TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Description | Reference(s) or source |
|---|---|---|
| A. keyseri 12B | Grows with o-phthalate and dibutylphthalate | 24, 43, 45 |
| A. keyseri 12B-C14 | Derivative of A. keyseri 12B containing pRE1 (cured of pRE2 and pRE3) | This study |
| E. coli JM109 | recA endA1 gyrA96 thi hsdR17 supE44 relA1 Δ(lac-proAB) (F′ traD36 proAB lacIqZΔM15) | 91 |
| E. coli BL21(DE3)(pLysS) | F−ompT hsdSB(rB mB−) gal dcm (DE3) pLysS. λDE3 prophage carries T7 RNA polymerase under lacUV5 control; Cmr; obtained from Novagen | 82 |
| pBBR1MCS2 | Kmr, multiple cloning site in lacZα | 48 |
| pBluescriptII KS | Apr, multiple cloning site in lacZα, between lac and T7 promoters; obtained from Stratagene Cloning Systems | 1 |
| pBluescriptII SK | Apr, multiple cloning site in lacZα, between lac and T7 promoters; obtained from Stratagene Cloning Systems | 1 |
| pLV59 | Encodes EcoRI restriction endonuclease and temperature-sensitive EcoRI methylase, Cmr, positive-selection cloning vector | 68 |
| pUCBM21 | Apr, derived from pUC18 with additional cloning sites inserted into lacZα; obtained from Boehringer Mannheim | 91 |
| pRE1 | 130-kbp plasmid from A. keyseri 12B and 12B-C14, encodes phthalate catabolism | This study |
| pRE752 | 14.1-kbp BglII fragment from pRE1 (map coordinates 19.4 to 33.5) inserted into pLV59 | This study |
| pRE754 | 7.79-kbp BglII fragment from pRE1 (map coordinates 10.5 to 18.3) inserted into pLV59 | This study |
| pRE755 | 8.07-kbp BglII fragment from pRE1 (map coordinates 2.4 to 10.5) inserted into pLV59, hybridized to pRE920 | This study |
| pRE761 | 1.15-kbp BglII fragment from pRE1 (map coordinates 18.3 to 19.4) inserted into pLV59 | This study |
| pRE790 | 2.4-kbp BglII fragment from pRE1 (map coordinates 0 to 2.4) inserted into pLV59, hybridized to pRE920 | This study |
| pRE824 | 8.14-kbp PstI fragment from pRE1 (from a mixture of plasmids from strain 12B) (map coordinates 17.2 to 25.4) inserted into pLV59, clone identified by screening on 2-trifluoromethylbenzoate-containing medium | This study |
| pRE826 | 8.14-kbp PstI fragment from pRE824 inserted into pUCBM21 | This study |
| pRE842 | 9.1-kbp HindIII fragment from pRE1 (from a mixture of plasmids from strain 12B) (map coordinates 16.3 to 25.8) inserted into pLV59; identified by hybridization to the PstI fragment of pRE824 | This study |
| pRE861 | 1.97-kbp HindIII-BglII fragment from pRE754 (map coordinates 16.3 to 18.3) inserted into HindIII-BamHI-digested pBluescriptII KS; carries pehA, lac orientation | This study |
| pRE871 | 9.1-kbp HindIII fragment from pRE842 inserted into pBluescriptII KS; lac orientation | This study |
| pRE899 | pRE871 with ClaI fragment (map coordinates 17.0 to 18.8) deleted; lacks phtB | This study |
| pRE920 | 16.5-kbp HindIII fragment from pRE1 (map coordinates-0.2 to 16.3) inserted into pLV59; identified by hybridization to a SmaI fragment (map coordinates 13.1 to 14.4) from pRE754 | This study |
| pRE995 | 5.4 kbp-ClaI-BglII fragment (map coordinates 5.1 to 10.5) from pRE755 inserted into ClaI-BamHI-digested pBluescriptII KS; carries most of the pcm operon, T7 orientation | This study |
| pRE1026 | 966-bp BspEI-XmaI fragment (map coordinates 23.0 to 24.0) from pRE824 inserted into XmaI-digested pBluescriptII SK; carries phtC, lac orientation | This study |
| pRE1043 | 1.86 kbp XhoI-BamHI fragment (map coordinates 6.1 to 8.0) from pRE920 inserted into pBluescriptII KS; carries pcmA, T7 orientation | This study |
| pRE1056 | 1.16-kbp PstI fragment (map coordinates 4.3 to 5.5) from pRE920 inserted into pBluescriptII SK; carries pcmF, possible dehydrogenase gene; T7 orientation | This study |
| pRE1058 | 1.19-kbp BssHI fragment (map coordinates 5.3 to 6.5) inserted into pBluescriptII SK; carries pcmB, lac orientation | This study |
| pRE1062 | 5.24-kbp SalI fragment (map coordinates 16.3 to 17.0 + 18.8 to 23.4) from pRE899 inserted into pBBR1MCS2; Kmr; carries phtAaAbAcAd downstream from lac promoter | This study |
| pRE1065 | 1.66-kbp NgoMIV fragment (map coordinates 7.4 to 9.0) from pRE920 inserted into XmaI-digested pBluescriptII SK; carries pcmC, T7 orientation | This study |
| pRE1066 | 9.1-kbp HindIII fragment from pRE842 inserted into pBBR1MCS2; Kmr; carries pht operon downstream from lac promoter | This study |
| pRE1089 | XbaI-digested 723-bp PCR product (map coordinates 16.4 to 17.1), made using Taq polymerase and primers ACG GTC TAG AAA GGA GGA AAG CAT GTC CGC G and TGC GTC TAG AGC GCT GGC ATG with pRE754 as template inserted into pBluescriptII SK; carries pehA, lac orientation | This study |
| pRE1096 | BspEI-XbaI-digested 3.8-kbp PCR product (map coordinates 13.2 to 17.1), made using Taq polymerase and primers TCA TTC CGG AGG AGA AGG GTA TGG ACG TAA and TGC GTC TAG AGC GCT GGC ATG with pRE754 as template, inserted into XmaI-XbaI-digested pBluescript II SK; carries ptrDABC pehA, T7 orientation | This study |