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MOLECULAR AND CELLULAR PATHOGENESIS

The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis

Petra C. F. Oyston, Nick Dorrell, Kerstin Williams, Shu-Rui Li, Michael Green, Richard W. Titball, Brendan W. Wren
Petra C. F. Oyston
Defence Evaluation and Research Agency, Salisbury, Wiltshire, SP4 0JQ, and
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Nick Dorrell
Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom
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Kerstin Williams
Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom
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Shu-Rui Li
Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom
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Michael Green
Defence Evaluation and Research Agency, Salisbury, Wiltshire, SP4 0JQ, and
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Richard W. Titball
Defence Evaluation and Research Agency, Salisbury, Wiltshire, SP4 0JQ, and
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Brendan W. Wren
Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, United Kingdom
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DOI: 10.1128/IAI.68.6.3419-3425.2000
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  • Fig. 1.
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    Fig. 1.

    Comparison of the deduced PhoP amino acid sequences ofY. pestis, Y. pseudotuberculosis (Y. ptb), Y. enterocolitica (Y. ent), E. coli, and Salmonella serovar Typhimurium (S. typhm), aligned by using Clustal V multiple sequence alignment software. Identical amino acids (●), conserved changes (‖), and the essential aspartic acid residue (∗) are shown.

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    Fig. 2.

    Survival of Y. pestis wild-type (GB) andphoP mutant (SAI2.2) strains after uptake by J774 macrophages. Results shown are the means of duplicate determinations in two separate experiments, with standard errors.

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    Fig. 3.

    2D gel electrophoresis protein expression profiles for the Y. pestis wild-type strain (a) and Y. pestis phoP mutant strain (b) grown at 28°C. Differences in protein expression are highlighted (0). Mr, molecular weights in thousands.

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    Fig. 4.

    2D gel electrophoresis protein expression profiles for the Y. pestis wild-type strain (A) and Y. pestis phoP mutant (B) grown at 37°C. Differences in protein expression are highlighted (0). Mr, molecular weight in thousands.

Tables

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  • Table 1.

    Bacterial strains and plasmids used in this studya

    Strain or plasmidRelevant characteristic(s)Source or reference
    Strains
     Y. pestis
      GBVirulent wild-type strain, bv. orientalis33
      SAI2.2Y. pestis GB phoPmutantThis study
     Y. pseudotuberculosis YPIII pIB1Virulent wild-type strain, serotype III5
     Y. enterocolitica 8081Virulent wild-type strain, serotype O85
     E. coli
      XL2-Blue MRF′Cloning strainStratagene
      CC118 λpirCloning strain29
      S17 λpir pNJ5000Triple mating strain with Tetr helper plasmid18
    Plasmids
     pUC19AprPharmacia
     pCVD442Apr Sucs, suicide vector9
     pNJ5000Tetr, for triple mating18
     pYP5pUC19 containing 262-bp PCRDOP gene fragment of Y. pestis phoPThis study
     pYPTB7pUC19 containing 262-bp PCRDOP gene fragment ofY. pseudotuberculosis phoPThis study
     pDA5pUC19 plus 4-kb DraI chromosomal fragment containingphoPThis study
     pSA2pUC19 and 1.5-kbSspI chromosomal fragment containing phoPThis study
     pSAI1pSA2 with 31-bp deletion inphoPThis study
     pSAI2Donor plasmid for conjugation; Apr Sucs, pSA1XbaI-PvuII fragment containing the mutatedphoP gene cloned into XbaI-SmaI pCVD442This study
    • ↵a Abbreviations: Apr, ampicillin resistant; Tetr, tetracycline resistant; Sucs, sucrose sensitive.

  • Table 2.

    Oligonucleotides used for PCR

    PrimerPCR methodStrandSequence (5′–3′)a, b
    P3PCRDOP+AATCTGCAGYTNMGNCAYCAYYTNAANGT
    P4PCRDOP−CCAAGCTTNARNACYTCNACYTTPTCYTG
    P9IPCRM+CCAGATCTGGATGGCTTAAGCCTTATC
    P10IPCRM−AAAGATCTTATCTGGGCCATGTTCCTG
    P24phoP specific−ACTTTATCTTGCCAGCTTT
    P27phoP specific+CGCGTTGTTGCGTCACCAT
    P41phoPamplification+ACCTATCACCAGATATTGGCGTG
    P42phoPamplification−CCCATCATCATTCTACTGATGTGCG
    • ↵a Underlined nucleotides representPstI (P3), HindIII (P4), and BglII (P9 and P10) restriction endonuclease sites.

    • ↵b R = A or G; Y = C or T; M = A or C; N = A, C, G, or T.

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The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis
Petra C. F. Oyston, Nick Dorrell, Kerstin Williams, Shu-Rui Li, Michael Green, Richard W. Titball, Brendan W. Wren
Infection and Immunity Jun 2000, 68 (6) 3419-3425; DOI: 10.1128/IAI.68.6.3419-3425.2000

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The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis
Petra C. F. Oyston, Nick Dorrell, Kerstin Williams, Shu-Rui Li, Michael Green, Richard W. Titball, Brendan W. Wren
Infection and Immunity Jun 2000, 68 (6) 3419-3425; DOI: 10.1128/IAI.68.6.3419-3425.2000
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KEYWORDS

Bacterial Proteins
macrophages
transcription factors
Yersinia pestis

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