1 contains structural genes encoding 90-kDa, 72-kDa, and 37-kDa polypeptides. Two-dimensional gel electrophoresis analysis of cells from Pgm+, spontaneous Pgm, and Hms transposon strains, as well as a spontaneous Pgm strain transformed with pHMS1, indicated that two families of surface-exposed polypeptides (of about 87 and 69-73 kDa) are associated with the Hms+ phenotype.

Key words

Yersinia pestis Hemin storage Iron Pigmentation Congo red 


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  1. Armstrong SK, Parker CD (1986) Heat-modifiable envelope proteins ofBordetella pertussis. Infect Immun 54:109–117Google Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1987) Current protocols in molecular biology. John Wiley & Sons, New YorkGoogle Scholar
  3. Beesley ED, Brubaker RR, Janssen WA, Surgalla MJ (1967) Pesticins. III. Expression of coagulase and mechanism of fibrinolysis. J Bacteriol 94:19–26Google Scholar
  4. Berkhoff HA, Vinal AC (1985) Congo red medium to distinguish between invasive and non-invasiveEscherichia coli pathogenic for poultry. Avian Dis 30:117–121Google Scholar
  5. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  6. Brubaker RR (1969) Mutation rate to nonpigmentation inPasteurella pestis. J Bacteriol 98:1404–1406Google Scholar
  7. Brubaker RR (1983) The Vwa+ virulence factor of yersiniae: the molecular basis of the attendant nutritional requirement for Ca2+. Rev Infect Dis 5[Suppl 4]:S748-S758Google Scholar
  8. Brubaker RR, Beesley ED, Surgalla MJ (1965)Pasteurella pestis: role of pesticin I and iron in experimental plague. Science 149:422–424Google Scholar
  9. Burrows TW (1973) Observations of the pigmentation ofYersinia pseudotuberculosis. Contr Microbiol Immunol 2:184–189Google Scholar
  10. Clark-Curtiss JE, Curtiss III R (1983) Analysis of recombinant DNA usingEscherichia coli minicells. Methods Enzymol 101:347–362Google Scholar
  11. Daskaleros PA, Payne SM (1987) Congo red binding phenotype is associated with hemin binding and increased infectivity ofShigella flexneri in the HeLa cell model. Infect Immun 55:1393–1398Google Scholar
  12. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation ofE. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145Google Scholar
  13. Dyer DW, West EP, Sparling PF (1987) Effects of serum carrier proteins on the growth of pathogenic neisseriae with hemebound iron. Infect Immun 55:2171–2175Google Scholar
  14. Griffiths E (1987) The iron-uptake systems of pathogenic bacteria. In: Bullen JJ, Griffiths E (eds) Iron and infection: molecular, physiological and clinical aspects. John Wiley & Sons, New York, pp 69–137Google Scholar
  15. Helms SD, Oliver JD, Travis JC (1984) Role of heme compounds and haptoglobin inVibrio vulnificus pathogenicity. Infect Immun 45:345–349Google Scholar
  16. Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298Google Scholar
  17. Humphreys GO, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463Google Scholar
  18. Jackson S, Burrows TW (1956a) The pigmentation ofPasteurella pestis on a defined medium containing haemin. Br J Exp Pathol 37:570–576Google Scholar
  19. Jackson S, Burrows TW (1956b) The virulence-enhancing effect of iron on non-pigmented mutants of virulent strains ofPasteurella pestis. Br J Exp Pathol 37:577–583Google Scholar
  20. Jacobs WR, Barrett JF, Clark-Curtiss JE, Curtiss III R (1986) In vivo repackaging of recombinant cosmid molecules for analyses ofSalmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries. Infect Immun 52:101–109Google Scholar
  21. Kado CI, Liu S-T (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365–1373Google Scholar
  22. Kay WW, Phipps BM, Ishigura EE, Trust TJ (1985) Porphyrin binding by the surface array virulence protein ofAeromonas salmonicida. J Bacterial 164:1332–1336Google Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685Google Scholar
