Many Streptomyces selection markers (e.g., tsr, apr, spec, PLX4032 research buy hyg, erm and kan) could be used in strains 2C and 4F. No antibacterial activity (e.g., against Bacillus subtilis, Escherichia coli or Staphyloccocus aureus) was detected in the

two strains (unpublished data). Thus, we found two promising cloning hosts, 2C and 4F. Table 2 Plasmids used in this study Plasmids Genotype or description Source or reference pTSC1 A 6996-bp plasmid of strain X4-3 This work pTSC2 A 7.5-kb plasmid of strain X3-3 This work pTSC3 A 50-kb plasmid of strain T6-1-4 This work pTSL1 A 16-kb linear plasmid of strain T6-1-4 This work pSP72 amp colEI-ori Life Technologies, Inc pBluescript II SK amp colEI-ori lacZ Stratagene, Inc pQC156 A 2.6-kb BclI-fragment of melC/tsr cloned in pSP72 (BglII) [46] pCWH1 A 7-kb KpnI fragment of pTSC1 cloned in pQC156 This work pCWH100 A 7-kb KpnI fragment of pTSC1 cloned in pBluescript II SK This work pIJ702 melC tsr pIJ101 origin [31] pZR10 A 8.9-kb Sau3A1-fragment of pFP11 origin cloned in pQC156 [33] pZR115 A 4.1-kb Sau3A1-fragment of pFP1

origin cloned in pQC156 [33] pZR205 Two fragments (PCR) of SLP1 rep/imp cloned in pQC156 [33] pZR51 A 2.2-kb HindIII fragment of pFRL2 origin cloned into pQC156 [32] pHAQ61 A 2.9-kb fragment of SAP1 origin cloned in pQC156 Zhang and Qin, unpublished data pYQ40 A 2-kb fragment of SCP2 origin cloned in pQC156 Yang and Qin, unpublished data pGP9 A 4.1-kb EcoRI/BglII fragment of pSHK1 Fulvestrant research buy origin cloned in pQC156

[32] pSET152 Streptomyces phage φC31-derived integration vector, apr r [38] pHAQ31 amp colEI-ori cos melC tsr [47] Cosmid N7-85 pHAQ31 (BamHI) containing c. 33 kb sequence (5510413-5543521 bp) from S. coelicolor A3(2) This work pCWH74 A 2.6-kb XbaI/NheI fragment containing the phiC31 integrase gene cloned in a pHAQ31-derived cosmid containing the actinorhodin biosynthetic gene cluster This work 024CAO-3 The anthramycin Thymidylate synthase biosynthetic gene cluster cloned into a cosmid CAO2 [22] Since 2C and 4F were classified in the genus Streptomyces, several mesophilic Streptomyces vectors were employed for transformation experiments. As shown in Table 3, pIJ702 (a pIJ101 derivative, [31]), pZR51 (pFRL2, [32]), pZR115 (pFP1, [33]) and pZR10 (pFP11, [33]) were able to transform both 2C and 4F. No transformants were obtained for SCP2 [34], SLP1[35], SAP1 [36] and pSHK1 [32] derivatives (pYQ40, pZR205, pHAQ61, and pGP9, respectively). pCWH1 could also transform S. lividans ZX7 [37] at high frequency (104/μg DNA). A Streptomyces integrating plasmid, pSET152 [38], could be introduced by conjugation from E. coli into many thermophilic Streptomyces strains (14 of 22 strains). Thus, pTSC1-derived pCWH1 can replicate in both thermophilic and mesophilic Streptomyces strains.