Table 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Description or relevant characteristicsa | Reference, source, or restriction enzyme |
|---|---|---|
| Strains | ||
| S. lydicus | ||
| NRRL2433 | Streptolydigin producer | 16 |
| SLMZ | S. lydicus with slgZ deletion; streptolydigin B producer | This study |
| SLMM | S. lydicus with slgM deletion | This study |
| Escherichia coli | ||
| DH10B | For subcloning | Invitrogen |
| ET12567(pUB307) | For S. lydicus conjugation | 12 |
| Plasmids | ||
| Slg4A8 | Source of slgZ and slgM | 16 |
| pOJPM | Source of slgM | 16 |
| pCR-BLUNT | For initial cloning and sequencing of PCR products | Invitrogen |
| pOJ260 | Source of oriT RK2 region | 2 |
| pOJ260P | For gene replacement in S. lydicus | 15 |
| pEM4T | For gene expression in S. lydicus | 14 |
| pLHyg | Source of hygromycin resistance gene hyg | 15 |
| pEFBA | Source of apramycin resistance gene aac(3)IV | 13 |
| pEFBAoriT | pEFBA containing oriT region from pOJ260 as an XbaI-SpeI fragment | This study |
| pΔslgZHyg | pEFBAoriT containing hyg as an SpeI-NheI fragment and PCR products HEI19/HEI20 and HEI21/HEI22 flanking aac(3)IV; used to generate mutant SLMZ | This study |
| pOJM | pOJ260P containing PCR product CRIS15/CRIS16; used to generate mutant SLMM | This study |
| pEM4TslgZ | pEM4T containing slgZ PCR product HEI23/HEI24 | This study |
| pEM4TslgM | pEM4T containing slgM as a BamHI-EcoRI fragment from pOJPM | This study |
| pEM4TslgZM | pEM4TslgM containing slgZ as a BamHI fragment | This study |
| pEM4HT | pEM4T containing hyg as an SpeI-NheI fragment | |
| pEM4HTslgZ | pEM4TslgZ containing hyg as an SpeI-NheI fragment | This study |
| pEM4HTslgM | pEM4TslgM containing hyg as an SpeI-NheI fragment | This study |
| pEM4HTslgZM | pEM4TslgZM containing hyg as an SpeI-NheI fragment | This study |
| Primers | ||
| HEI19 | AACTAGTCCTTCACCGCCCTGGCCC | SpeI |
| HEI20 | AAAATGCATGTGAGGGCGGCAGCTGGC | NsiI |
| HEI21 | AAGGATCCCGAAGATCCCCGGCGTGG | BamHI |
| HEI22 | AAGATATCGTCGAGGAACGCGTGCGG | EcoRV |
| CRIS15 | AATCTAGAACGTGGGCGACGACACGG | XbaI |
| CRIS16 | AGAATTCCTGGTAGCCGCCGTCCG | EcoRI |
| HEI23 | AAGGATCCGGCAAGTACCGCGGCGCC | BamHI |
| HEI24 | AAGGATCCGGCAGACAGCCCGCTGCC | BamHI |
a
Underlining in the primer sequences indicates restriction sites.