Bacterial strains, plasmids, and general methods
Strains used in this work are listed in Table 1. Plasmid isolation, transformation of E. coli DH5α and PCR amplification followed Sambrook et al. . Streptomyces culture, plasmid isolation and preparation of protoplasts and transformation of Streptomyces lividans ZX7 followed Kieser et al. . Plasmid trans-conjugation from E. coli ET12567/pUZ8002 into thermophilic Streptomyces strains followed Bierman et al. . KpnI-treated pTSC1 was cloned in pBluescript II SK to obtain pCWH100 and was sequenced by primer-walking at Shanghai Invitrogen Inc. Sequence comparisons were done with software from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST. The complete nucleotide sequence of pTSC1 was deposited in the GenBank database under no. GU271942.
Isolation and identification of thermophilic Streptomyces strains
Samples of garden soil, weed compost and swine manure were collected from Shanghai city, Hunan, Hubei and Fujian provinces in the summers of 2005 and 2006. The samples were dried at 100°C for 1 h and cultivated on SC medium (starch 10 g, casein 0.3 g, KNO3 2 g, MgSO4.7H2O 0.05 g, FeSO4.7H2O 0.01 g, CaCO3 0.02 g, agar 18 g, H2O to 1000 ml, pH7.2)  at 50°C for 3-5 d. Thermophilic Streptomyces strains were cultured in TSB (Oxoid tryptone soya broth powder, 30 g, H2O to 1000 ml) liquid medium at 45°C for 1 d and genomic DNA was isolated followed the Kirby mix procedure . 16S rRNA genes were amplified by PCR with primers (5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TCAGGCTACCTTGTTACGACTT-3'). PCR conditions were: template DNA denatured at 95°C for 5 min, then 95°C 30 s, 55°C 30 s, 72°C 2 min, for 35 cycles. PCR products were cloned in pBluescript II SK and sequenced with its T7 and T3 primers.
Strains were inoculated on MS (mannitol 20 g, soya flour 20 g, agar 20 g, H2O to 1000 ml, pH7) medium covered with cellophane disks. After 2 days incubation at 42°C, the cells were fixed with fresh 2% glutaraldehyde (pH7.2) and 1% osmium tetroxide. Spores were examined with a JSM-6360LV scanning electron microscopy (Jeol).
Isolation of plasmids from thermophilic Streptomyces strains
Isolating plasmid from thermophilic Streptomyces strains followed the protocol of Kieser  with sight modification. Strains were cultured in TSB liquid medium at 42°C overnight and mycelium was harvested by spinning at 4000 rpm for 15 min. About 50 μl mycelium was suspended in 350 μl TES buffer (25 mM Tris-HCL pH8, 25 mM EDTA pH8, 0.3 M sucrose, 2 mg/ml lysozyme, 5 μg/ml pre-boiled RNase A) and incubated at 37°C for 30 min. 44 μl of 10% SDS was added and mixed immediately by rotating and then 4 μl of 10 mg/ml proteinase K was added, followed by incubation for 60 min. 225 μl of 0.3 N NaOH/2% SDS was added and mixed immediately by vortexing, incubated at 70°C for 15 min and then cooled. 200 μl acid phenol/chloroform was added and vortexed and centrifuged at 12000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube containing 55 μl un-buffered sodium acetate and 500 μl isopropanol was added. After mixing and centrifugation at 12000 rpm for 10 min and all liquid was removed using a pipette. The pellet was washed twice with 1 ml 70% ethanol, air dried and dissolved in 50 μl TE buffer.
Growth curve of thermophilic Streptomyces strains in liquid culture
About 1.5 × 107 spores were inoculated into 50 ml TSB liquid medium supplemented with 0.01% antifoam289 (Sigma A 5551) and cultured at 30, 37, 45 and 50°C. 1 ml culture was harvested at each time-point and wet mycelium was harvested by centrifugation at 12000 rpm for 5 min. After drying for 10 min in a vacuum, the pellet was weighed with a fine balance (min. 10 mg). Growth curves were drawn with an average of three weighings at each time-point.
