TABLE 1.
Microorganisms and plasmids used in this study
| Strain or plasmid | Relevant characteristic | Source or referencea |
|---|---|---|
| Escherichia coli | ||
| DH10B | φ80dlacZΔM15/araD139 Δ(ara-leu)7697 ΔlacX74 galU galK rpsL deoR endA1 nupG recA1 mcrA Δ(mrr hsdRMS mcrBC) | 10 |
| DH5α | φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1 | 10 |
| DH5α/λpir | λpir/φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 hsdR17 deoR thi-1 supE44 gyrA96 relA1 | 30 |
| GeneHogs(trfA) | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15/araD139 Δ(ara-leu)7697 ΔlacX74 galU galK rpsL deoR endA1 nupG recA1 trfA AmprfhuA::IS2 | Invitrogen |
| BW26678 | laclqrrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78/pKD46 | 6 |
| GM119 | dam-3 dcm-9 metB1 galK2 galT27 lacY1 tsx-78 supE44 thi-1 mel-1 tonA31 | 52 |
| WM3780 | dam-3 dcm-9 metB1 galK2 galT27 lacY1 tsx-78 supE44 thi-1 mel-1 tonA31 attHK::pJK202Δ(oriR6K-aadA)::Frt | This study |
| WM3321 | BL21(DE3)/pNR69 | 58 |
| Streptomyces viridochromogenes | Wild type (DSM 40736) | DSM |
| Streptomyces lividans | ||
| 66 | Wild type (NRRL B-16148) | ARS culture collection |
| WM 4366 | S. lividans with pJVD9 integrated into φC31 attB site | This study |
| WM 4367 | S. lividans with pJVD9/fosmid 5-9G integrated into φC31 attB site | This study |
| WM4368 | S. lividans with pJVD9/fosmid 5-9GΔ(orf416-orf571′)::kan integrated into φC31 attB site | This study |
| Bacillus subtilis | ||
| ATCC 6633 | PTT-sensitive bioassay tester strain | ATCC |
| WM 4445 | Spontaneous PTTr mutant | This study |
| Plasmids | ||
| pAM34 | Ampr Strr cloning vector, IPTG-dependent ColE1 oriR, lac1q | 8 |
| pAH144 | Strr, oriR6K, HKattP integrating cloning vector | 11 |
| RP4 | Broad-host-range plasmid | 36 |
| pJK190 | 53,981-bp BamHI-BglII fragment of RP4 cloned into BamHI-cut pAH144 | This study |
| pJK202 | Deletion of two adjacent BstBI fragments of pJK190 | This study |
| pMP45 | PCR template for oriR6K amplification in pJVD1 construction | 38 |
| pOJ436 | Aprr, oriT, φC31 int, φC31 attP, cosvector | 5 |
| pWM357 | oriF, dual cosvector, Cmr | 60 |
| pJVD1 | pWM357 unique ClaI site cut and ligated to ClaI site-flanked oriR6K and HKattB cassette PCR product cloned from pMP45 with primers oriR6K/HK attB FOR and oriR6K REV | This study |
| pJVD8 | pAH144 cut with NotI/NcoI to remove oriR6K, T4 DNA polymerase blunted, and ligated to T4 DNA polymerase-blunted BamHI pAM34 fragment containing replicon, lac1q, Ampr Strr | This study |
| pJVD9 | pJVD8 SmaI unique site cut and ligated to pOJ436 DraI fragment containing Aprr, oriT, φC31 int, φC31 attP | This study |
| fosmid 5-9G | S. viridochromogenes genomic DNA cloned into pJVD1 fosmid vector; contains PTT biosynthetic genes | This study |
| pJVD9/fosmid 5-9G | pJVD9 fosmid 5-9G cointegrant via HKattP/attB site-specific recombination | This study |
| pJVD9/fosmid 5-9GΔ(orf416-orf571′)::kan | pJVD9-fosmid 5-9G cointegrant via HKattP/attB site-specific recombination with orf416-orf571′ replaced by Kanr marker | This study |
| pWM303 | Pir-dependent cloning vector | 29 |
| pPB37 | oriR6K, bla-containing PCR product from pWM303 obtained with primers ori-BglII and bla-BamHI: circularized after cutting with BamHI and BglII | This study |
| pJK78 | Unique XbaI site in pPB37 destroyed by cutting with XbaI followed by treatment with T4 DNA polymerase and dNTPs | This study |
| pJK90 | AscI-cut PCR product obtained from GeneJumperOriV template and primer MuR-A/E/B (anneals to both sides of replicon) cloned into AscI site of pJK78 | This study |
| Supercos1 | Kmr cosmid vector | Stratagene |
| pJK91 | Replacement of BamHI fragment from pJK90 with BamHI-cut PCR product obtained from Supercos1 template and primers Supercos-aphF and Supercos-aphR | This study |
| pIJ702 | ThiorStreptomyces cloning vector | 23 |
| pJK92 | NotI/XbaI-cut PCR product obtained from pIJ702 template with primers Tsr-L and Tsr-R cloned into NotI/XbaI sites of pJK91 | This study |
| pJK93 | AscI-cut PCR product obtained from pJK93 template and primer MuR-A/M (anneals to both sides of replicon) cloned into AscI site of pJK90 | This study |
| pJK94 | Unique BglII site in pJK93 destroyed by cutting with BglII followed by treatment with T4 DNA polymerase and dNTPs | This study |
| pJK95 | AscI-cut PCR product obtained from pJK94 template and primer MuR-A/E/B (anneals to both sides of replicon) cloned into AscI site of pJK90: donor plasmid for mini-Mu-JK4790 | This study |
a
DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; ARS, U.S. Department of Agriculture Agricultural Resource Service, Peoria, Ill.; ATCC, American Type Culture Collection, Manassas, Va.