TABLE 1.
Bacterial strains and plasmids used in this studya
| Strain or plasmid | Relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| Y. pestis | ||
| KIM6+ | Pgm+ Hms+ Pla+ | 14 |
| KIM6 | Pgm− (Δpgm; Hms−) Pla+ | 14 |
| KIM6-2008 | Kmr Hms− (hmsH2008::mini-kan) Pla+ | 36 |
| KIM6-2011 | Kmr Hms− (hmsF2011::mini-kan) Pla+ | 36 |
| KIM6-2012 | Kmr Hms− (hmsR2012::mini-kan) Pla+ | 36 |
| KIM6-2051+ | Kmr Hms− (hmsT2051::mini-kan) Pla+ | 25 |
| KIM6-2057.1 | Hms− (ΔhmsR46, in-frame deletion) Pla+ | 29 |
| KIM6-2058.1 | Hms− (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 |
| KIM6-2097 | Apr Pgm− (Δpgm; Hms−) inv::pHinv hmsH::lacZ Pla+ | This study |
| KIM10+ | Hms+ Pla− (cured of pPCP1) | 40 |
| E. coli | ||
| BL21 | Strain for expression of GST fusion proteins | Pharmacia Biotech |
| DH5α | Cloning strain | 2 |
| DH5α (λpir) | Strain for maintenance of R6K ori suicide plasmids | S. C. Straley |
| M15 (pREP4) | His tag expression strain | 62; Qiagen, Inc. |
| Plasmids | ||
| pAHMS16 | 4.4 kb, AprhmsT+, 1.9-kb KpnI-FspI fragment from pAHMS14 into KpnI-HincII sites of pUC18 | 25 |
| pBluescript II KS+ | 2.9 kb, Apr, high-copy-number cloning vector | Stratagene |
| pBSlacZMCS | 7.1 kb, Apr, high-copy-number cloning vector with rrnBT1 transcriptional terminator and lacZ gene from pEU730 | This study |
| pCVD442 | 6.2 kb, AprsacB, R6K ori, suicide vector | 11 |
| pCVDHmsH49 | Apr suicide plasmid encoding hmsH49 from pNPM49 | This study |
| pEU730 | 15.2 kb, Smr Spcr, low-copy-number vector with promotorless lacZ | 15 |
| pGEX-2T | 5.0 kb, AprlacIq GST fusion protein plasmid | Pharmacia Biotech |
| pGEX2T-HmsT | 5.6 kb, Apr, GST-′HmsT expression vector, 588-bp hmsT PCR product ligated into SmaI-BamHI sites of pGEX-2T | This study |
| pGstHmsS | 5.2 kb, Apr, GST-′HmsS expression vector, 231-bp hmsS PCR product ligated into BamHI-EcoRI sites of pGEX-2T | This study |
| pHinv | 11.2 kb, Apr, 4.5-kb EagI fragment from pHmsHlacZ ligated into corresponding site in pSucinv, lacZ expression driven by hmsH promoter region | This study |
| pHMS1.2 | 13.4 kb, AprhmsHFRS+, 9.7-kb SalI-HindIII fragment from pHMS1 into pBR322 | 36 |
| pHMS1.2SL | 13.4 kb, Apr, hmsHFRS with disrupted stem-loop in hmsF | This study |
| pHMS9 | 16.4 kb, Kmr, hmsHFRS with disrupted stem-loop in hmsF, 4.3-kb PmlI-SalI fragment from pHMS1.2SL ligated into corresponding sites of pHMS1 | This study |
| pHmsHlacZ | 7.4 kb, Apr, 314-bp PCR product from pHMS1.2 ligated into PstI-Asp718 sites of pBSlacZMCS | This study |
| pHmsTlacZ | 7.5 kb, Apr, 418-bp PCR product from pAHMS16 ligated into PacI-PmeI sites of pBSlacZMCS | This study |
| pKD3 | 2.8 kb, Cmr, template plasmid | 9 |
| pKD4 | 3.3 kb, Kmr, template plasmid | 9 |
| pKD46 | 6.3 kb, Apr, Red recombinase expression plasmid | 9 |
| pKRP10 | 2.9 kb, Apr Cmr, cam cassette plasmid | 42 |
| pLG338 | 7.3 kb, Kmr Tcr, low-copy-number cloning vector | 55 |
| pNPM49 | ∼16.4-kb, KmrhmsFRS+hmsH, spontaneous mutant | 30 |
| pQE30 | 3.5 kb, Apr, His6 tag expression vector | Qiagen, Inc. |
| pQE30H.6 | 5.6 kb, Apr, His6 tag-′HmsH expression vector; 2.2-kb NcoI-XhoI fragment from pHMS1.2 ligated into HindIII-SalI sites of pQE30 | This study |
| pQE32 | 3.5 kb, Apr, His6 tag expression vector | Qiagen, Inc. |
| pQE32F.2 | 6.0 kb, Apr, His6 tag ′HmsF expression vector; 2.5-kb PmlI-PvuII fragment from pHMS1.2 ligated into SmaI site of pQE32 | This study |
| pRI203 | 9.0 kb, Apr, encodes Y. pseudotuberculosis inv | 22; V. L. Miller |
| pSUC1 | 4.7 kb, Apr, suicide vector derived from pCVD442, sacB, R6K ori | 12 |
| pSucinv | 6.7 kb, Apr, suicide vector with 2.0-kb StuI-XhoI fragment of Y. pseudotuberculosis inv gene from pRI203 ligated into SmaI-SalI sites of pSUC1 | This study |
| pSucinvII | 6.1 kb, Cmr, suicide vector with Y. pseudotuberculosis inv, pSucinv with bla replaced by 0.8-kb EcoRI-HincII fragment from pKRP10 encoding cam | This study |
| pTinvII | 10.6 kb, Cmr, 4.6-kb EagI fragment from pHmsTlacZ ligated into corresponding site in pSucinvII, lacZ expression driven by hmsT promoter region | This 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.