TABLE 1.

Bacterial strains and plasmids

Strain or plasmidRelevant phenotypeaDescription (reference or source)
E. coli strains
    DH5αMCRRecAFrom Gibco BRL
    HB101(RP1)RecA StrrHB101 containing IncPα plasmid RP1 (21)
    BL21-Codon Plus (DE3) RILCmrCells enable high-level expression of heterologous proteins in E. coli (Stratagene)
B. thetaiotaomicron 5482A strains
    BT4001RifrSpontaneous rifampin-resistant mutant
    BT4007Rifr Tcr EmrBT4001 containing wild-type CTnDOT
Plasmids
    pMJF2Apr (Emr)Cloning vector to create a uidA fusion, also an E. coli-Bacteroides shuttle vector (6)
    pAFD1Apr (Emr)E. coli-Bacteroides shuttle vector containing ermF
    pC-COWApr Tcr Cmr (Cmr)E. coli-Bacteroides shuttle vector with IS4351-cat and Bacteroides plasmid pB8-51 that is compatible with pAFD1and pMJF2-based vectors (9)
    pCR-BluntKnr3.5-kb cloning vector for PCR product (Invitrogen)
    pET28bKnrPlasmid for the overexpression of His6-tagged proteins in E. coli (Novagen)
    pGFK24.1Apr0.3-kb PCR product containing the upstream region of the orf2c operon cloned into pCR2.1(K. Moon, unpublished)
    pGFK43.11Apr (Cmr)Plasmid containing uidA fused to 300 bp upstream region of the orf2c start codon cloned into the NruI-SphI site of pC-COW (K. Moon, unpublished)
    pGFK70.1Apr1.2-kb PCR product containing RteCHis6 cloned into pCR2.1 (K. Moon, unpublished)
    pGFK76.5Apr (Cefr)Plasmid containing an in-frame fusion of His6-tagged rteC to the tetQ promoter (K. Moon, unpublished)
    pJPARK5Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −67 and −70 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK7Apr (Emr)1.4-kb Sph-SstI fragment from pGFK76.5 containing wild-type pPQ-rteC; His6-tagged cloned into SphI-SstI site of pAFD1(this study)
    pJPARK18Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −63 and −66 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK19Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −59 and −62 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK20Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −55 and −58 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK21Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −51 and −54 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK22Apr0.3-kb PCR product containing 20-bp mutated sequence between positions −46 and −65 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
    pJPARK26Knr760-bp PCR product containing wild-type rteC cloned into pCR-Blunt (this study)
    pJPARK28Apr0.3-kb PCR product containing 7-bp mutated sequence between positions −43 and −49 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
    pJPARK32Knr760-bp NdeI-XhoI fragment from pJPARK26 cloned into NdeI-XhoI site of pET28b, creating His6-tagged rteC (this study)
    pJPARK37Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 7-bp mutated sequence between positions −43 and −49 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK38Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 20-bp mutated sequence between positions −46 and −65 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK39Apr Tcr Cmr (Cmr)uidA-fused 0.3-kb PCR product containing 4-bp mutated sequence between positions −35 and −38 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
    pJPARK49Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (K174A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK50Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (R178A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK51Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (Y180A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK63Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (D175A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK64Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (E189A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK65Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (K184A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK66Apr (Emr)1.2-kb PCR product containing RteCHis6 mutant (K184A) cloned into NcoI-SmaI site of pJPARK7 (this study)
    pJPARK76Apr0.3-kb PCR product containing 5-bp mutated sequence between positions −53 and −57 upstream of the orf2c transcriptional start site cloned into pCR2.1 (this study)
    pJPARK78Apr Tcr Cmr (Cmr)uidA fused 0.3-kb PCR product containing 5-bp mutated sequence between positions −53 and −57 upstream of the orf2c transcriptional start site cloned into the NruI-SphI site of pC-COW (this study)
  • a Parentheses indicate resistances expressed in Bacteroides.