TABLE 1.
Strains, phage, plasmids, and cosmids used in this study
| Strain, phage, or plasmid | Relevant characteristicsa | Reference or source |
|---|---|---|
| S. hygroscopicus strains | ||
| 5008 | Wild-type producer of validamycin | 27 |
| YU-1 | Nonproducer of validamycin generated by gene replacement of a ca. 30-kb region including acbC homolog with aac(3)IV | This work (Fig. 3) |
| JXH-1 | As above, but replacing a 563-bp DNA fragment internal to valA | This work (Fig. 5) |
| S. lividans 66 ZX1 | rec-46 str-6 pro-2 | 30 |
| E. coli strains | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araΔ139 D(ara, leu)1697 galU galK λ−rpsL nupG | GIBCO-BRL |
| ET12567(pUZ8002) | dam dcm hsdS/pUZ8002 | 21 |
| LE392 | supE44 supF58 hsdR514, used for infection with in vitro-packaged cosmids | 3 |
| BL21Gold(DE3)/pLysS | F−ompT hsdSB (rB− mB−) gal dcm(DE3) pLysS (Cmr) | 18 |
| XL-1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac | Stratagene |
| Pellicularia sasakii | Fungal indicator strain sensitive to validamycin A | SJUSC |
| Plasmids | ||
| pAS8/7 | pIJ6021 with a 1,264-bp NdeI-EcoRI fragment containing acbC in the corresponding sites | 25 |
| pBlueScript II SK(+) | bla lacZ orif1 | Stratagene |
| pHZ1358 | Cosmid used for the construction of 5008 genomic library; pIJ101 derivative; tsr Ltz−sti+oriT | 26 |
| pHZ2229 | pBlueScript II SK(+) carrying a ca. 6-kb sequenced BamHI fragment (Fig. 2) from cosmid 3G8 | This study |
| pHZ2234 | Construct after religation of BamHI-digested 3G8 | This study |
| pHZ2236 | Insertion in BamHI site of pHZ2234 of an aac(3)IV gene sandwiched between the two 2.1-kb genomic DNA fragments | This study |
| pIJ2925 | bla lacZ | 12 |
| pJTU504 | Insertion of a 1.33-kb XbaI-EcoRI fragment from pJTU690 in pBlueScript II SK(+) | This study |
| pJTU514 | pJTU504 derivative with the insertion of aac(3)IV in EcoRI site | This study |
| pJTU515 | Insertion of 1.5-kb PvuII-HindIII fragment from pJTU696 into EcoRV-HindIII sites of pJTU514 | This study |
| pJTU516 | Insertion of a 4.23-kb BglII fragment from pJTU515, | This study |
| which contains a linked 1.33-kb (left arm)-1.4-kb aac(3)IV-2.1-kb (right arm) for valA inactivation, in BamHI site of pHZ1358 | ||
| pJTU690 | Part of the 6-kb sequenced BamHI fragment (Fig. 2) including the 1.33-kb XbaI-EcoRI fragment inserted into pBlueScript II SK(+) | This study |
| pJTU696 | Part of the 6-kb sequenced BamHI fragment (Fig. 2) including the 1.5-kb PvuII-HindIII fragment inserted into pBlueScript II SK(+) | This study |
| pKMW5 | pUC18 derivative carrying the strD and strE genes of the streptomycin pathway | 24 |
| pRSET-B | bla reppUC, T7 promoter | Invitrogen |
| pUC18 | bla lacZ | 28 |
| Phage | ||
| φC31 | Wild type; c+attP+int+ | 31 |
a
Ltz (lethal zygosis), pock formation caused by plasmid transfer; mel, tyrosinase gene for melanin production; oriT, origin of transfer of plasmid RK2; tsr, thiostrepton resistance gene; aac(3)IV, apramycin resistance gene; sti, origin for second-strand synthesis of the multicopy plasmid pIJ101 (6); vph, viomycin resistance gene; xylE, catechol 2,3-dioxygenase from Pseudomonas putida; Cmr, chloramphenicol resistance gene.