  24. Lawlor KM, Daskaleros PA, Robinson RE, Payne SM (1987) Virulence of iron transport mutants ofShigella flexneri and utilization of host iron compounds. Infect Immun 55:594–599Google Scholar
  25. O'Farrell PH, O'Farrell PZ (1977) Two-dimensional polyacrylamide gel electrophoretic fractionation. Methods Cell Biol 26:407–420Google Scholar
  26. Payne SM, Finkelstein RA (1977) Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect Immun 18:94–98Google Scholar
  27. Perry RD, Brubaker RR (1979) Accumulation of iron by yersiniae. J Bacteriol 137:1290–1298Google Scholar
  28. Perry RD, Harmon PA, Bowmer WS, Straley SC (1986) A low-Ca2+ response operon encodes the V antigen ofYersinia pestis. Infect Immun 54:428–434Google Scholar
  29. Perry RD, Pendrak ML, Schuetze P (1990) Identification and cloning of a hemin-storage locus involved in the pigmentation phenotype ofYersinia pestis. J Bacteriol 172:5929–5937Google Scholar
  30. Pidcock KA, Wooten JA, Daley BA, Stull TL (1988) Iron acquisition byHaemophilus influenzae. Infect Immun 56:721–725Google Scholar
  31. Price SB, Straley SC (1989)lcrH, a gene necessary for virulence ofYersinia pestis and for the normal response ofY. pestis to ATP and calcium. Infect Immun 57:1491–1498Google Scholar
  32. Prpie JK, Robins-Browne RM, Davey RB (1983) Differentiation between virulent and aviruientYersinia enterocolitica isolates by using Congo red agar. J Clin Microbiol 18:486–490Google Scholar
  33. Sikkema DJ, Brubaker RR (1987) Resistance to pesticin, storage of iron, and invasion of HeLa cells by yersiniae. Infect Immun 55:572–578Google Scholar
  34. Sikkema DJ, Brubaker RR (1989) Outer membrane peptides ofYersinia pestis mediating siderophore-independent assimilation of iron. Biol Metals 2:174–184Google Scholar
  35. Stoebner JA, Payne SM (1988) Iron-regulated hemolysin production and utilization of heme and hemoglobin byVibrio cholerae. Infect Immun 56:2891–2895Google Scholar
  36. Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low-copy-number plasmid vectors for cloning inEscherichia coli. Gene 18:335–341Google Scholar
  37. Straley SC, Bowmer WS (1986) Virulence genes regulated at the transcriptional level by Ca2+ inYersinia pestis include structural genes for outer membrane proteins. Infect Immun 51:445–454Google Scholar
  38. Straley SC, Brubaker RR (1982) Localization inYersinia pestis of peptides associated with virulence. Infect Immun 36:129–135Google Scholar
  39. Stugard CE, Daskaleros PA, Payne SM (1989) A 101-kilodalton heme-binding protein associated with Congo red binding and virulence ofShigella flexneri and enteroinvasiveEscherichia coli strains. Infect Immun 57:3534–3539Google Scholar
  40. Surgalla MJ, Beesley ED (1969) Congo red-agar plating medium for detecting pigmentation inPasteurella pestis. Appl Microbiol 18:834–837Google Scholar
  41. Une T, Brubaker RR (1984) In vivo comparison of avirulent Vwa and Pgm or Pst+ phenotypes of yersiniae. Infect Immun 43:895–900Google Scholar
  42. Van Asbeck BS, Verhoef J (1983) Iron and host defense. Eur J Clin Microbiol 2:6–10Google Scholar
  43. Ward CG, Hammond JS, Bullen JJ (1986) Effect of iron compounds on antibacterial function of human polymorphs and plasma. Infect Immun 51:723–730Google Scholar
  44. Way JC, Davis MA, Morisato D, Roberts DE, Kleckner N (1984) New Tn10 derivatives for transposon mutagenesis and for construction oflacZ operon fusions by transposition. Gene 32:369–379Google Scholar
  45. Weinberg ED (1978) Iron and infection. Microbiol Rev 42:45–66Google Scholar
  46. West SEH, Sparling PF (1985) Response ofNeisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production. Infect Immun 47:388–394Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Michael L. Pendrak
    • 1
  • Robert D. Perry
    • 1
  1. 1.Department of Microbiology and ImmunologyLouisiana State University Medical Center ShreveportShreveportUSA

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