TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant phenotypea | Description (reference) |
|---|---|---|
| E. coli | ||
| DH5α MCR | recA | Gibco BRL |
| HB101 (RP1) | recA Strr | HB101 containing IncPα plasmid RP1 (25) |
| BL21 (DE3) | F−gal dcm (DE3) | E. coli with the bacteriophage T7 promoter-based expression system that carries the lambda DE3 lysogen (Invitrogen) (31) |
| B. thetaiotamicron 5482A | ||
| BT4001 | Rifr | Spontaneous rifampin mutant of B. thetaiotamicron 5482A (24) |
| BT4007 | Rifr Tcr Emr | B. thetaiotaomicron 4001 that contains wild-type CTnDOT |
| BT4001ΩQAB | Rifr Tcr | B. thetaiotamicron BT4001 that carries the CTnDOT region comprising tetQ, rteA, and rteB inserted into the chromosome via an NBU 2 minielement (40) |
| BT4001ΩQABC | Rifr Tcr | B. thetaiotamicron BT4001 that carries the CTnDOT region comprising tetQ, rteA, rteB, and rteC inserted into the chromosome via an NBU1 minielement (40) |
| BT4104ΩRDB1 | Thy− Tpr Tcr Emr ΩrteA | Chromosomal disruption of rteA in BT4104 (16) |
| Plasmids | ||
| pMJF2 | Apr (Emr) | A cloning vector to create a uidA fusion, also an E. coli-Bacteroides shuttle vector (11) |
| pLYL05 | Apr (Cefr) | An E. coli-Bacteroides shuttle vector containing cfxA (27, 34) |
| pLYL02 | Apr (Emr) | Same as pMJF2 with the uidA gene in opposite orientation to facilitate cloning (this report) |
| pC-COW | Apr Tcr Cmr (Cmr) | An E. coli-Bacteroides shuttle vector with IS4351-cat and Bacteroides plasmid pB8-51 that is compatible with pMJF2-based vectors (37) |
| pCR2.1 | Apr | A 3.9-kb cloning vector for PCR products (Invitrogen) |
| pYS32 | Apr (Emr) | A 2.9-kb HindIII fragment of pYS33 that contains uidA fused to the 1.0-kb promoter region of the orf2c operon cloned into pC-COW (33) |
| pGFK1 | Apr (Emr) | 1.2-kb BspEI-XmnI fragment of region of CTnDOT cloned into pLY02 to generate the fusion of uidA to the 188-bp N-terminal coding sequence of orf3 and its 1.0-kb upstream sequence (this study) |
| pGFK10 | Apr (Emr) | 2.7-kb SphI-SmaI PCR product containing 300 bp upstream of orf2c start codon cloned into the SphI-SmaI sites of pMJF2 (this study) |
| pGFK34 | Apr (Emr) | 0.3-kb SphI-SmaI PCR product containing 300 bp upstream of orf2c start codon cloned into the SphI-SmaI sites of pMJF2 (this study) |
| pGFK35 | Apr (Emr) | Plasmid containing a mutated sequence (AAAC) in the putative −7 region from pGFK34 and cloned into the SphI-SmaI site of pMJF2 (this study) |
| pGFK36 | Apr (Emr) | A plasmid containing a mutated sequence (TTTC) in the putative −7 region from pGFK34 and cloned into the SphI-SmaI site of pMJF2 (this study) |
| pGFK38 | Apr (Emr) | A plasmid containing a mutated sequence (TATG) in the putative −7 region from pGFK34 and cloned into the SphI-SmaI site of pMJF2 (this study) |
| pGFK43 | Apr (Cmr) | A plasmid containing a 20-bp mutated sequence between bp-70 and -50 upstream of the orf2c transcriptional start site from pGFK34 and cloned into the SphI-SmaI site of pMJF2 (this study) |
| PGFK63 | Apr (Emr) | 0.3-kb SphI-SmaI PCR product containing 300 bp upstream of orf2c start codon cloned into the SphI-SmaI site of pMJF2 (this study) |
| pGFK65 (pPc-rteC) | Apr (Cefr) | A clone containing rteC with its own promoter cloned into pLYL05 (this study) |
| pGFK59.3 (pPQ) | Apr (Cefr) | 0.2-kb SphI-SstI PCR product containing the tetQ promoter with an NcoI site cloned into SphI-SstI pLYL05 (this study) |
| pGFK67 (pPQ-rteC) | Apr (Cefr) | 1.2-kb NcoI-SstI PCR product containing the rteC coding region plus the tetQ promoter region cloned into the NcoI-SstI site on pGFK59.3 to produce an in-frame fusion of rteC to the tetQ promoter (this study) |
| pGFK69 (pPQ-rteC; His6 tagged) | Apr (Cefr) | 1.2-kb NcoI-SmaI PCR product containing a His6 tag on the C-terminal end of RteC cloned into the NcoI-SmaI site of pGFK59.3 (this study) |
| pET27b | Knr | A plasmid for the overexpression of His6-tagged proteins in E. coli (Novagen) |
| pGFK90.1 | Knr | 0.7-kb NcoI-XhoI rteC coding region cloned into the NcoI-XhoI site of pET27b (this study) |
a
Phenotypes in parentheses are expressed only in Bacteroides, and phenotypes outside parentheses are expressed in E. coli. Abbreviations: Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Rifr, rifampin resistance; Tcr, tetracycline resistance; Tpr, trimethoprim resistance, Knr, kanamycin resistance; Thy−, thymidine auxotroph.