TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidKey propertiesReference or source
Y. pestis strains
    KIM6+Pgm+ Lcr15
    KIM6-2109+Pgm+ Lcr; ΔluxS::kan ΔypsIR ΔytbIRA. Bobrov, S. W. Bearden, J. D. Fetherston, and R. D. Perry
    CO99-3015.S1CO92 Pgm+ Lcr; pFra pPCP1; Lcr derivative of CDC strain CO99-3015 of virulent Y. pestis (a subclone of CO92)Scott Bearden, CDC, Ft. Collins, CO
    CO99-3015.S4invA::promoterless lacZ Lcr; made from CO99-3015.S1 by single-crossover integration of pSucinv::lacZSpencer Leigh
    CO99-3015.S5CO92 Δpgm; pCD2 pFra pPCP1; Δpgm derivative of CDC strain CO99-3015 of virulent Y. pestis (a subclone of CO92); used to make pCD2ApScott Bearden, CDC, Ft. Collins, CO
    CO99-3015.S6CO92 Apr Δpgm; pCD2 pFra pPCP1; contains pCD2ApSpencer Leigh
    CO99-3015.S10CO92 Apr Pgm+ Lcr; pFra pPCP1; invA::PyadBC-lacZ; single-crossover integration of pSucinv::yadBC-lacZ into CO99-3015.S1This study
    CO92.S1Pgm+ Lcr; pFra pPCP1; Lcr derivative of virulent Y. pestis CO92; molecular group 1.ORILuther Lindler
    CO92.S2CO92 Pgm+ Lcr; pFra Δcaf1; F1 derivative made by Red/Flp-mediated allelic exchange into CO92.S1; entire caf1 coding sequence and upstream 12 bp deletedThis study
    CO92.S6CO92 Apr Pgm+ Lcr+; pCD2Ap pFra pPCP1; reconstituted virulent strain made by introduction of pCD2Ap into CO92.S1This study
    CO92.S7CO92 Apr Pgm+ Lcr+; pCD2Ap pFra Δcaf1 pPCP1; reconstituted conditionally virulent F1 strain made by introduction of pCD2Ap into CO92.S2aThis study
    CO92.S8CO92 Pgm+ Lcr; pFra pPCP1 ΔyadBC; derived from CO92.S1 by allelic exchangeThis study
    CO92.S9CO92 Apr Pgm+ Lcr+; pCD2Ap pFra pPCP1 ΔyadBC; potentially virulent derivative of CO92.S8 containing pCD2ApThis study
    CO92.S10CO92 Pgm+ Lcr; pFra Δcaf1 pPCP1 ΔyadBC; F1 derivative of CO92.S8 made by Red/Flp-mediated allelic exchange; entire caf1 coding sequence and upstream 12 bp deletedThis study
    CO92.S11CO92 Apr Pgm+ Lcr+; pCD2Ap pFra Δcaf1 pPCP1 ΔyadBC; F1 potentially conditionally virulent derivative of CO92.S10 containing pCD2ApaThis study
    CO92.S12CO92 Kmr Pgm+ Lcr; pFra Δcaf1 pPCP1::Kan-fsPla ΔyadBC ΔdegP; Pla DegP strain derived from CO92.S11 by allelic exchange and plasmid exchangeThis study
    CO92.S15CO92 Pgm+ Lcr; pFra pPCP1; yadBC CO92.S8 strain reconstituted to yadBC+ by allelic exchangeThis study
    CO92.S16Reconstituted virulent CO92 Apr Pgm+ Lcr+; pCD2Ap pFra pPCP1; reconstituted virulent strain made by introducing pCD2Ap into CO92.S15This study
    CO92.S17CO92 Pgm+ Lcr; pFra pPCP1 ΔyadC; derived from CO92.S1 by allelic exchangeThis study
    CO92.S18CO92 Apr Pgm+ Lcr+; pCD2Ap pFra pPCP1 ΔyadC strain made Lcr+ by introducing pCD2Ap into CO92.S17This study
E. coli strains
    DH5αF Δφ80dlacZΔM15 endA1 recA1 hsdR17(rM mK+) supE44 thi-1 gyrA96 Δ(lacZYA-argF)U169Life Technologies
    DH5α λpirrecA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR Δ(lacZYA-argF)U169 λpir+Lab stock
    S17-1Smr Spr Tra+; pro thi hsdR recA; chromosomal integration of RP4-2-Tc::Mu-Kan::Tn711
