TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Descriptiona | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | Strain used for gene cloning | Clontech |
| BL21(DE3)pLysS | F− ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS; Cmr | Seebio Biotech |
| C. hutchinsonii strains | ||
| ATCC 33406 | Wild type | ATCC |
| Δ3237 strain | Unmarked deletion mutant of CHU_3237 | This study |
| C3237 strain | Complementation of Δ3237 mutant with pCH3237 | This study |
| Δ3202–3190 strain | Deletion mutant of large genomic fragment from CHU_3202 to CHU_3190 | This study |
| Δ0804–0819 strain | Deletion mutant of large genomic fragment from CHU_0804 to CHU_0819 | This study |
| Δ0834–0841 strain | Deletion mutant of large genomic fragment from CHU_0834 to CHU_0841 | This study |
| Δ0428–0449 strain | Deletion mutant of large genomic fragment from CHU_0428 to CHU_0449 | This study |
| 0344::cat strain | Disruption mutant of CHU_0344 made with pSJHC as template | This study |
| Plasmids | ||
| pEP4351 | Cmr Tcr (Emr) Mob+ | 49 |
| pLYL03 | Apr (Emr) Mob+ | 50 |
| pKD3 | Plasmid carrying two FRT sites; Cmr Apr | 19 |
| pCP20 | Plasmid carrying FLP recombinase; Cmr Apr | 38 |
| pBJ113 | Plasmid carrying galK | 37 |
| pSKSO8 | oriC; Apr (Emr) | 13 |
| pSK1284gfp | Plasmid carrying gfp and promoter of CHU_1284; Apr (Emr) | 13 |
| pEPLO293cfxA | Plasmid carrying cfxA | 12 |
| pET-His | Used for recombinant CHU_0344 expression; Apr | Seebio Biotech |
| pSJHS | Gene-targeting template plasmid carrying ermF; Apr (Emr) | This study |
| pSJHC | Gene-targeting template plasmid carrying cat under the control of the ompA promoter from F. johnsoniae; Apr (Cmr) | This study |
| pTKS | Unmarked deletion template plasmid carrying ermF flanked by two FRT sites cloned from pKD3 with primers KSfrtF and KSfrtR; Apr (Emr) | This study |
| pTSK | Similar to pTKS except for cloning the FRT sites with primers frtSKF and frtSKR; Apr (Emr) | This study |
| pCFXKS | Similar to pTKS except for carrying cfxA instead of ermF; Apr (Cfr) | This study |
| pCFXSK | Similar to pTSK except for carrying cfxA instead of ermF; Apr (Cfr) | This study |
| pSKSO8TG | Promoterless gfp and a Rho-independent terminator inserted into pSKSO83; oriC; Apr (Emr) | This study |
| pCHF | Plasmid carrying flp cotranscribed with cat under the control of the ompA promoter from F. johnsoniae; promoter of CHU_1284 and galK inserted and gfp evicted from pSKSO8TG; oriC; Apr (Cmr) | This study |
| pCH | Similar to pCHF except for the flp gene; oriC; Apr (Cmr) | This study |
| pCH3237 | A 4.4-kbp fragment spanning CHU_3237 amplified with primers C3237F and C3237R and ligated into SacI and SalI sites of pCH; oriC; Apr (Cmr) | This study |
a
Antibiotic resistance phenotypes: Apr, ampicillin; Cmr, chloramphenicol; Emr, erythromycin; Cfr, cefoxitin; Kmr, kanamycin. Phenotypes in parentheses are expressed in C. hutchinsonii, and phenotypes not in parentheses are expressed in E. coli.