Protoplast preparation and transformation of thermophilic Streptomyces strains
Protoplast preparation, regeneration and transformation of the thermophilic Streptomyces strains 2C and 4F followed standard Streptomyces protocols [6, 45] with slight modifications. About 1 × 109 spores were inoculated into 50-ml YEME liquid medium (yeast extract powder 3 g, peptone 5 g, malt extract powder 3 g, glucose 10 g, with 25% sucrose, H2O to 1000 ml, pH7, supplemented with 0.5% glycine for 2C and 0.3% for 4F) at 45°C for ~7 h. Mycelium was harvested, washed once with 10.3% sucrose, and 1 mg/ml lysozyme solution in P buffer was added at 30°C (ca. 15 min for 2C and 30 min for 4F) to make protoplasts. After transformation, regeneration of protoplasts was achieved on R2YE medium at 45°C for ca. 9 h, to be selected by antibiotics.
Construction of plasmids for transformation of thermophilic Streptomyces strains
Plasmids used in this work are listed in Table 2. Sizes of circular plasmids pTSC1, pTSC2 and pTSC3 and linear plasmid pTSL1 from thermophilic Streptomyces strains were measured by electrophoresis with known DNA markers (i.e. 1-kb supercoiled ladder and sequenced circular/linear plasmids). pQC156  containing Streptomyces selection markers melC/tsr was cloned in an E.coli plasmid pSP72. KpnI-treated pTSC1 was cloned in pQC156 to obtain pCWH1. The mesophilic Streptomyces replicons, including circular plasmids SCP2 , pFP1 and pFP11, linear plasmids SAP1 , pFRL2  and pSHK1 , and integrating plasmid SLP1 , were cloned in pQC156 to yield pYQ40, pZR115, pZR10, pQC578, pHAQ61, pZR51, pGP9 and pZR205, respectively. These plasmids were introduced by protoplast transformation into thermophilic Streptomyces strains.
Cloning and heterologous expression of the actinorhodin gene cluster in thermophilic Streptomyces
pHAQ31  contained an E.coli replication origin and two cos sites of Supercos1  and Streptomyces selection markers melC/tsr genes . pHAQ31-derived cosmid N7-85 contained the whole actinorhodin biosynthetic gene cluster (5510413-5543521 bp) from S. coelicolor A3(2). A 3.4-kb XbaI/NheI fragment containing the phage фC31 integrase gene of pSET152 was cloned in a XbaI site of N7-85. The resulting plasmid, pCWH74, was introduced by conjugation from E. coli into thermophilic Streptomyces strains , which were cultured on R2YE (sucrose 103 g, K2SO4 0.25 g, MgCl2.6H2O 10.12 g, glucose 10 g, Difco Casaminoacids 0.1 g, trace element solution 2 ml, Difco yeast extract 5 g, TES 5.73 g, agar 22 g, H2O to 1000 ml, after autoclave and add 0.5% KH2PO4 5 ml, 5 M CaCl2.2H2O 4 ml, 20% L-proline 15 ml, 1N NaOH 7 ml) and MS media at 30, 37 and 45°C to detect blue actinorhodin pigment. To quantitate the production of actinorhodin, about 1 × 106 spores of M145 and 4F containing pCWH74 were inoculated into 50 ml R2YE liquid medium (lacking KH2PO4 and CaCl2) at 30 and 37°C; 1 ml culture was harvested in a time-course and treated with KOH, whereupon absorption at OD640 indicated actinorhodin production .
Heterologous expression of the anthramycin biosynthetic gene cluster in thermophilic Streptomyces
An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster (EU195114.1, 1-33150 bp) (kindly provided by Prof. Brian Bachmann) was introduced by conjugation from E. coli into strain 4F . Detection of anthramycin production followed Hu et al. . After culturing in AP1 (corn starch 10 g, 2% peptonized milk, yeast extract powder 30 g, H2O to 1000 ml, pH7) medium at 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Anthramycin was first isolated on a HPLC column (Zorbax eclips 1.8 μm XDB-C18) and then mass spectrometry was performed using 6520 Agilent Accurate-Mass Q-TOF LC/MS. Anthramycin was separated by using a Zorbax eclips 1.8 μm XDB-C18 with a linear water-acetonitrile gradient containing 10 mM ammonium acetate (0.2 ml/min). The electrospray needle of the mass spectrometer was at 4000 V, the voltage of the skimmer was set to 65 V, Oct RF Vpp750V, collision ev 45 V, nebulizer pressure at 45 psig, and drying gas N2 350°C 9 L/min.