TABLE 2

Plasmids and strains used in this studya

Plasmids
and strains
DescriptionSource or
reference(s)
Plasmids
    pJET1.2/bluntE. coli plasmid used for high-efficiency blunt-end cloning of PCR products (Ampr)Thermo Scientific
    pBDF019pJET1.2 derivative containing the slun21380 gene (fevR) and its upstream (217-bp) and
downstream (360-bp) regions (Ampr)
This study
    pSET152Integrative vector transmissible by conjugation from E. coli to Streptomyces spp. [lacZα,
ori (pUC18), aac(3)IV (Aprar), oriT (RK2), attP (ØC31), int (ØC31)]
42
    pBDF021pSET152 derivative containing the insertion of pBDF019 cloned in EcoRI and XbaI sites (Aprar)This study
    pBDF027pJET1.2 derivative containing an internal fragment (791 bp amplified with fevR+118f_XbaI
and fevR+909r_PstI primers) of the slun21380 (fevR) coding sequence (Ampr)
This study
    pSET151Nonreplicating plasmid in Streptomyces spp. [lacZα, oriT (RK2), xylE, tsr (Thior), bla (Ampr),
ori (pUC19)]
42, 43
    pBDF028pSET151 derivative containing the insertion of pBDF027 cloned in XbaI and PstI sites
(Ampr, Thior)
This study
    pUZ8002Nontransmissible plasmid supplying transfer functions for mobilization of oriT-containing
vectors from E. coli to Streptomyces spp. (Kanr)
41
    pAU3-45pSET152 derivative with a thiostrepton resistance gene inserted into the blunted NheI
restriction site [lacZα, ori (pUC18), aac(3)IV (Aprar), oriT (RK2), attP (ØC31), int (ØC31),
tsr (Thior)]
44
    pBDF029pBDF022 derivative containing the apramycin resistance cassette from pIJ773 cloned in
PstI and HindIII sites (Thior, Ampr, Aprar)
This study
    pIJ773pBluescript II SK(+)-based plasmid containing the apramycin resistance cassette [oriT (RK2)
+ aac(3)IV (Aprar)] flanked by FRT (FLP recognition target) recombination sites
43
    pBDF022pHJL401 derivative containing the insert of pBDF019 cloned in EcoRI and XbaI sites
(Thior, Ampr)
This study
    pHJL401Intermediate-copy-number (∼10 copies) vector in Streptomyces spp. [lacZα, ori (pUC19),
ori (SCP2), tsr (Thior), bla (Ampr)]
42
Strains
    E. coli DH5αGeneral cloning hostGibco-BRL
    E. coli ET12567Nonmethylating (dam, dcm, hsdS null mutants) host for transfer of plasmid DNA into
Streptomyces spp., used together with pUZ8002 (Cmlr, Tetr)
45
    Streptomyces lunaelactis
MM109T
Wild-type and reference strain of Streptomyces lunaelactis25
    Streptomyces lunaelactis
MM91
Wild-type strain Streptomyces lunaelactis MM9126
    Streptomyces lunaelactis
MM109BD1 (ΩfevR)
MM109 derivative with pBDF028 inserted to disrupt gene slun21380 (fevR) (Thior)This study
    Streptomyces lunaelactis
MM109BD2 (fevR+)
MM109T derivative with pBDF021 inserted to add an additional copy of slun21380
(fevR) controlled by its native promoter (Aprar)
This study
    Streptomyces lunaelactis
MM109BD3
Complementation of strain MM109BD1 (ΩfevR) with pBDF029 (Thior, Aprar)This study
  • a Ampr, ampicillin resistance; lacZα, LacZ galactosidase alpha subunit coding sequence; ori (pUC18/pUC19), origin of replication of the pUC18/pUC19 plasmid (E. coli); aac( 3)IV, apramycin resistance marker; Aprar, apramycin resistance; oriT (RK2), origin of conjugative transfer from plasmid RK2; attP (ØC31), phage attachment site of the ØC31 integrase [= int (ØC31)]; xylE, catechol 2,3-dioxygenase (reporter gene); tsr, thiostrepton resistance gene; Thior, thiostrepton resistance; bla, ampicillin resistance gene; dam, dcm, hsdS, genotype of methylase-deficient E. coli strain; Cmlr, chloramphenicol resistance; Tetr, tetracycline resistance.