TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristics | Reference and/ or source |
|---|---|---|
| Strains | ||
| R. meliloti | ||
| Rm1021 | SU47 str-21 | 32 |
| Pn1 | Rm1021 Δ(phnH–J):Ω Spr | This study |
| E. coli | ||
| BL21(DE3) | E. coli B F−dcm ompThsdS (rB−mB−) gal λ(DE3) [Cam] | 41, Stratagene |
| DH5α | supE44 ΔlacU169 (φ80 lacZΔM15) hsdR1 recA1 endA1 gyrA96 thi-1 relA1 | 13, GIBCO BRL |
| GM1674 | F′ proAB+ lacIqZΔM15 Δ(pro-lac) XIII dam-J dcm-6 thi-1 tsx63 λ− | 17 |
| Plasmids | ||
| pHP45Ω | pBR322 derivative containing Smr-Spr region flanked by Ω repeats | 37 |
| pJQ200SK | Mobilizable suicide vector with p15A origin of replication; contains RP4 origin of transfer and sacB from B. subtilis for counterselection; Gmr | 38 |
| pRK2013 | ColE1 replicon with RK2 tra genes; helper plasmid used for mobilizing P- and Q-group plasmids; Nmr Kmr | 9 |
| pRSETB | ColE1 expression vector T7 promoter; Ampr | 19, Invitrogen |
| pRMP1 | pTZ19R carrying 3.9-kb XbaI/BamHI fragment containing PCR-amplified R. meliloti phnG–K | This study |
| pRMP2 | Same as pRMP1 but with the 3.9-kb fragment cloned in pTZ18R | This study |
| pRMP3 | pTZ19R carrying the same fragment as pRMP1+2 except with a 1.8-kb deletion from ClaI to NdeI and the deleted sequence replaced by the Smr-Spr fragment from pHP45Ω | This study |
| pRMP4 | pJQ200SK carrying the 4.1-kb insert from pRUPn2 cloned into the XbaI site of the MCSa | This study |
| pTHGB | pRSETB carrying R. meliloti PCR-amplified phnG cloned into the BglII/HindIII site of the MCS; this fuses PhnG in frame to a His tag | This study |
| pTHHB | pRSETB carrying R. meliloti PCR-amplified phnH cloned into the BglII/HindIII site of the MCS; this fuses PhnH in frame to a His-tag | This study |
| pTHIB | pRSETB carrying R. meliloti PCR-amplified phnI cloned into the BglII/HindIII site of the MCS; this fuses PhnI in frame to a His tag | This study |
| pTHJB | pRSETB carrying R. meliloti PCR-amplified phnJ cloned into the BglII/HindIII site of the MCS; this fuses PhnJ in frame to a His tag | This study |
| pTHKB | pRSETB carrying R. meliloti PCR-amplified phnK cloned into the BglII/HindIII site of the MCS; this fuses PhnK in frame to a His tag | This study |
| pTHGC | pRSETB carrying R. meliloti PCR-amplified phnG cloned into the NdeI/HindIII site of the MCS; this does not fuse PhnG to the His tag | This study |
| pTHHC | pRSETB carrying R. meliloti PCR-amplified phnH cloned into the NdeI/HindIII site of the MCS; this does not fuse PhnH to the His tag | This study |
| pTHIC | pRSETB carrying R. meliloti PCR-amplified phnI cloned into the NdeI/HindIII site of the MCS; this does not fuse PhnI to the His tag | This study |
| pTHJC | pRSETB carrying R. meliloti PCR-amplified phnJ cloned into the NdeI/HindIII site of the MCS; this does not fuse PhnJ to the His tag | This study |
| pTHKC | pRSETB carrying R. meliloti PCR-amplified phnK cloned into the NdeI/HindIII site of the MCS; this does not fuse PhnK to the His tag | This study |
| pTZ19R/18R | Phagemid, pUC replicon carrying f1(−) origin and T7 promoter. pTZ19R and -18R have pUC19 and -18 polylinkers, respectively | 31, Pharmacia |
a
MCS, multiple cloning site.