Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jun;175(11):3536–3545. doi: 10.1128/jb.175.11.3536-3545.1993

Multiple effects of lcrD mutations in Yersinia pestis.

G V Plano 1, S C Straley 1
PMCID: PMC204754  PMID: 8501057

Abstract

Plasmid pCD1 of Yersinia pestis contains a low-calcium response stimulon responsible for the temperature- and calcium-regulated expression and secretion of proteins involved in virulence, which include the V antigen and Yops. We have previously shown that insertional inactivation of the bicistronic lcrDR operon abolished the calcium requirement for growth at 37 degrees C and reduced expression of the V antigen and Yops. In this study, we constructed and characterized three mutants having nonpolar lcrD deletions. All three mutants lost the two main low-calcium response properties: a calcium requirement for growth at 37 degrees C and strong expression of the V antigen and Yops. The effects on virulence gene expression occurred at both the levels of transcription and secretion. The growth, transcription, and secretion defects could be at least partially complemented for two of the lcrD mutants by providing lcrD in trans. A third mutant could not be complemented, and a plasmid carrying this mutation had a dominant negative effect over normal LcrD function. In the three mutants, the amount of mutant LcrD protein detectable in immunoblots was inversely related to the amount of complementation. Taken together, these data indicate that LcrD function involves the interaction of LcrD with another molecule.

Full text

PDF
3536

Images in this article

3540Image
on p.3540
3541Image
on p.3541
3541Image
on p.3541
3542Image
on p.3542
3543Image
on p.3543
3544Image
on p.3544

