TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant properties | Reference or source |
|---|---|---|
| E. coli | ||
| BMH 71-18 mutS | thi supE Δ(lac-proAB) [mutS::Tn10] [F′ proAB laqIqZΔM15] | Promega |
| DH5α | end-1 hsdR17 (rK− mK+) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacIZYA-argF)U169 deoR[φ80dlacΔ(lacZ)M15] | GIBCO-BRL |
| Y. pestisa | ||
| KIM5-3001 (wt) | Smr; pCD1 (LCR+), pPCP1(Pla+) pMT1 | 38 |
| KIM5-3001.16 (ΔyscO) | Smr; pCD1 yscO(Δ6–151),b pPCP1(Pla+), pMT1 | This study |
| KIM6-3001 | Smr; pCD1− (LCR−), pPCP1(Pla+) | 38 |
| KIM8-3002 (wt Pla−) | Smr; pCD1, pPCP1−(Pla−), pMT1 | 75 |
| KIM8-3002.3 (ΔyscO Pla−) | Smr; pCD1 yscO(Δ6–151),b pPCP1−(Pla−), pMT1 | This study |
| Plasmids | ||
| pBluescript II SK+ | Apr; cloning vector | Stratagene |
| pBluescript II SK− | Apr; cloning vector | Stratagene |
| pYP-F2 | Apr; BamHI F fragment cloned from pBGCD1 carrying yscN′OPQRS, with frameshift mutation in yscR, was cloned into pBluescript II SK− with the insert oriented with lac promoter | 20 |
| pYscOP.2 | Apr; 2.38-kb Bpu 1102I fragment of pYP-F2 carrying yscO and yscP was filled in with Klenow and cloned into XhoI-digested/Klenow blunt-ended pBluescript II SK+ with the insert oriented with the lac promoter | This study |
| pYscOP | Apr; same as pYscOP.2 with insert oriented with the T7 promoter | This study |
| pYscO.2 | Apr; SphI-KpnI (KpnI site in vector) digestion of pYscOP.2 followed by blunt ending with T4 polymerase and religation resulting in the elimination of yscP and carrying yscO | This study |
| pYscO | Apr; ClaI (one site in vector) digest of pYscOP followed by religation of plasmid, carries yscO | This study |
| pΔyscO | Apr; AvrIIc-digested pyscOP.2 was filled in with Klenow and religated, resulting in a deletion of yscO (Δ6–151)b | This study |
| pYscN′ΔOP | Apr; 400-bp SacII fragment of pYP-F2 ligated with ∼5.0-kb SacII fragment of pΔyscO carrying yscN′, yscO (Δ6–151),b and yscP | This study |
| pYscP | Apr; 1.8-kb AvaI fragment of pYP-F1 (20) carrying yscP, filled in with Klenow and cloned into EcoRV site of pBluescript II SK+ with insert oriented with the T7 promoter | This study |
| pHTV | Apr; expression vector carrying lcrV; translationally fused to a leader encoding 19 residues, including 6 histidines | 21, 45 |
| pTRCM.2 | Apr; expression vector carrying yopM behind the trc promoter | 56 |
| pGEX-3X | Apr; GST fusion expression vector using the tac promoter | Pharmacia |
| pGST-YscO | Apr; 1.9-kb MluI-EcoRI (vector site) fragment of pyscO filled in with Klenow following MluI digestion and directionally cloned into SmaI-EcoRI-digested pGEX-3X (expresses fusion protein of GST and aa 13–154 of YscO) | This study |
| pUK4134 | Apr; suicide vector oriR6K oriT cos rpsL | 65 |
| pUKΔyscO | Apr; ∼2.3-kb XbaI (in vector site)-AgeI fragment of pyscN′ΔOP filled in with Klenow and cloned into the EcoRV site of pUK4134, carries yscO (Δ16–151)b | This study |
| pCVD442 | Apr Sucs; suicide vector | 16 |
| pCVD442ΔyscO | Apr Sucs; same insert as in pUKΔyscO cloned into the SmaI site of pCVD442 | This study |
a
All Y. pestis strains are Pgm− (76).
b
Numbers in parentheses give the amino acids deleted from the protein product.
c
AvrII sites introduced by site-directed mutagenesis (see Materials and Methods).