TABLE 1.
Bacterial strains and plasmids used in this studya
| Strain or plasmid | Relevant characteristic(s) | Reference or Source |
|---|---|---|
| E. coli | ||
| DH5α | Cloning host | 2 |
| DH5α (λpir) | Strain for propagating plasmids with R6K origins, derived from DH5α | S. C. Straley |
| Y. pestis | ||
| KIM6+ | Pgm+ (Hms+ Ybt+) Lcr− | 25 |
| KIM6 | Pgm− (Δpgm − Hms− Ybt−) Lcr− | 25 |
| KIM6-2046.1 | Hms+ Ybt− (irp2::kan2046.1) Lcr− Kmr | 25 |
| KIM6-2055 | Hms+ Ybt− (ybtA::kan2055) Lcr− Kmr | 23 |
| KIM6-2085+ | Hms+ Ybt− (ΔybtD2085) Lcr− | This study |
| KIM6-2086 | Hms+ Ybt− (irp1-2086) Lcr− | This study |
| KIM6-2086.1+ | Pgm+ (Hms+ Ybt+) Lcr−; derived from KIM6-2086 by replacement of the irp1-2086 mutation with irp1+ | This study |
| Plasmids | ||
| pACYC184 | 4.2-kb cloning vector; Cmr Tcr | 17 |
| pEU730 | 15.2-kb low-copy-number vector with promoterless lacZ; Spcr Smr | 28 |
| pEUYbtP | 15.4-kb low-copy-number ybtP::lacZ reporter | 24 |
| plasmid, Spcr; iron-, Fur-, and YbtA-regulated expression of β-galactosidase | ||
| pKNG101 | 6.8-kb suicide vector; sacB+, R6K origin; Sucs Smr | 44 |
| pWSK29 | 5.4-kb low-copy-number cloning vector; Apr | 74 |
| pET22b-HMWP1-TEmut | 9.49-kb NcoI/XhoI fragment of irp1, with altered bp, in pET22b; Apr, 15.0 kb, irp1-2086 (HMWP1-S2908A) | Z. Suo and C. T. Walsh |
| pEUYbtD1 | 15.4-kb low-copy-number ybtD::lacZ reporter plasmid; 182-bp AscI/KpnI fragment containing ybtD promoter region cloned into AscI/KpnI sites of pEU730; Spcr Smr; fusion of ybtD 167-bp promoter to lacZ | This study |
| pEUYbtD2 | 15.6-kb low-copy-number ybtD::lacZ reporter plasmid; 355-bp AscI/KpnI fragment containing ybtD promoter region cloned into AscI/KpnI sites of pEU730; Spcr Smr; fusion of ybtD 342-bp promoter to lacZ | This study |
| pIrp1TE2 | 1.37-kb PvuII/XhoI fragment from pET22b-HMWP1-TEmut cloned in SmaI/SalI sites of suicide vector pKNG101; Sucs Smr, 8.2 kb, irp1-2086 (HMWP1-S2908A) | This study |
| pKNGIRP1 | 1,703-bp PCR product from pPSN3 ligated into the | This study |
| BamHI/XbaI site of pKNG101; sacB+, R6K origin; Sucs Smr, 8.5 kb; irp1-TE+ | ||
| pPSN3 | 9,994-bp SalI fragment encompassing the irp1-ybtE region in pBLG2; Apr; 15.8 kb | 24 |
| pYBTD1 | 4.44-kb XhoI/BglII fragment from Y. pestis KIM6+ genomic DNA cloned into XhoI/BamHI sites of pWSK29; Apr; 9.8 kb; ybtD+ | This study |
| pYBTD2 | 774-bp EcoRV/AsuII fragment within ybtD removed from pYBTD1; Apr; 9.0 kb; ΔybtD | This study |
| pYBTD3 | 3.7-kb XbaI/ApaI fragment from pYBTD2 cloned into XbaI/ApaI sites of suicide vector pKNG101; Suc Smr; ΔybtD | This study |
| pYBTD4 | 0.93-kb BamHI/XbaI PCR product of ybtD and its putative promoter region ligated into Bam HI/XbaI sites of pACYC184; Cmr; 10.5 kb; ybtD+ | This study |
a
Y. pestis strains with a plus sign possess an intact 102-kb pgm locus containing the genes for hemin storage (hms) and the Ybt system. All other Y. pestis strains contain a mutation within the pgm locus due to either a deletion or insertion of an antibiotic resistance cassette. Strains synthesizing the siderophore Ybt are designated Ybt+, while those affected in Ybt production are Ybt−. Lcr− indicates the absence of the low-calcium-response virulence plasmid pCD1. Abbreviations: Apr, Kmr, Spcr, Smr, Tcr, and Cmr, resistance to ampicillin, kanamycin, spectinomycin, streptomycin, tetracycline, and chloramphenicol, respectively.