Molecular Biology > Cloning and Recombination Methods

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Isolation and Confirmation of Yersinia pestis Mutants Exempt from Select Agent Regulations

Robert D. Perry1,  Scott W. Bearden2

1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky
2Centers for Disease Control and Prevention, Division of Vector‐Borne Infectious Diseases, Fort Collins, Colorado


Publication Name: 
Current Protocols in Microbiology
Unit Number: 
Unit 5B.2
DOI: 
10.1002/9780471729259.mc05b02s11
Online Posting Date: 
November, 2008
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

This unit describes protocols for Yersinia pestis to confirm plasmid profiles, construct and confirm a pgm mutation, and cure the low-calcium response (Lcr) plasmid encoding a type III secretion system (TTSS). Strains lacking either the chromosomal pgm locus or the Lcr plasmid can be safely studied under BSL-2 conditions and are exempt from Select Agent regulations in the U.S. Curr. Protoc. Microbiol. 11:5B.2.1-5B.2.12. © 2008 by John Wiley & Sons, Inc.

Keywords: plague; plasmid isolation; low-calcium response; type III secretion; pigmentation; biofilm

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Plasmid DNA
  • Alternate Protocol: Rapid Lysis Plasmid Isolation
  • Basic Protocol 2: Isolation and Demonstration of a pCD-Negative Strain
  • Basic Protocol 3: Isolation and Demonstration of a pgm Mutation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Plasmid DNA

 Materials
  • Y. pestis liquid culture
  • B & D solution 1 (see recipe), ice cold
  • B & D solution 2 (see recipe)
  • B & D solution 3 (see recipe), ice cold
  • Phenol/CHCl3 solution (see recipe)
  • 95% ethanol
  • 1× TE-RNase (see recipe)
  • 10× DNA loading buffer (see recipe)
  • DNA size standard
  • 0.7% (w/v) agarose gel
  • 1× TBE running buffer (appendix 2A)
  • Gel-Star stain (use according to manufacturer's instructions) or 1% ethidium bromide solution
  • 1 M NaCl
  • 1.5- to 2.0-ml microcentrifuge tubes
  • Electrophoresis gel box
  • Electrophoresis power supply
  • UV light box
  • Gel photographic equipment

Alternate Protocol: Rapid Lysis Plasmid Isolation

 Additional Materials (also see Basic Protocol 1)
  • 18- to 24-hr Y. pestis culture on SBA or TBA plate
  • TE buffer (appendix 2A)
  • Kado lysis buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (appendix 2A)
  • 0.6% SeaKem Gold agarose gel in 1× TBE (or other high-quality agarose)
  • Inoculating loop, sterile
  • 60°C heating block or water bath
  • Refrigerated microcentrifuge

Basic Protocol 2: Isolation and Demonstration of a pCD-Negative Strain

 Materials
  • Y. pestis strain
  • Heart infusion broth (HIB, Difco)
  • TBA-MgOX plates (see recipe)
  • CR plates (see recipe in unit 5B.1)
  • 37°C incubator
  • Inoculating loop, sterile

Basic Protocol 3: Isolation and Demonstration of a pgm Mutation

 Materials
  • Y. pestis strain
  • Heart infusion broth (HIB, Difco)
  • 33 mM K-PO4 buffer
  • CR plates (see recipe in unit 5B.1)
  • pgm primers 1 (CCCCGCCAGATCCTTACC) and 2 (GGCAGACGGACC ATCCAG)
  • psn primers 1 (ATTCAGGATGGCCTGCTG) and 2 (GACGATTAA CGAACCGGA)
  • 25 mM MgCl2
  • 10× dNTP stocks (2 mM each of dATP, dCTP, dGTP, and dTTP)
  • Taq polymerase and 10× buffer
  • 10× DNA loading dye
  • DNA standards
  • 1% (w/v) agarose gel
  • Ethidium bromide
  • 26° to 30°C incubator
  • Spectrophotometer
  • Dilution tubes
  • DNA thermal cycler
  • Electrophoresis power supply
  • Electrophoresis gel box
  • UV light box
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 5B.2.1
    Y. pestis plasmid profiles of KIM11+ (lane 1), KIM6+ (lane 2), KIM5 (lane 3), and A1122 (lane 4). Plasmids were isolated using Basic Protocol 1, separated by electrophoresis through a 0.7% agarose gel, and visualized with ethidium bromide on a UV light box. Molecular mass markers and plasmid names are indicated. Plasmids are closed circular forms that migrate faster than the linear molecular mass markers. Although KIM11+ lacks pMT1 (100 kb), all or a portion of the plasmid is integrated into the chromosome. A1122 harbors a duplicated pPCP1 plasmid of ~20 kb termed pEL101 (Perry and Fetherston, 1997). Depending upon the plasmid preparation, nicked forms of pPCP1 (migrating similar to pEL101) and contaminating, sheared chromosomal DNA (migrating at or slightly above the highest marker) can be seen.

