TABLE 1.

Bacterial strains, plasmids and primers useda

Strain, plasmid, or primerRelevant characteristics or sequenceSource and/or reference or use
Strains
    Y. pestis
        KIM6(pKD46)+Pgm+ Lcr Pla+ SpeA+ SpeC+ Apr pKD4614; this study
        KIM6-2110(pKD46)+Pgm+ Lcr Pla+ SpeA+speC::kan2110) Apr pKD46This study
        KIM6-2111(pKD46)+Pgm+ Lcr Pla+ SpeC+speA::cam2111) Apr pKD46This study
        KIM6-2112(pKD46)+Pgm+ Lcr Pla+speA::cam2111) (ΔspeC::kan2110) pKD46This study
    E. coli
        BL21(DE3)F ompT hsdSB(rB mB) gal dcm (DE3)Novagen
        Top10F′F′ [lacIq Tn10 (Tetr)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupGInvitrogen
Plasmids
    pET-28a-c5.4 kb, Kmr, six-histidine-tagged fusion protein plasmidNovagen
    pET-28b-speA7.4 kb, Kmr, His-ADC expression vector, 2-kb speA PCR product ligated into EcoRI-XhoI sites of pET-28b3
    pET-28b-speC7.6 kb, Kmr, His-ODC expression vector, 2.2-kb speC PCR product ligated into BamHI-XhoI sites of pET-28bThis study
    pBAD/Hisab4.1 kb, Apr, six-histidine-tagged fusion protein plasmidInvitrogen
    pBAD/HisbADC6.1 kb, Apr, His-ADC expression vector, 2-kb speA PCR product ligated into SacI-HindIII sites of pBAD/HisbThis study
    pKD32.8 kb, Cmr, template plasmid10
    pKD43.3 kb, Kmr, template plasmid10
    pKD466.3 kb, Apr, Red recombinase expression plasmid10
Primers
    ODC_KOf5′-ATGACACTATTAAAAATAGCAACAAGCGCCCGCCTGGCTTCTT ATGTAGAGTGTAGGCTGGAGCTGCTTC-3′For insertion of Km or Cm cassette into speC
    ODC_KOr5′-TTGCGTAATCACATAACCAAAGATACGCCGCCAAGACACAT CACCTTTTTCATATGAATATCCTCCTTAGT-3′For insertion of Km or Cm cassette into speC
    ADC-KO2f5′-TTCTTTACGCTCCATGCAGGAGGTAGCCATGAATGATCGAAAT GCCAGCAGTGTAGGCTGGAGCTGCTTC-3′For insertion of Km or Cm cassette into speA
    ADC-KO2r5′-CAGGAACTGCGCCTGCAATTCGGTATCCAGATCGGTTTCTTTT ACCTGACCATATGAATATCCTCCTTAGT-3′For insertion of Km or Cm cassette into speA
    YpADCecofbTCCGAATTCGATGTCTGATGATAACTTGATTAGCCGTCCGCloning into expression vector for speA
    YpADCXhorTAACTCGAGTTCGTCTTCAAGATATGTGTAGCCGCloning into expression vector for speA
    ODCBamf5′-GGATCCGATGACACTATTAAAAATAGCAACAAGCGCCCGCC-3′Cloning into expression vector for speC
    ODCXhor5′-CTCGAGTTGCGTAATCACATAACCAAAGATACGCCGCC-3′Cloning into expression vector for speC
    YPadcSacfb5′-TCCGAGCTCGATGTCTGATGATAACTTGATTAGCCGTCCG-3′Clone speA for insertion into the pBAD/Hisb expression vector for complementation
    YPadcHindr5′-TAAAAGCTTTTCGTCTTCAAGATATGTGTAGCCG-3′Clone speA for insertion into the pBAD/Hisb expression vector for complementation
    YPADC+50f5′-CGTCAGTATCGCAATAGAGGCG-3′For sequencing from 50 bp outside speA
    YPADC+50r5′-CAAACCTGAGTTTGCCC-3′For sequencing from 50 bp outside speA
    YPODC+50r5′-GGCAGCCTCATACCTGACCG-3′For sequencing from 50 bp outside speC
    YPODC+50f5′-GGCCTCGGCTGATGGAGCGGAG-3′For sequencing from 50 bp outside speC
  • a All Y. pestis strains are avirulent due to the lack of the low-calcium response plasmid pCD1. Strains with a plus sign possess an intact 102-kb pgm locus containing the genes for hemin storage (hms) and yersiniabactin (Ybt) iron transport system. Abbreviations: Apr, Cmr, and Kmr, resistance to ampicillin, chloramphenicol, and kanamycin, respectively.