TABLE 4 .
Bacterial strains and plasmids used in this study
| Strain or plasmid | Description | Resistance trait(s)a | Source |
|---|---|---|---|
| E. faecalis strains | |||
| LS4828 | Penicillin-resistant clinical isolate | Penr Ampr | This study |
| JH2-2 | Penicillin-sensitive isolate | Pens Amps | 19 |
| CH19 | β-Lactamase-producing isolate | 24 | |
| OG1X | Transformation recipient strain | Pens Spes | 25 |
| E. faecium D366 | Pens | 26 | |
| E. coli strains | |||
| DH5α | 27 | ||
| BL21 Star(DE3) | |||
| Plasmids | |||
| pBSU101 | Shuttle vector allowing expression of egfp in both Gram-negative and Gram-positive organisms | Sper | 11 |
| pRIH141 | Promoter region of pbp4 from LS4828 inserted upstream of egfp between EcoRI and BamHI sites of pBSU101 | Sper | This study |
| pRIH143 | Promoter region of pbp4 from JH2-2 inserted upstream of egfp between EcoRI and BamHI sites of pBSU101 | Sper | This study |
| pET-RP1B | Expression vector allowing amino-terminal His tag fusions | Kanr | 20 |
| pWP101 | pbp4Δ35 from LS4828 cloned into pET-RP1B for overexpression in E. coli | Kanr | This study |
| pWP102 | pbp4Δ35 from JH2-2 cloned into pET-RP1B for overexpression in E. coli | Kanr | This study |
| pRIH145 | pWP102 with Ala617 mutated to Thr | Kanr | This study |
| pRIH146 | pWP102 with Val223 mutated to Ile | Kanr | This study |
a
Abbreviations: Pen, penicillin; Spe, spectinomycin; Kan, kanamycin.