TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Relevant characteristicsa | Reference or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| S17-1 | Contains plasmid RP4 integrated in the chromosome | 25 |
| XL1-Blue MRF′ | Δ(mcrA)183 Δ(mcrcB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac[F′ proAB lacIqZΔM15Tn10(Tetr)] | Stratagene |
| DH5α | F−recA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR (lacZYA-argF)U196 φ80 dlacZΔM15 | Bethesda Research Laboratories |
| DH10B | F− ΔmcrA Δ(mrr-hsdRMS-mcrBD) φ80dlacZφM15 ΔlacX74 deoR recA1 Δara-139 Δ(ara leu)7697 galU galK λ− rpsL endA1 nupG | Gibco BRL |
| S. roseosporus | ||
| A21978.6 | Produces daptomycin | ATCC 31568 |
| A21978.65 | Produces elevated levels of daptomycin | Lilly Culture Collection |
| MM91 | A21978.6Ω91 (draI–E::Tn5099) | This work |
| MM92 | A21978.6Ω92 (draI–E::Tn5099) | This work |
| MM93 | A21978.6Ω93 (draI–C::Tn5099) | This work |
| MM94 | A21978.6Ω94 (draI–C::Tn5099) | This work |
| MM95 | A21978.6Ω95 (draI–C::Tn5099) | This work |
| MM114 | A21978.65::pRHB152 | This work |
| MM123 | A21978.65::pRHB153 | This work |
| MM138 | A21978.6::pRHB154 | This work |
| MM139 | A21978.6::pRHB155 | This work |
| MM140 | A21978.65::pRHB157 | This work |
| MM131 | A21978.6::pRHB166 | This work |
| MM132 | A21978.6::pRHB168 | This work |
| MM133 | A21978.6::pRHB169 | This work |
| MM134 | A21978.6::pRHB170 | This work |
| MM135 | A21978.6::pRHB172 | This work |
| MM136 | A21978.6::pRHB173 | This work |
| MM137 | A21978.6::pRHB174 | This work |
| Plasmids | ||
| pOJ260 | 3.5-kb plasmid containing AmrreppUC MCS lacZα oriT | 5 |
| pKC1471 | 3.0-kb cosmid containing Sprrepp15A MCS | 16 |
| pBluescript II KS− | Apr ColE1 rep | Stratagene |
| pCRII | 3.9-kb TA cloning vector | Invitrogen |
| pCZA213 | 11.2-kb plasmid containing Tn5099 | 27 |
| pRHB146 | 3.4-kb pUCBM21 derivative containing oriT | 20 |
| pRHB152 | pRHB146 containing Tn5099 and 7.1 kb of S. roseosporus flanking DNA from MM91 | This work |
| pRHB153 | pRHB146 containing Tn5099 and 6.0 kb of S. roseosporus flanking DNA from MM93 | This work |
| pRHB154 | pRHB146 containing Tn5099 and 5.0 kb of S. roseosporus flanking DNA from MM92 | This work |
| pRHB155 | pRHB146 containing Tn5099 and 5.0 kb of S. roseosporus flanking DNA from MM94 | This work |
| pRHB157 | pRHB146 containing Tn5099 and 7.0 kb of S. roseosporus flanking DNA from MM95 | This work |
| pRHB159 | pKC1471 containing 55-kb insert of S. roseosporus DNA | This work |
| pRHB160 | pKC1471 containing 45-kb insert of S. roseosporus DNA | This work |
| pRHB161 | pKC1471 containing 46-kb insert of S. roseosporus DNA | This work |
| pRHB162 | pKC1471 containing 45-kb insert of S. roseosporus DNA | This work |
| pRHB166 | pOJ260 containing 14-kb EcoRV fragment from pRHB160 | This work |
| pRHB168 | pOJ260 containing 5.2-kb EcoRI left-end fragment from pRHB159 | This work |
| pRHB169 | pOJ260 containing 2.3-kb EcoRI left-end fragment from pRHB161 | This work |
| pRHB170 | pOJ260 containing 3.9-kb EcoRI left-end fragment from pRHB162 | This work |
| pRHB172 | pOJ260 containing 7-kb EcoRI fragment from pRHB160 | This work |
| pRHB173 | pOJ260 containing 4.5-kb KpnI-ScaI fragment from pRHB161 inserted in KpnI-DraI site | This work |
| pRHB174 | pOJ260 containing 10-kb ScaI fragment from pRHB161 inserted in EcoRV site | This work |
| pUCBM21 | pUC19 with expanded MCS | Boehringer Mannheim Biochemicals |
| pRHB599 | pBluescript II KS− containing 2.7-kb KpnI-EcoRI fragment from pRHB614 | This work |
| pRHB602 | pBluescript II KS− containing 2.5-kb EcoRI fragment from pRHB614 | This work |
| pRHB603 | pBluescript II KS− containing 0.5-kb EcoRI fragment from pRHB614 | This work |
| pRHB680 | pBluescript II KS− containing 0.6-kb ScaI fragment from pRHB613 | This work |
| pRHB678 | pCRII containing 362-bp internal fragment of S. roseosporus masC homolog | This work |
| pRHB588 | pK1139 containing 0.36-kb EcoRI fragment from pRHB678 containing internal masC sequence | This work |
| pIF175 | Mini-γδ plasmid for deletions and inversions | 36 |
| M13mp19 | Vector for DNA sequencing | 38 |
a
Amr, apramycin resistance; reppUC, pUC replicon for E. coli; oriT, RK2 origin of transfer; MCS, multiple cloning site; Spr, spectinomycin resistance.