TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristics | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | Cloning host | 3 |
| DH5α(λpir) | Strain for propagating plasmid with R6K origins; derived from DH5α | S. C. Straley |
| 1017 | ent::Tn5 Kmr; derived from HB101 | 14 |
| Y. pestis strainsa | ||
| KIM6+ | Lcr− Pgm+ (Ybt+) Hmu+ Yfe+ Yfu+ | 18; this study |
| KIM6 | Lcr− Pgm− (Δpgm Ybt−) Hmu+ Yfe+ Yfu+ | 18; this study |
| KIM6-2030 | Lcr− Pgm− (Δpgm Ybt−) Hmu+ Yfe+ Yfu+ Fur− (fur::kan-9) | 44 |
| KIM6-2031.1+ | Lcr− Pgm+ (Ybt+) Hmu+ Yfe− (ΔyfeAB2031.1) Yfu+ | 5 |
| KIM6-2031.1 | Lcr− Pgm− (Δpgm Ybt−) Hmu+ Yfe− (ΔyfeAB2031.1) Yfu+ | 5 |
| KIM6-2082+ | Lcr− Pgm+ (Ybt+) Hmu+ Yfe+ Yfu− (ΔyfuA2082); derived from KIM6+ | This study |
| KIM6-2082 | Lcr− Pgm− (Δpgm Ybt−) Hmu+ Yfe+ Yfu− (ΔyfuA2082); derived from KIM6 | This study |
| KIM6-2082.1+ | Lcr− Pgm+ (Ybt+) Hmu+ Yfe− (ΔyfeAB2031.1) Yfu− (ΔyfuA2082); derived from KIM6-2031.1+ | This study |
| KIM6-2082.1 | Lcr− Pgm− (Δpgm Ybt−) Hmu+ Yfe− (ΔyfeAB2031.1) Yfu− (ΔyfuA2082); derived from KIM6-2031.1 | This study |
| KIM5-2031.12+ | Lcr+ (pCD1Ap, 'yadA::bla) Pgm+ (Ybt+) Hmu+ Yfe− (ΔyfeAB2031.1) Yfu+ Apr; derived from KIM6-2031.1+ | This study |
| KIM5-2082.3+ | Lcr+ (pCD1Ap, 'yadA::bla) Pgm+ (Ybt+) Hmu+ Yfe+ Yfu− (ΔyfuA2082) Apr; derived from KIM6-2082+ | This study |
| KIM5(pCD1Ap)+ | Lcr+ (pCD1Ap, 'yadA::bla) Pgm+ (Ybt+) Hmu+ Yfe+ Yfu+ Apr; derived from KIM6+ | This study |
| Plasmids | ||
| pACYC184 | 4.2-kb cloning vector; Cmr Tcr | 12 |
| pBluescript II KS+ | 3.0-kb cloning vector; Apr | 3 |
| pBGL2 | 4.8-kb cloning vector; Apr Tcr | 35 |
| pBR322 | 4.4-kb cloning vector; Apr Tcr | 3 |
| pEU730 | 15.2-kb single copy reporter vector; promoterless lacZ; Spr | 19 |
| pKNG101 | 6.8-kb suicide vector; SacB+ R6K origin; Smr | 27 |
| pRL494e | 3.7-kb vector with ampicillin resistance cassette (bla); Apr Kmr | 15 |
| pWSK129 | 6.7-kb low-copy-number cloning vector; Kmr | 52 |
| pCD1 | 70.5-kb Lcr plasmid of KIM5 | 36 |
| pCD1Ap | 70.5-kb pCD1 with bla cassette inserted into 'yadA; 71.7-kb Lcr+ Apr | This study |
| pBGCD3 | 9.98-kb BglII fragment from pCD1 ligated into pBGL2; 14.8 kb 'yadA'/'yadA, Apr Tcr | This study |
| pWSKYadA | 3.7-kb BglII-SmaI fragment from pBGCD3 ligated into the BamHI-EcoRV sites of pWSK129; 10.4 kb, yadA'/'yadA, Kmr | This study |
| pACYCYadA | 1.9-kb ScaI-HindIII fragment from pWSKYadA ligated into the EcoRV-HindIII sites of pACYC184; 6.0 kb, 'yadA, Cmr | This study |
| pACYCYadAp | 1.2-kb EcoRV fragment containing bla cassette from pRL494e ligated into pACYCYadA; 7.2 kb, 'yadA::bla, Cmr Apr | This study |
| pKNGYadAp | 3.6-kb XbaI-SalI fragment from pACYCYadAp ligated into pKNG101; 10.1 kb, 'yadA::bla, R6K origin, Smr | This study |
| pYFU238 | 8.9-kb Sau3AI Y. pestis chromosomal fragment ligated into BamHI site of pLG338; 16.2 kb, yfuAB+yfuC', Kmr | 35; this study |
| pYFU1 | 9.8-kb XhoI-PstI Y. pestis chromosomal fragment ligated into pBluescript II KS+; 12.8 kb, Yfu+ Apr | This study |
| pYFU2 | 6.7-kb BglII-SalI fragment from pYFU238 ligated into BamHI-SalI sites of pWSK129; 13.4 kb, Yfu− (yfuAB+yfuC') Kmr | This study |
| pYFU3 | Deletion of 1.8- and 0.2-kb BamHI fragments in yfuA from pYFU2; 11.4 kb, Yfu− (ΔyfuA2082 [YfuABC−]) Kmr | This study |
| pYFU3.1 | 4.7-kb XbaI-SalI fragment from pYFU3 ligated into pKNG101; 11.2 kb, ΔyfuA2082 (YfuABC−), Smr | This study |
| pYFU4 | 310-bp yfuABC promoter ligated into pEU730; 15.5-kb yfu::lacZ fusion; Spr | This study |
| pYFU5 | 10.1-kb PvuII-XbaI fragment from pYFU1 cloned into pBR322; 14.5 kb, Yfu+ Apr Kmr | This study |
| pYFU6 | 6.7-kb XbaI-XhoI fragment from pYFU2 cloned into pBR322; 10.7 kb, Yfu− (yfuAB+yfuC) Apr Kmr | This study |
a
Strains designated by a “+” suffix possess an intact pgm locus (i.e., Ybt+ Hms+); other strains have either deletion of the 102-kb pgm chromosomal locus or a mutation within the locus (Ybt− or Hms−). KIM6 strains lack the pCD1 plasmid (Lcr−), while KIM5 strains possess pCD1 or pCNAp (Lcr+); all strains possess the hemin utilization locus (Hmu+).