Selected References

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

  1. Andrews G. P., Maurelli A. T. mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect Immun. 1992 Aug;60(8):3287–3295. doi: 10.1128/iai.60.8.3287-3295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BURROWS T. W., BACON G. A. The basis of virulence in Pasteurella pestis: the development of resistance to phagocytosis in vitro. Br J Exp Pathol. 1956 Jun;37(3):286–299. [PMC free article] [PubMed] [Google Scholar]
  3. Barve S. S., Straley S. C. lcrR, a low-Ca2(+)-response locus with dual Ca2(+)-dependent functions in Yersinia pestis. J Bacteriol. 1990 Aug;172(8):4661–4671. doi: 10.1128/jb.172.8.4661-4671.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ben-Gurion R., Shafferman A. Essential virulence determinants of different Yersinia species are carried on a common plasmid. Plasmid. 1981 Mar;5(2):183–187. doi: 10.1016/0147-619x(81)90019-6. [DOI] [PubMed] [Google Scholar]
  5. Bergman T., Håkansson S., Forsberg A., Norlander L., Macellaro A., Bäckman A., Bölin I., Wolf-Watz H. Analysis of the V antigen lcrGVH-yopBD operon of Yersinia pseudotuberculosis: evidence for a regulatory role of LcrH and LcrV. J Bacteriol. 1991 Mar;173(5):1607–1616. doi: 10.1128/jb.173.5.1607-1616.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bliska J. B., Guan K. L., Dixon J. E., Falkow S. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1187–1191. doi: 10.1073/pnas.88.4.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brubaker R. R. The V antigen of yersiniae: an overview. Contrib Microbiol Immunol. 1991;12:127–133. [PubMed] [Google Scholar]
  9. Bölin I., Portnoy D. A., Wolf-Watz H. Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect Immun. 1985 Apr;48(1):234–240. doi: 10.1128/iai.48.1.234-240.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cornelis G. R., Sluiters C., Delor I., Geib D., Kaniga K., Lambert de Rouvroit C., Sory M. P., Vanooteghem J. C., Michiels T. ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions. Mol Microbiol. 1991 May;5(5):1023–1034. doi: 10.1111/j.1365-2958.1991.tb01875.x. [DOI] [PubMed] [Google Scholar]
  11. Cornelis G., Sluiters C., de Rouvroit C. L., Michiels T. Homology between virF, the transcriptional activator of the Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator. J Bacteriol. 1989 Jan;171(1):254–262. doi: 10.1128/jb.171.1.254-262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fenselau S., Balbo I., Bonas U. Determinants of pathogenicity in Xanthomonas campestris pv. vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):390–396. doi: 10.1094/mpmi-5-390. [DOI] [PubMed] [Google Scholar]
  13. Ferber D. M., Brubaker R. R. Plasmids in Yersinia pestis. Infect Immun. 1981 Feb;31(2):839–841. doi: 10.1128/iai.31.2.839-841.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Forsberg A., Viitanen A. M., Skurnik M., Wolf-Watz H. The surface-located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis. Mol Microbiol. 1991 Apr;5(4):977–986. doi: 10.1111/j.1365-2958.1991.tb00773.x. [DOI] [PubMed] [Google Scholar]
  15. Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goguen J. D., Yother J., Straley S. C. Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants. J Bacteriol. 1984 Dec;160(3):842–848. doi: 10.1128/jb.160.3.842-848.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gough C. L., Genin S., Zischek C., Boucher C. A. hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria. Mol Plant Microbe Interact. 1992 Sep-Oct;5(5):384–389. doi: 10.1094/mpmi-5-384. [DOI] [PubMed] [Google Scholar]
  18. Haddix P. L., Straley S. C. Structure and regulation of the Yersinia pestis yscBCDEF operon. J Bacteriol. 1992 Jul;174(14):4820–4828. doi: 10.1128/jb.174.14.4820-4828.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jobling M. G., Holmes R. K. Construction of vectors with the p15a replicon, kanamycin resistance, inducible lacZ alpha and pUC18 or pUC19 multiple cloning sites. Nucleic Acids Res. 1990 Sep 11;18(17):5315–5316. doi: 10.1093/nar/18.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LAWTON W. D., ERDMAN R. L., SURGALLA M. J. BIOSYNTHESIS AND PURIFICATION OF V AND W ANTIGEN IN PASTEURELLA PESTIS. J Immunol. 1963 Aug;91:179–184. doi: 10.21236/ad0299868. [DOI] [PubMed] [Google Scholar]
  22. Lambert de Rouvroit C., Sluiters C., Cornelis G. R. Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol. 1992 Feb;6(3):395–409. [PubMed] [Google Scholar]