    View Image
  • Figure 5B.2.2
    Pgm+ (KIM6+; left) and Pgm– (KIM6; right) colonies on a CR plate. Cells were streaked for isolated colonies and incubated 48 hr at 30°C. White spots on CR+ colonies are reflected light, not spontaneous pgm mutants arising within the colony.

    View Image
  • Figure 5B.2.3
    PCR products from KIM6+ Pgm+ (lanes 1 and 2) and KIM6 pgm cells (lanes 3 and 4). PCR products obtained using Basic Protocol 3 and pgm primers (lanes 1 and 3) or psn primers (lanes 2 and 4) were separated by electrophoresis through a 1% agarose gel, and visualized with ethidium bromide on a UV light box. The expected 402-bp product from Pgm+ cells with the psn primers (lane 2) and 2020-bp product from pgm cells with the pgm primers (lane 3) were observed. Outer lanes contain molecular mass markers with selected mass sizes indicated.

    View Image

Literature Cited

Literature Cited
    Anisimov, A.P., Dentovskaya, S.V., Titareva, G.M., Bakhteeva, I.V., Shaikhutdinova, R.Z., Balakhonov, S.V., Lindner, B., Kocharova, N.A., Senchenkova, S.N., Holst, O., Pier, G.B., and Knirel, Y.A. 2005. Intraspecies and temperature-dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B. Infect. Immun. 73:7324-7331.
    Bearden, S.W., Fetherston, J.D., and Perry, R.D. 1997. Genetic organization of the Yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis. Infect. Immun. 65:1659-1668.
    Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.
    Brubaker, R.R. 1969. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 98:1404-1406.
    Brubaker, R.R. 2005. Influence of Na+, dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis. Infect. Immun. 73:4743-4752.
    Fetherston, J.D., Schuetze, P., and Perry, R.D. 1992. 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. 6:2693-2704.
    Fetherston, J.D., Bertolino, V.J., and Perry, R.D. 1999. YbtP and YbtQ: Two ABC transporters required for iron uptake in Yersinia pestis. Mol. Microbiol. 32:289-299.
    Geoffroy, V.A., Fetherston, J.D., and Perry, R.D. 2000. Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore Yersiniabactin but have different effects on regulation. Infect. Immun. 68:4452-4461.
    Inglesby, T.V., Dennis, D.T., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Koerner, J.F., Layton, M., McDade, J., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., Schoch-Spana, M., and Tonat, K. 2000. Plague as a biological weapon: Medical and public health management. J. Amer. Med. Assoc. 283:2281-2290.
    Jones, H.A., Lillard, J.W. Jr., and Perry, R.D. 1999. HmsT, a protein essential for expression of the haemin storage (Hms+) phenotype of Yersinia pestis. Microbiology 145:2117-2128.
    Kado, C.I. and Liu, S.-T. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 45:1365-1373.
    Kirillina, O., Fetherston, J.D., Bobrov, A.G., Abney, J., and Perry, R.D. 2004. HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. Mol. Microbiol. 54:75-88.
    Kutyrev, V.V., Protsenko, O.A., Smirnov, G.B., Boolgakova, E., Kukleva, L.M., Zudina, I.V., Vidyaeva, N.A., and Koozmichenko, I. 2003. Yersinia pestis from natural foci. Adv. Exp. Med. Biol. 529:313-316.
    Lathem, W.W., Crosby, S.D., Miller, V.L., and Goldman, W.E. 2005. Progression of primary pneumonic plague: A mouse model of infection, pathology, and bacterial transcriptional activity. Proc. Natl. Acad. Sci. U.S.A. 102:17786-17791.
    Perry, R.D., Pendrak, M.L., and Schuetze, P. 1990. Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis. J. Bacteriol. 172:5929-5937.
    Perry, R.D. and Fetherston, J.D. 1997. Yersinia pestis—Etiologic agent of plague. Clin. Microbiol. Rev. 10:35-66.
    Straley, S.C. and Starnbach, M.N. 2000. "Yersinia: Strategies that thwart immune defenses". In Effects of Microbes on the Immune System (M.W. Cunningham and R.S. Fujinami, eds.) pp. 71-92. Lippincott Williams & Wilkins, Philadelphia.
    Surgalla, M.J. and Beesley, E.D. 1969. Congo red-agar plating medium for detecting pigmentation in Pasteurella pestis. Appl. Microbiol. 18:834-837.
    Une, T. and Brubaker, R.R. 1984. In vivo comparison of avirulent Vwa– and Pgm– or Pstr phenotypes of Yersiniae. Infect. Immun. 43:895-900.
    Welkos, S., Pitt, M.L.M., Martinez, M., Friedlander, A., Vogel, P., and Tammariello, R. 2002. Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague. Vaccine 20:2206-2214.
    Zahorchak, R.J. and Brubaker, R.R. 1982. Effect of exogenous nucleotides on Ca2+ dependence and V antigen synthesis in Yersinia pestis. Infect. Immun. 38:953-959.
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.