TABLE 1.
Bacterial strains and plasmids used in these experiments
| Bacterial strain or plasmid | Resistance phenotype or genotypea | Description (reference or source) |
|---|---|---|
| Strains | ||
| E. faecium | ||
| D344R | Apr Eryr Tcr | High-level ampicillin-resistant clinical isolate (26) |
| D344SRF | Aps Eryr Tcr Fusr Rifr | pbp5-negative, ampicillin-susceptible derivative of D344R (16) |
| C68 | Apr Eryr Tcr Vmr | Clinical isolate initially used to define sequences downstream of pbp5 (4) |
| GE-1 | Aps Kanr Fusr Rifr | pbp5-negative recipient for enterococcal mating experiments (4) |
| E. coli DH10B | ΔM15 ΔlacX74 deoR recA1 ara Δ139 Δ(ara leu)7697 galU galK λ-rpsL endA1 nupG | Transformation-competent E. coli (Invitrogen, Carlsbad, Calif.) |
| Plasmids | ||
| pIndigoBAC-5 | Cmr | Cloning vector for large DNA fragments (Epicentre Technologies, Madison, Wis.) |
| pBCSK(−) | Cmr | Cloning vector for smaller DNA fragments (Stratagene, La Jolla, Calif.) |
| pCR-XL-Topo | Cmr | Linearized vector with TA overhang for cloning PCR products (Invitrogen, Carlsbad, Calif.) |
| pACYC184 | Cmr Tcr | Cloning vector for smaller fragments (21) |
| pCWR796 | Cmr | Ca.-40-kb BglII fragment from D344R containing intact Tn5386 cloned into BamHI site of pIndigoBAC-5 (this study) |
| pCWR808 | Cmr | Ca.-40-kb BgIII fragment from D344SRF containing intact Tn5386 cloned into BamHI site of pIndigoBAC-5 (this study) |
| pCWR845 | Cmr Tcr | Ca.-25 to -30-kb BgIII fragment from D344R containing intact Tn916 cloned into BamHI site of pIndigoBAC-5 (this study) |
| pCWR850 | Cmr | 1.2-kb HincII fragment containing the left junction of Tn916 in D344R cloned into pBCSK(−) (this study) |
| pCWR854 | Cmr | 3.5-kb HincII fragment containing the right junction of Tn916 cloned into pACYC184 (this study) |
| pCWR815 | Cmr | 6-kb BglII/HincII fragment containing the left junction of Tn5386 cloned into pBCSK(−)—4.4 kb of Tn5386 (this study) |
| pCWR869 | Cmr | 3.5-kb EcoRV fragment of PCR product containing the right junction of Tn5386 in D344R (identical to fragment from D344S)—2.7 kb of Tn5386 cloned into pBCSK(−) (this study) |
| pCWR822 | Cmr | 4.7-kb HincII fragment containing the left junction of Tn5386 in D344SRF—4.4 kb of Tn5386 (this study) |
a
Ap, ampicillin; Cm, chloramphenicol; Ery, erythromycin; Fus, fusidic acid; Kan, kanamycin; Rif, rifampin; Tc, tetracycline; and Vm, vancomycin.