  23. Leung K. Y., Straley S. C. The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. J Bacteriol. 1989 Sep;171(9):4623–4632. doi: 10.1128/jb.171.9.4623-4632.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Michiels T., Vanooteghem J. C., Lambert de Rouvroit C., China B., Gustin A., Boudry P., Cornelis G. R. Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol. 1991 Aug;173(16):4994–5009. doi: 10.1128/jb.173.16.4994-5009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Michiels T., Wattiau P., Brasseur R., Ruysschaert J. M., Cornelis G. Secretion of Yop proteins by Yersiniae. Infect Immun. 1990 Sep;58(9):2840–2849. doi: 10.1128/iai.58.9.2840-2849.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  29. Perry R. D., Harmon P. A., Bowmer W. S., Straley S. C. A low-Ca2+ response operon encodes the V antigen of Yersinia pestis. Infect Immun. 1986 Nov;54(2):428–434. doi: 10.1128/iai.54.2.428-434.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Plano G. V., Barve S. S., Straley S. C. LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol. 1991 Nov;173(22):7293–7303. doi: 10.1128/jb.173.22.7293-7303.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Portnoy D. A., Blank H. F., Kingsbury D. T., Falkow S. Genetic analysis of essential plasmid determinants of pathogenicity in Yersinia pestis. J Infect Dis. 1983 Aug;148(2):297–304. doi: 10.1093/infdis/148.2.297. [DOI] [PubMed] [Google Scholar]
  33. Price S. B., Cowan C., Perry R. D., Straley S. C. The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes. J Bacteriol. 1991 Apr;173(8):2649–2657. doi: 10.1128/jb.173.8.2649-2657.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Price S. B., Straley S. C. lcrH, a gene necessary for virulence of Yersinia pestis and for the normal response of Y. pestis to ATP and calcium. Infect Immun. 1989 May;57(5):1491–1498. doi: 10.1128/iai.57.5.1491-1498.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Protsenko O. A., Anisimov P. I., Mozharov O. T., Konnov N. P., Popov Iu A. Vyiavlenie i kharakteristika plazmid chumnogo mikroba, determiniruiushchikh sintez pestitsina I, antigena fraktsiia I i ékzotoksina "myshinogo" toksina. Genetika. 1983 Jul;19(7):1081–1090. [PubMed] [Google Scholar]
  36. Ramakrishnan G., Zhao J. L., Newton A. The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis. J Bacteriol. 1991 Nov;173(22):7283–7292. doi: 10.1128/jb.173.22.7283-7292.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reisner B. S., Straley S. C. Yersinia pestis YopM: thrombin binding and overexpression. Infect Immun. 1992 Dec;60(12):5242–5252. doi: 10.1128/iai.60.12.5242-5252.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rimpiläinen M., Forsberg A., Wolf-Watz H. A novel protein, LcrQ, involved in the low-calcium response of Yersinia pseudotuberculosis shows extensive homology to YopH. J Bacteriol. 1992 May;174(10):3355–3363. doi: 10.1128/jb.174.10.3355-3363.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rosqvist R., Forsberg A., Wolf-Watz H. Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun. 1991 Dec;59(12):4562–4569. doi: 10.1128/iai.59.12.4562-4569.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sanders L. A., Van Way S., Mullin D. A. Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. J Bacteriol. 1992 Feb;174(3):857–866. doi: 10.1128/jb.174.3.857-866.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Skryzpek E., Straley S. C. LcrG, a secreted protein involved in negative regulation of the low-calcium response in Yersinia pestis. J Bacteriol. 1993 Jun;175(11):3520–3528. doi: 10.1128/jb.175.11.3520-3528.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Skrzypek E., Haddix P. L., Plano G. V., Straley S. C. New suicide vector for gene replacement in yersiniae and other gram-negative bacteria. Plasmid. 1993 Mar;29(2):160–163. doi: 10.1006/plas.1993.1019. [DOI] [PubMed] [Google Scholar]
  43. Sodeinde O. A., Goguen J. D. Genetic analysis of the 9.5-kilobase virulence plasmid of Yersinia pestis. Infect Immun. 1988 Oct;56(10):2743–2748. doi: 10.1128/iai.56.10.2743-2748.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sodeinde O. A., Sample A. K., Brubaker R. R., Goguen J. D. Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins. Infect Immun. 1988 Oct;56(10):2749–2752. doi: 10.1128/iai.56.10.2749-2752.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Une T., Brubaker R. R. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of yersiniae. Infect Immun. 1984 Mar;43(3):895–900. doi: 10.1128/iai.43.3.895-900.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Viitanen A. M., Toivanen P., Skurnik M. The lcrE gene is part of an operon in the lcr region of Yersinia enterocolitica O:3. J Bacteriol. 1990 Jun;172(6):3152–3162. doi: 10.1128/jb.172.6.3152-3162.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yother J., Goguen J. D. Isolation and characterization of Ca2+-blind mutants of Yersinia pestis. J Bacteriol. 1985 Nov;164(2):704–711. doi: 10.1128/jb.164.2.704-711.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zahorchak R. J., Brubaker R. R. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in Yersinia pestis. Infect Immun. 1982 Dec;38(3):953–959. doi: 10.1128/iai.38.3.953-959.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zahorchak R. J., Charnetzky W. T., Little R. V., Brubaker R. R. Consequences of Ca2+ deficiency on macromolecular synthesis and adenylate energy charge in Yersinia pestis. J Bacteriol. 1979 Sep;139(3):792–799. doi: 10.1128/jb.139.3.792-799.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES