TABLE 1.
Bacterial strains and plasmids
| Plasmid or strain | Description | Reference or source |
|---|---|---|
| E. coli | ||
| pWHM3 | Apr (Thior) E. coli-S. erythraea vector used for making insertions in the S. erythraea chromosome | 34 |
| pAIX-5 | Apr pUC19 containing a 5.5-kb XhoI-XhoI fragment from the eryAI gene to the eryCIV gene | |
| pLitmus | Apr pUC19-based E. coli shuttle vector used for subcloning S. erythraea DNA | New England Biolabs |
| pDPE148A | Apr pLitmus29 containing a 300-bp AvrII fragment containing the S. erythraea rrn terminator | This study |
| pDPE149 | Apr pLitmus29 containing a cloned S. erythraea terminator | This study |
| pDPE27 | Apr pBR322-based E. coli shuttle vector containing the S. erythraea eryCVI, eryBVI, eryCIV, and eryCV genes | This study |
| pDPE45 | Apr pUC18 containing an 2.9-kb HindIII/SstI fragment of S. erythraea DNA extending from an internal SstI site in eryAI to a HindIII site of eryBIV | This study |
| pDPE46 | Apr pDPE45 containing the JVI replicon of Streptomyces phaeochromogenes | 1, this study |
| pARR1 | Apr pDPE45 containing a 300-bp SpeI/XbaI rrn terminator sequence from pDPE148A cloned into the SpeI site | This study |
| pARR2 | Apr (Thior) pWHM3 containing a 4.0-kb HindIII/SstI fragment used to make the eryBIV transcriptional mutant | This study |
| pARR3 | Apr pDPE45 with a 1.59-kb HindIII/NcoI fragment deleted | This study |
| pARR4 | Apr pARR3 containing a 300-bp EcoRI/SstI rrn terminator sequence from pDPE148A | This study |
| pARR5 | Apr pARR4 containing a 1.1-kb SstI/PstI eryAI fragment from pAIX-5 | This study |
| pARR8 | Apr (Thior) pWHM3 containing a 3.3-kb fragment from pARR5 containing the rrn terminator within the eryAI gene | This study |
| pARR9 | Apr; same as pARR5 but containing most of the eryBV gene | This study |
| pARR16 | Apr (Hygr) pJV1-based vector used for constructing eryAI S. erythraea CA340 insertion mutant | This study |
| pARR17 | Apr (Hygr); same as pARR16 but containing an eryAI/rrn terminator cassette | This study |
| pARR47 | Apr pUC18 containing a 200-bp EcoRI/SmaI PCR fragment from the eryAI promoter region; used to introduce half a SmaI site at the eryAI −10 hexamer | This study |
| pARR48 | Apr pARR47 containing an additional 400-bp PCR fragment cloned into the SmaI site; used to create a complete SmaI site at the eryAI −10 hexamer | This study |
| pARR49 | Apr pARR3 containing the mutated eryAI −10 hexamer cloned as a 600-bp BclI/ClaI fragment | This study |
| pARR50 | Apr Thior pWHM3-based vector containing the mutated eryAI −10 hexamer plus additional 1.4-kb eryAI and eryBIV DNA; used to make the eryAI −10 hexamer insertional mutant | This study |
| pKAS132 | Apr pDPE27 containing the rrn terminator cloned into the eryBVI gene | This study |
| pKAS133 | Apr pDPE27 containing the rrn terminator cloned into the eryCIV gene | This study |
| pKAS134 | Apr (Thior) pWHM3 containing a 4.6-kb HindIII/SphI fragment from pKAS132; used for integrating the terminator in the eryBVI gene | This study |
| pKAS135 | Apr (Thior) pWHM3 containing a 4.8-kb HindIII/SphI fragment from pKAS133; used for integrating the terminator in the eryCIV gene | This study |
| pDPE205 | Apr; E. coli shuttle vector containing the rrn terminator cloned into the eryBIV gene | This study |
| pDPE206 | Apr (Thior) pWHM3 containing a 3.0-kb SstI/HindIII fragment from pDPE205; used for integrating the terminator into the eryBIV gene | This study |
| pDPE212 | Apr (Thior) pWHM3 containing a terminator cassette in eryCVI; used for integrating the terminator in eryCVI | This study |
| pDPE218 | Apr (Thior) pWHM3 containing a subcloned rrn terminator within the eryBIII gene; used for integrating the terminator in the eryBIII gene | This study |
| pSAM14-2 | Apr (Hygr), integrative plasmid containing a hygromycin rrn terminator cassette in the eryAIII gene; contains the JV1 replicon | This study |
| S. erythraea | ||
| NRRL2338 | Wild type; erythromycin producer. | |
| eryAI::trrn | Thios NRRL2338 mutant containing an integrated rrn terminator in the eryAI gene obtained from pARR8 | This study |
| eryAIII::trrn | Hygr NRRL2338 mutant containing an integrated rrn terminator in the eryAIII gene obtained from pSAM14-2 | This study |
| eryBV::trrn | Thios NRRL2338 mutant containing an integrated rrn terminator in the eryBV gene obtained from pARR2 | This study |
| eryBVI::trrn | Thios NRRL2338 mutant containing an integrated rrn terminator in the eryBVI gene obtained from pKAS134 | This study |
| eryCIV::trrn | Thios NRRL2338 mutant containing an integrated rrn terminator in the eryCVI gene obtained from pDPE212 | This study |
| eryCVI::trrn | Thios NRRL2338 mutant containing an integrated rrn terminator in the eryCVI gene obtained from pKAS135 | This study |
| ARR50 | ThiosS. erythraea NRRL2338 containing a substituted SmaI site at the eryAI −10 hexamer region by gene replacement | This study |
| CA340 | NRRL2338 derivative; industrially-improved erythromycin producer | |
| eryAI::trrn | HygsS. erythraea CA340 mutant containing an integrated rrn terminator in the eryAI gene obtained from pARR17 | This study |
| eryAIII::trrn | Hygr CA340 mutant containing an integrated rrn terminator in the eryAIII gene obtained from pSAM14-2 | This study |