TABLE 1.

Bacterial strains and plasmids used in this studya

Strain or plasmidRelevant characteristicsSource or reference
Strains
    Y. pestis
        KIM6+Pgm+ Hms+ Pla+14
        KIM6Pgmpgm; Hms) Pla+14
        KIM6-2008Kmr Hms (hmsH2008::mini-kan) Pla+36
        KIM6-2011Kmr Hms (hmsF2011::mini-kan) Pla+36
        KIM6-2012Kmr Hms (hmsR2012::mini-kan) Pla+36
        KIM6-2051+Kmr Hms (hmsT2051::mini-kan) Pla+25
        KIM6-2057.1HmshmsR46, in-frame deletion) Pla+29
        KIM6-2058.1Hms (hmsH49) Pla+This study
        KIM6-2093 (pKD46)+Apr Cmr Hms+ ΔY2360::cam Pla+This study
        KIM6-2095 (pKD46)+Apr Kmr Hmsc Δlon clpPX::kan Pla+This study
        KIM6-2096+Cmr Hms+ inv::pTinvII hmsT::lacZ Pla+This study
        KIM6-2097Apr Pgmpgm; Hms) inv::pHinv hmsH::lacZ Pla+This study
        KIM10+Hms+ Pla (cured of pPCP1)40
    E. coli
        BL21Strain for expression of GST fusion proteinsPharmacia Biotech
        DH5αCloning strain2
        DH5α (λpir)Strain for maintenance of R6K ori suicide plasmidsS. C. Straley
        M15 (pREP4)His tag expression strain62; Qiagen, Inc.
Plasmids
    pAHMS164.4 kb, Apr hmsT+, 1.9-kb KpnI-FspI fragment from pAHMS14 into KpnI-HincII sites of pUC1825
    pBluescript II KS+2.9 kb, Apr, high-copy-number cloning vectorStratagene
    pBSlacZMCS7.1 kb, Apr, high-copy-number cloning vector with rrnBT1 transcriptional terminator and lacZ gene from pEU730This study
    pCVD4426.2 kb, Apr sacB, R6K ori, suicide vector11
    pCVDHmsH49Apr suicide plasmid encoding hmsH49 from pNPM49This study
    pEU73015.2 kb, Smr Spcr, low-copy-number vector with promotorless lacZ15
    pGEX-2T5.0 kb, Apr lacIq GST fusion protein plasmidPharmacia Biotech
    pGEX2T-HmsT5.6 kb, Apr, GST-′HmsT expression vector, 588-bp hmsT PCR product ligated into SmaI-BamHI sites of pGEX-2TThis study
    pGstHmsS5.2 kb, Apr, GST-′HmsS expression vector, 231-bp hmsS PCR product ligated into BamHI-EcoRI sites of pGEX-2TThis study
    pHinv11.2 kb, Apr, 4.5-kb EagI fragment from pHmsHlacZ ligated into corresponding site in pSucinv, lacZ expression driven by hmsH promoter regionThis study
    pHMS1.213.4 kb, Apr hmsHFRS+, 9.7-kb SalI-HindIII fragment from pHMS1 into pBR32236
    pHMS1.2SL13.4 kb, Apr, hmsHFRS with disrupted stem-loop in hmsFThis study
    pHMS916.4 kb, Kmr, hmsHFRS with disrupted stem-loop in hmsF, 4.3-kb PmlI-SalI fragment from pHMS1.2SL ligated into corresponding sites of pHMS1This study
    pHmsHlacZ7.4 kb, Apr, 314-bp PCR product from pHMS1.2 ligated into PstI-Asp718 sites of pBSlacZMCSThis study
    pHmsTlacZ7.5 kb, Apr, 418-bp PCR product from pAHMS16 ligated into PacI-PmeI sites of pBSlacZMCSThis study
    pKD32.8 kb, Cmr, template plasmid9
    pKD43.3 kb, Kmr, template plasmid9
    pKD466.3 kb, Apr, Red recombinase expression plasmid9
    pKRP102.9 kb, Apr Cmr, cam cassette plasmid42
    pLG3387.3 kb, Kmr Tcr, low-copy-number cloning vector55
    pNPM49∼16.4-kb, Kmr hmsFRS+ hmsH, spontaneous mutant30
    pQE303.5 kb, Apr, His6 tag expression vectorQiagen, Inc.
    pQE30H.65.6 kb, Apr, His6 tag-′HmsH expression vector; 2.2-kb NcoI-XhoI fragment from pHMS1.2 ligated into HindIII-SalI sites of pQE30This study
    pQE323.5 kb, Apr, His6 tag expression vectorQiagen, Inc.
    pQE32F.26.0 kb, Apr, His6 tag ′HmsF expression vector; 2.5-kb PmlI-PvuII fragment from pHMS1.2 ligated into SmaI site of pQE32This study
    pRI2039.0 kb, Apr, encodes Y. pseudotuberculosis inv22; V. L. Miller
    pSUC14.7 kb, Apr, suicide vector derived from pCVD442, sacB, R6K ori12
    pSucinv6.7 kb, Apr, suicide vector with 2.0-kb StuI-XhoI fragment of Y. pseudotuberculosis inv gene from pRI203 ligated into SmaI-SalI sites of pSUC1This study
    pSucinvII6.1 kb, Cmr, suicide vector with Y. pseudotuberculosis inv, pSucinv with bla replaced by 0.8-kb EcoRI-HincII fragment from pKRP10 encoding camThis study
    pTinvII10.6 kb, Cmr, 4.6-kb EagI fragment from pHmsTlacZ ligated into corresponding site in pSucinvII, lacZ expression driven by hmsT promoter regionThis study
  • a All Y. pestis strains are avirulent due to lack of the low-calcium response plasmid pCD1. Strains with a plus sign possess an intact 102-kb pgm locus containing the genes for the hemin storage (hms) and yersiniabactin (Ybt) iron transport system. All other Y. pestis strains have either a pgm deletion or a mutation within the pgm locus. Apr, Cmr, Kmr, Smr, Spcr, and Tcr indicate resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, and tetracycline, respectively.