TABLE 1.
Bacterial strains and plasmids used
| Strain or plasmid | Relevant characteristic(s)a | Source or reference(s) |
|---|---|---|
| E. coli strains | ||
| DH5α | Cloning host | 3 |
| HB101 1017 | ent::Tn5, Kmr | 11 |
| M15(pREP4) | pREP4 (lacI, Kmr); host for inducing protein expression from pQE30-derived clones | 69; Qiagen, Inc. |
| SY327 (λpir) | Host for propagating pCVD442-derived clones | 36 |
| Y. pestis strainsb | ||
| KIM5-2044.11 | Pgm− Hum− (ΔhmuP′RSTUV-2044.1) Lcr+ YopJ− [pCD1::Mu dI1734-73 (yopJ::Mu dI1734)], Kmr; derived from KIM6-2044.1 | This study |
| KIM5-2044.21+ | Pgm+ Psa− (Δpsa2053.1) Hmu− (ΔhmuP′RSTUV-2044.1) Lcr+ YopJ− [pCD1::Mu dI1734-73 (yopJMu dI1734)], Kmr; derived from KIM6-2044.2+ | This study |
| KIM5-2060.21+ | Pgm+ Psa− (Δpsa2053.1) Hmu− (ΔhmuP′RSTUV-2060.1) Lcr+ YopJ− [pCD1::Mu dI1734-73 (yopJ::Mu dI1734)], Kmr; derived from KIM6-2060.2+ | This study |
| KIM5-3173 | Pgm− Hmu+ Lcr+ YopJ− [pCD1::Mu dI1734-73 (yopJ::Mu dI1734)], Kmr | 48 |
| KIM6+ | Pgm+ Lcr− Psa+ Hmu+ | 45, 55, 56 |
| KIM6 | Pgm− (Δpgm [Δhms Δybf+) Lcr− Psa+ Hmu+ | 44, 46, 55, 56 |
| KIM6-(pRT240)+ | Pgm+ Lcr− Psa+ Hmu+ pRT240 [LacZ(Con), Apr] | 58 |
| KIM6-2030+ | Pgm+ Lcr− Psa+ Fur− (fur9::kan) Hmu+ Kmr | 58 |
| KIM6-2044.1+ | Pgm+ Lcr− Psa+ Hmu− (Δorf-XYhmuP′RSTUV-2044.1) | 24 |
| KIM6-2044.1 | Pgm− Lcr− Psa+ Hmu− (Δorf-XYhmuP′RSTUV-2044.1); Δpgm mutant from KIM6-2044.1+ | This study |
| KIM6-2044.2+ | Pgm+ Lcr− Psa− (Δpsa2053.1) Hmu− (ΔhmuP′RSTUV-2044.1); derived from KIM6-2044.1(pCVDPSA1)+ | This study |
| KIM6-2053.1+ | Pgm+ Lcr− Psa− (Δpsa2053.1) Hmu+ | 4 |
| KIM6-2060.1+ | Pgm+ Lcr− Psa+ Hmu− (ΔhmuP′RSTUV-2060.1); derived from KIM6(pHMU61)+ | This study |
| KIM6-2060.2+ | Pgm+ Lcr− Psa− (Δpsa2053.1) Hmu− (ΔhmuP′RSTUV-2060.1); derived from KIM6-2053.1(pHMU61)+ | This study |
| KIM6-2061.1+ | Pgm+ Lcr− Psa+ Hmu− (in-frame ΔhmuR-2061.1); derived from KIM6(pHMU47)+ | This study |
| KIM6-2062.1+ | Pgm+ Lcr− Psa+ Hmu− (in-frame ΔhmuS-2062.1); derived from KIM6(pHMU64)+) | This study |
| KIM6-2063.1+ | Pgm+ Lcr− Psa+ Hmu− (hmuT::cat-2063.1); derived from KIM6(pHMU48)+ | This study |
| Plasmids | ||
| pACYC184 | 4.2-kb cloning vector; Cmr Tcr | 3 |
| pBluescriptII KS+ | 3.0-kb cloning vector; Apr | Stratagene |
| pBR322 | 4.4-kb cloning vector; Apr Tcr | 3 |
| pCD1::Mu dl1734-73 | Modified Lcr plasmid pCD1 (yopJ::Mu dI1734); Lcr+ YopJ−, Kmr | 62 |
| pCVD442 | 6.2-kb suicide vector, pir-dependent replication; Apr, SacB+ | 13 |
| pCVDPSA1 | Δpsa suicide plasmid construct; Apr | 4 |
| pEU730 | 15.2-kb cloning vector, promoterless lacZ; Spcr | 17 |
| pKRP10 | 3.7-kb plasmid carrying Cmr cassette; Apr Cmr | 49 |
| pQE30 | 3.5-kb His-tagged protein expression cloning vector; Apr | Qiagen, Inc. |
| pHMU4 | 10.7-kb EcoRI-BglII fragment cloned into pBR322; Apr, Hmu+ (orfXY+ hmuP′RSTUV+) | 24 |
| pHMU6 | 6.2-kb EcoRI-SalI fragment cloned into pBR322; Apr, Hmu− (orfXY+ hmuP′RST+ hmuU′) | 24 |
| pHMU7 | 8.6-kb EcoRI-PstI fragment cloned into pBR322; Tcr, Hmu+ (orfXY+ hmuP′RSTUV+) | 24 |
| pHMU23 | Mu dI1734 insertion into pHMU4; Apr Kmr, Hmu− (hmuR::Mu dI1734) | 24 |
| pHMU28 | 2.1-kb EcoRI-EcoRV fragment from pHMU6 cloned into pBluescriptII KS+; Apr | This study |
| pHMU30 | 2.6-kb AseI fragment from pHMU6 cloned into pACYC184; Cmr Tcr, hmuP′R+ | This study |
| pHMU35 | 7.8-kb PvuI-PstI fragment from pHMU4 cloned into pBR322 lacking bla promoter; Tcr, hmuP′RSTUV+ | This study |
| pHMU37 | 5.4-kb EcoRI-NruI fragment from pHMU6 cloned into pBR322; Tcr, hmuP′RS+ hmuT′ | This study |
| pHMU39 | 0.7-kb ClaI-EcoRV fragment from pHMU30 and 1.0-kb DraI-BamHI fragment from pHMU30 cloned sequentially into pBluescriptII KS+; Apr, in-frame ΔhmuR-2061.1 | This study |
| pHMU43 | ∼1.9-kb fragment containing hmuR (minus signal sequence region) cloned into pQE30; Apr, His-tagged HmuR | This study |
| pHMU44 | 123-bp AseI-NdeI fragment from pHMU28, blunt ended and cloned into pEU730; Spcr, p1::lacZ reporter fusion | This study |
| pHMU46 | 1.7-kb PvuII-SalI fragment from pHMU6 cloned into pBluescriptII KS+ and 0.8-kb SmaI fragment of pKRP10 cloned within NruI site of construct; Apr Cmr, hmuT::cat-2063.1 | This study |
| pHMU47 | 1.8-kb SalI-SacI fragment from pHMU39 cloned into pCVD442; Apr, in-frame ΔhmuR-2061.1 | This study |
| pHMU48 | 2.5-kb XhoI-SacI fragment from pHMU46 cloned into pCVD442; Apr Cmr, hmuT::cat-2063.1 | This study |
| pHMU51 | 2.1-kb DraI-HpaI fragment from pHMU6 cloned into pBR322; Apr, hmuS+ hmuT′ | This study |
| pHMU52 | 1.0-kb PCR fragment of hmuS from pHMU6 cloned into pEQ30; Apr, His-tagged HmuS | This study |
| pHMU53 | 1.23-kb BstBI-EcoRV fragment from pHMU51 replaced with 0.26-kb BstBI-EcoRV PCR fragment generated with gene splicing by overlap extension technology, as reported by Ho et al. (23); Apr, in-frame ΔhmuS | This study |
| pHMU55 | 130-bp PCR product generated from end of hmuR to start of hmuS with pHMU6 and cloned into pEU730; Spcr, p2::lacZ reporter fusion | This study |
| pHMU60 | 5.9-kb deletion consisting of 1.1-kb SspI fragment and 4.8-kb SspI-SwaI fragment from pHMU7; Tcr; ΔhmuP′RSTUV-2060.1 | This study |
| pHMU61 | 1.8-kb NdeI-SspI fragment from pHMU60 cloned into pCVD442; Apr, ΔhmuP′RSTUV-2060.1 | This study |
| pHMU62 | 3.7-kb SspI-SalI fragment from pHMU6 cloned into pBR322; Apr, hmuST+ hmuU′ | This study |
| pHMU63 | 1.23-kb BstBI-EcoRV fragment from pHMU62 replaced with 0.26-kb BstBI-EcoRV fragment from pHMU53; Apr, in-frame ΔhmuS-2062.1 | This study |
| pHMU64 | 2.7-kb SspI-NheI fragment from pHMU63 cloned into pCVD442; Apr, in-frame ΔhmuS-2062.1 | This study |
| pHMU66 | 2.1-kb PmlI-Bsu36I fragment from pHMU4 replaced with ∼1.1-kb PmlI-Bsu36I fragment from pHMU63; Apr, in-frame ΔhmuS hmuP′RSTUV+ | This study |
a
Pgm, pigmentation (102-kb chromosomal locus containing genes of hemin storage system [hms] and siderophore biosynthetic and transport system [ybt/psn] [44]); Lcr, low Ca2+ response (associated with pCD1); psa, gene encoding pH 6 antigen; Psa, pH 6 antigen, Hmu, hemin utilization; LacZ(Con), constitutive expression of β-galactosidase; ent, enterobactin gene.
b
KIM5 strains contain a form of pCD1 (Lcr plasmid), while KIM6 strains have been cured of the Lcr plasmid. A “+” after strain name designates a Pgm+ phenotype.