    BW20767RP4-2-Tc::Mu-Kan::Tn7 integrant, leu-63::IS10 recA1 zbf-5 creB510 hsdR17 endA1 thi uidA(ΔMluI)::pir+28
    HB101recA23 hsdS20 supE44 (rB mB) ara-14 lacY1 galK2 xyl-5 rpsL20 mtl-1 proA2 leu1
    MG4(pKDT17)E. coli MG4 with reporter plasmid pKDT1739
Plasmids
    pKD3Template plasmid; cat flanked by FRT sites; Cmr Apr9
    pKD4Template plasmid; kan flanked by FRT sites; Kmr Apr9
    pKD46Red recombinase expression plasmid; Apr9
    pCP20Suicide plasmid with temperature-sensitive replication and thermally induced expression of FLP recombinase; Apr Cmr9
    pLD55Suicide vector with oriR6Kγ and fusaric acid selection; Apr Tcr28
    pLD55ΔyadBCFragment Y1-4 (∼1.5 kb) inserted into SpeI site of pLD55; deletes bp 1 to 2904 of yadBC; Apr TcrThis study
    pLD55yadBC-LEntire yadBC operon flanked by 560 bp upstream and 551 bp downstream cloned into pLD55 (4,146-bp insertion); restores yadBC to a ΔyadBC mutant or the individual genes to single yadB or yadC mutantsThis study
    pWSK295.4 kb, Apr Kmr, low-copy-number cloning vector48
    pYadBC5.8-kb BglII fragment (bp −2352 to 3494) cloned into BglII site of pWSK29; 11.2 kb; Apr KmrThis study
    pBAD244.5 kb, Apr, expression vector using ParaBAD to drive gene expression17
    pBAD24YadBC3.0-kb yadBC cloned into EcoRI/HindIII-cut pBAD24; 7.5 kb; AprThis study
    pLD55ΔDegPFragment ΔdegPUp1-ΔdegPdown2 (ca. 2 kb) inserted into XhoI/SacI site of pLD55; deletes 1,523 bp, from −50 bp upstream of degP through degP and 27 bp downstream; AprThis study
    pPCP1::Kan10.6 kb; Kmr; kan gene from pKD4 inserted into pPCP1 at bp 2135 to 2221 in the intergenic region downstream from the IS100 ATP-binding protein (YPPCP1.02); construction deleted sequence between 2135 and 2221This study
    pPCP1::Kan-fsPlaDerivative of pPCP1::Kan in which a frameshift in pla was created by filling in the BamHI site at codon 79; Kmr PlaThis study
    pBSlacZMCSCloning vector with rrnBT1 transcriptional terminator and lacZ gene from pEU730; Apr36
    pBS-PyadBC-lacZ207-bp Y. pestis yadBC promoter region (bp −205 to 3 of yadBC) inserted into KpnI/Acc65I-digested pBSlacZMCSThis study
    pSucinvSuicide vector with R6K ori and sacB selection containing the 2-kb StuI/XhoI fragment (contains bp 709 to 2706 [amino acids 237 to 902] of Y. pseudotuberculosis invA; Apr38
    pSucinv::yadBC-lacZ4.4-kb PyadBC-lacZ EagI fragment of pBS-PyadBC-lacZ inserted into EagI site of pSucinvThis study
    pSucinv::lacZPromoterless lacZ cloned into the EagI site within invA in pSucinv; AprSpencer Leigh
    pCD2ApLcr virulence plasmid of Y. pestis CO99-3015.S5 into which the bla gene from pRL494e was inserted within the yadA pseudogene (pCD2 yadA::bla); AprbSpencer Leigh
    pGEX-3XlacIq lacP-gst; AprPharmacia
    pGEX-YadB25-150378-bp EcoRI/SmaI yadB fragment (amplified by BA11 and BA22) inserted into EcoRI/SmaI-digested pGEX-3XThis study
    pGEX-YadC137-409819-bp EcoRI/SmaI yadB fragment (amplified by CA11 and CA22) inserted into EcoRI/SmaI-digested pGEX-3XThis study
  • a Full virulence is conditional on the intranasal route of infection (10).

  • b See reference 13.