Table 1 Bacterial strains and plasmids used in this study

Strain

 E. coli

  DH5α

F⁻, supE44, ΔlacU169 (φ80lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1

Life technologies

  GB05-red

An E. coli DH10B derive by deletion of fhuA, ybcC and recET, and insertion of the P_BAD-gbaA cassette at the ybcC locus

 S. cellulosum

  So0157-2

Epothilone producing strain

This lab

 M. xanthus

  DK1622

Wild type

[36, 37]

  DZ2

Wild type

  SW504

dif mutant, contains a difA in frame deletion

[22, 23]

Plasmid

 pACYC184

p15A ori; Cm^r

Novagen

 pKK843

With an 843-bp epothione promoter epop inserted in front of the promoter-less cat gene (epop-cat); Cm^r

 pKK-aphII

With an aphII promoter fragment inserted in front of the promoter-less cat gene (aphII-cat); Cm^r

 pMiniHimar-lacZ

Himar1 transposon (IR-Tpase-IR), lacZ; Km^r

[13, 14]

 pSWU30

Site specific integration vector with Mx8 attB

integration site (Mx8); Tet^r

 p15A-epop-cat

Ligating the epop-cat to the p15A ori; Cm^r

This study

 p15A-aph-cat

Ligating the aphII-cat to the p15A ori; Cm^r

This study

 pTp-epop

Ligating the IR-Tpase-IR to p15A-epop-cat; Cm^r

This study

 pTp-aph

Ligating the IR-Tpase-IR to p15A-aph-cat; Cm^r

This study

 pMx8-epop

Ligating Mx8 to p15A-epop-cat; Cm^r

This study

 pMx8-aph

Ligating Mx8 to p15A-aph-cat; Cm^r

This study

 Cosmid10

A plasmid from a Cosmid library of So0157-2 genome, containing foremost part of epothilone gene cluster (epoA, epoP, epoB, epoC and part of epoD); Amp^r, Neo^r

This study

 Fosmid3B11

A plasmid from a fosmid library of So0157-2 genome, containing latter part of epothilone gene cluster (partial epoC, epoD, epoE, epoF); Cmr

This study

 pSL1180

Cloning vector; Amp^r

Pharmacia

 pSL-UCD

Ligating the 2.0-kb epoup fragment, 2.2-kb epoCov fragment, and the 2.2-kb epodown fragment into pSL1180; Amp^r

This study

 pBJ113

Gene replacement vector with KG cassette; Km^r

 pGEM-Teasy

Cloning vector, Ampr

Promega

 pGEM-galk

Ligating the 1.7-kb galk gene to pGEM-Teasy; Ampr

This study

 pSL-ETG

Ligating the 3.2-kb TG cassette to pSL-UCD; Amp^r, Tet^r

This study

 pCVD442

A suicide plasmid; Amp^r

 pSL-ETGS

Ligating the 1.8-kb sacB gene to pSL-ETG; Amp^r, Tet^r

This study

 pSET152

Conjunction Vector; Apra^r

This study

 p15A-apra-cm

Ligating 1.7-kb apra fragment to p15A-aph-cat; Apra^r, Cmr

This study

 p15A-apra-cm-Tp

Ligating IR-Tpase-IR to p15A-apra-cm; Apra^r, Cmr

This study

 p15A-apra-cm-Mx8

Ligating Mx8 to p15A-apra-cm; Apra^r, Cmr

This study

 p15A-recTp

Ligating the 11.5-kb MunI-SpeI fragment from pSL-ETGS to p15A-apra-cm-Tp; Apra^r, Cmr, Tet^r

This study

 p15A-recMx8

Ligating the 11.5-kb MunI-SpeI fragment from pSL-ETGS to p15A-apra-cm-Mx8; Apra^r, Cmr, Tet^r

This study

 p15A-TP-fos

Recombination between the linearized Fosmid3B11 and p15A-recTp; Apra^r, Cmr, Tet^r

This study

 p15A-Mx8-fos

Recombination between the linearized Fosmid3B11 and p15A-recMx8; Apra^r, Cmr, Tet^r

This study

 p15A-Tp-CF

Recombination between the linearized Cosmid10 and p15A-TP-fos; Apra^r, Cmr

This study

 p15A-Mx8-CF

Recombination between the linearized Cosmid10 and p15A-TP-fos; Apra^r, Cmr

This study