Table 1. Strains and plasmids used in this work.
| Bacterial strain or plasmid | Description* | Source or reference |
|---|---|---|
| E. coli DH5α | supE44 ΔlacU169(φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | NEB |
| E. coli ET12567 (pUZ8002) | dam-13::Tn9(Cmr) dcm-6 hsdM; harbors conjugative plasmid pUZ8002; Cmr, Kmr | C.P.Smith, UMIST, UK; |
| E. coli WM6026 | lacIq rrnB3 ΔlacZ4787 hsdR514 ΔaraBAD567 ΔrhaBAD568 rph-1 attλ::pAE12 (ΔoriR6K-cat::Frt5) ΔendA::Frt uidA(ΔMluI)::pir attHK::pJK1006Δ (oriR6K-cat::Frt5 trfA::Frt) | [58] |
| E. coli BW25113 (pKD46) | (F-(φ80dΔ(lacZ)M15 recA1 endA1 gyrA96 thi1 deoR (lacZYA-argF)U169); λ bet- exo -gam; aph (Kmr) | J. Beckwith, HMS |
| S. ghanaensis ATCC14672 | Wild-type moenomycin producer | ATCC |
| S. ghanaensis B38.3 | Markerless deletion of moeH5; produces NoA | [21] |
| S. ghanaensis HIM5 | Himar1 insertion into ssfg_01967 gene of B38.3 | This work |
| Bacillus cereus ATCC19637 | Moenomycin-sensitive test culture | ATCC |
| pTES | Amr; phiC31-based Streptomyces integrative vector; expression of cloned gene from ermEp* | [59] |
| pKC1132 | Amr; conjugative suicide vector for gene disruption | [14] |
| pIJ6902 | AmrThr; phiC31-based Streptomyces integrative vector; expression of cloned gene from tipAp |
[60] |
| pHYG | pLitmus-derived vector containing hygromycin resistance cassette hyg; HyrApr | J. Salas, Universidad de Oviedo |
| patt-shyg | hygromycin cassette flanked by B-CC and P-GG sites for IMES recombination system | [61] |
| pGUS | Promoter probe vector, contains promoterless gusA; derived from pSET152 |
[20] |
| pGUSHL4aadA | pGUS derivative for translational fusions | [20] |
| padpAscript | pGUS derivative, transcriptional fusion of adpAp (0.5 kb KpnI-XbaI fragment) to gusA, AmrSpr |
[8] |
| padpAtransl | pGUSHL4aadA derivative, adpA-gusA translational fusion, AmrSpr | [8] |
| padpAcontr | pGUSHL4aadA, promoterless adpAgh-gusA fusion, AmrSpr | [8] |
| pmiaB | pKC1132 carrying 5 kb BamHI-HindIII fragment centred on ssfg_01967, Amr | This work |
| pmiaBhyg | pmiaB derivative, replacement of with hyg cassette from patt-shyg, AmrHyr | This work |
| pBluescriptIIKS | Apr; general purpose cloning vector; blue–white selection | ThermoScientific |
| pOOB120 | pBluescriptIIKS carrying 1.8 kb adpA-adpA fragment | This work |
| pOOB121 | pOOB120 with TTA codon within adpA replaced with CTC | This work |
| padpAtransl-CTC | pGUSHL4aadA carrying adpAp-adpA from pOOB121 | This work |
| pOOB106 | pTES carrying gene ssfg_01967 under ermEp* promoter | This work |
| pOOB106a | pOOB106 with aac(3)IV marker changed to hyg | This work |
| pOOB108L | pIJ6902 carrying gene ssfg_01967 under tipAp promoter | This work |
| pOOB113a | pOOB108L with aac(3)IV marker changed to hyg | This work |
*Cmr, Kmr, Amr, Spr, Thr, Hyr, Apr: chloramphenicol, kanamycin, apramycin, spectinomycin, thiostrepton, hygromycin and ampicillin resistance, respectively