TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Characteristicsa | Source or reference |
|---|---|---|
| P. versutus UW225 | Rifr; pTAV1-less derivative of wild-type UW1 | 6 |
| E. coli strains | ||
| TG1 | Host strain for plasmids | 37 |
| DH5α | Host strain for pRK2013 | 37 |
| M15 | Kmr; host strain for pQE30 and pQE30/repB; carries repressor plasmid pREP4 | Qiagen |
| Plasmids | ||
| pTAV320 | Kmr; 5.6-kb mini-replicon of pTAV1, composed of two PstI fragments (2.1 and 2.2 kb) of pTAV1 plus Kmr cassette | 5 |
| pRK415 | Tcr; mobilizable broad-host-range vector; origin of replication (oriV) and origin of transfer (oriT) of RK2 | 17 |
| pRK2013 | Kmr; helper plasmid carrying RK2 tra genes | 12 |
| pRK415/M1 | Tcr; pRK415 derivative with 322-bp PCR fragment (inc1) (amplified with IGSL and IGSR primers) of pTAV320, containing 15 bp of repB, complete igs, and 95 bp of repC (coordinates 2497 to 2818)b | This study |
| pRK415/M2 | Tcr; pRK415 derivative with 394-bp PCR fragment (inc2) (amplified with ZACL and ZACR primers) of pTAV320, containing 139 bp of repC and 255 bp downstream of the gene (coordinates 3797 to 4190) | This study |
| PRK415/M3 | Tcr; pRK415 derivative with 2,208-bp PstI fragment of pTAV320, containing proximal part of the replicon with repA and 680 bp of repB (coordinates 1 to 2208) | This study |
| pRK415/M4 | Tcr; pRK415 derivative with 1,323-bp BamHI-PstI fragment of pTAV320, containing 487 bp of terminal part of repA and 676 bp of repB (coordinates 881 to 2204) | This study |
| pRK415/M5 | Tcr; pRK415 derivative with 849-bp PstI-BamHI fragment of pTAV320, containing the promoter region and 446 bp of repA (coordinates 1 to 849) | This study |
| pRK415/M6 | Tcr; pRK415 derivative with 219-bp HindIII-Bsu36I fragment of pTAV320, containing 150 bp of igs and 69 bp of repC (coordinates 2574 to 2793) | This study |
| pRK415/M7 | Tcr; derivative of pRK415 with 370-bp PstI-HindIII fragment of pTAV320, containing 307 bp of terminal part of repB and 63 bp of the intergenic region (igs) (coordinates 2204 to 2574) | This study |
| pRK415/M8 | Tcr; pRK415 derivative with 710-bp Bsu36I-BglII and 241-bp BglII-PstI fragments of pTAV320, containing internal part of repC and terminal region of the mini-replicon, respectively (coordinates 2793 to 3503 and 4097 to 4338) | This study |
| pRK415/M11 | Tcr; pRK415 derivative with 1,143-bp Bsu36I-PvuII fragment of pTAV320, containing 1,143 bp of repC (coordinates 2793 to 3936) | This study |
| pRK415/M20 | Tcr; pRK415 derivative with 2,204-bp PstI-PstI fragment of pTAV320, containing mutated repA (not in-frame insertion resulting from AatII digestion and Klenow fragment end filling) and 676 bp of repB (coordinates 1 to 2204) | This study |
| pRK415/M21 | Tcr; pRK415 derivative with 594-bp BglII fragment of pTAV320, containing 432 bp of repC and 162 bp downstream of the gene (coordinates 3503 to 4097) | This study |
| pRK415/AB | Tcr; pRK415 containing 2,737-bp BssHII-Bsu36I restriction fragment of pTAV320 carrying repA and repB genes, igs, and 69 bp of repC (coordinates 56 to 2793) | This study |
| pTAV202 | Kmr; mini-replicon of the second replicator region of plasmid pTAV1, containing 4-kb SalI-PstI restriction fragment of pTAV1 plus Kmr cassette; unable to replicate in E. coli | 3 |
| pABW1 | Kmr; mobilizable cloning vector; ColE1 origin; oriT of RK2; unable to replicate in P. versutus | 7 |
| pABW3 | Kmr; cloning-mobilizable E. coli-P. versutus shuttle vector composed of pABW1 and pTAV202 | This study |
| pABW3/BGL | Kmr; pABW3 containing 594-bp BglII fragment of pTAV320 (coordinates 3503 to 4097) | This study |
| pQE30 | Apr; plasmid for construction and expression of fusion His6-tagged proteins; origin of ColE1 | Qiagen |
| pQE30/repB | Apr; pQE30 containing 982-bp PCR-amplified (with primers REPBL and REPBR) repB gene of pTAV320 (coordinates 1528 to 2510) | This study |
| pREP4 | Kmr; 3.7 kb; origin p15A; lacIq | Qiagen |
| pGB2 | Spr; cloning vector; pSC101 origin | 11 |
| pDBG1 | Spr; pGB2 containing inc2 region of pTAV320 | This study |
| pDBG2 | Spr; pGB2 containing inc2 region of pTAV320 in the orientation opposite that of pDBG1 | This study |
a
For plasmid derivative descriptions we used the nucleotide numbers of the sequence of pTAV320 as the coordinates.
b
GenBank accession number U60522.