Bacterial strains, plasmids, and general methods
Strains and plasmids used in this study are listed in Table 1. Streptomyces lividans ZX7  was the host for plasmid propagation and conjugal transfer. Streptomyces culture, isolation of plasmid and genomic DNA, preparation of protoplasts and transformation, and pulsed-field gel electrophoresis followed Kieser et al. . Plasmid conjugation from E. coli ET12567 (pUZ8002) into Streptomyces strains followed Bierman et al. . Plasmids pSP72 and pFX144 were used as cloning vectors. E. coli strain DH5α was used as cloning host. Plasmid isolation, transformation of E. coli and PCR amplification followed Sambrook et al. .
Isolation and identification of endophytic Streptomyces
Isolation of endophytic actinomycetes from Chinese medicinal herbs followed Cao et al. . The plant samples were submerged sequentially in 75% ethanol for 5 min, 0.9% sodium hypochlorite for 10 min, 10% sterile sodium bicarbonate for 10–20 min (10 min for leaf, 20 min for stem) and then washed by sterile water three times. The samples were cut into 1-cm2 pieces and were inserted in different media (e.g. TSB [Tryptone Soya Broth powder 30 g, agar 20 g/L] S [glucose 10 g, tryptone 4 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.1 g, CaCl2·2H2O 0.1 g, Ferric citrate reserving solution (1% (w/v) citric acid, 1% (w/v) ferric citrate) 1 ml, trace element solution (H3BO31.5 g, MnSO4·H2O 0.49 g, ZnSO4·7H2O 0.6 g, CuSO4·5H2O 0.1 g, (NH4)6(Mo7O2)4·4H2O 0.2 g, CoSO4·7H2O 0.01 g) 1 ml, agar 20 g/L] and Gause’s synthetic agar [soluble starch 20 g, KNO3, 1 g, NaCl 0.5 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, agar 20 g/L]) containing 25 ppm K2Cr2O4, 15 ppm nalidixic acid and 25 ppm nystatin. After incubation at 30°C for four weeks, actinomycete colonies were picked. Actinomycete strains were identified as Streptomyces strains by PCR amplification (primers: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-TCAGGCTACCTTGTTACGACTT3′) and sequencing of the 16S rRNA genes. The sequence of the 16S rRNA gene of Y27 was deposited in the GenBank under accession number JN207128.1.
Cloning and sequencing of Streptomyces plasmid pWTY27
pWTY27 DNA was digested with restriction endonucleases ApaI, BamHI, BclI, BglII, ClaI, EcoRI, HindIII, KpnI, MluI, NcoI, NheI, PstI, SacI, XbaI and XhoI to make a restriction map, and the unique SacI-digested plasmid DNA was cloned into pSP72 to obtain pYQ1. Shotgun cloning and sequencing of pYQ1 were performed on an Applied Biosystems Genetic Analyzer model 377 at the Chinese Human Genome Center in Shanghai. Analysis of Streptomyces protein coding regions was performed with “FramePlot 4.0 beta” (http://nocardia.nih.go.jp/fp4/), and ATG or GTG or TTG was used as start codons. Sequence comparisons and protein domain searching were done with software from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Blast.cgi). DNA secondary structures (e.g. direct repeats and inverted repeats) were predicted with “DNA folder” (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) and “Clone manager version 9” (http://www.scied.com/pr_cmpro.htm). The GenBank accession number for the complete nucleotide sequence of pWTY27 is GU226194.2.
Identification of a locus of pWTY27 for replication in Streptomyces lividans
Apramycin resistant transformants in S. lividans ZX7 were obtained for plasmid pWT24 carrying a 5.4-kb fragment (13942–14288/1–5114 bp of pWTY27). Various segments of the 5.4-kb sequence were PCR amplified and cloned in pFX144 to obtain plasmids pWT147, pWT219, pWT217 and pWT222. Sequences of 95 bp (1073–1167 and 259 bp (2433–2691 of pWT24 were deleted by digesting with MluI and NotI to obtain pWT34 and pWT33, respectively. These pWTY27-derived plasmids were constructed in E. coli DH5α and introduced by transformation into S. lividans ZX7. To compare transformation frequencies of plasmids in different experiments, we used 0.1 ng DNA (diluted from a concentrated solution) of Streptomyces plasmid pIJ702  each time and took 1 × 106 transformants per μg DNA as a control frequency.
Reverse transcription PCR assay
Strain Y27 was inoculated into tryptone soya broth (TSB, Oxoid) liquid medium, and RNA was isolated following Kieser et al. . The RNA samples were treated with DNase I (RNase-free, Takara) to remove possible contaminating DNA and reverse-transcribed into cDNA by using SuperScriptTM III Reverse Transcriptase (Invitrogen). Two primers (5′-GTGAATCTTGGGCTCGCCCTTG-3′/5′- GCCGAGAAGTGCATCCGCAAC-3′; the expected size of the PCR product is 302 bp) were used to allow amplification of segments extending from each replication gene into its immediate neighbor. PCR conditions were: template DNA denatured at 95°C for 5 min, then 95°C 30 s, 58°C 30 s, 72°C 30 s, for 30 cycles.
Electrophoretic mobility shift assay (EMSA)
The repA gene (621–2198 bp) of pWTY27 was cloned into the EcoRI and HindIII sites of E. coli plasmid pET28b to obtain pWT111, which was then introduced into E. coli BL21 (DE3). 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a log-phase culture at 16°C for 12 h to induce over-expression of the cloned gene. The 6His-tagged RepA protein was eluted in buffer containing imidazole and was purified to ~90% homogeneity by Ni2+ column chromatography following the supplier’s instructions (Qiagen). The 300-bp sequence (321–620) was PCR-amplified and end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (New England BioLabs). The DNA-binding reaction was performed at room temperature for 10 min in buffer (20 mM Tris–HCl at pH7.5, 100 mM NaCl, 1 mM ATP and 10% glycerol). PolydIdC DNA was used as non-specific competitor and unlabeled probe as specific competitor. The reaction complexes were separated on a 5% native polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 120 V for 1 h. Gels were dried and analyzed using the Phosphorimager (Fuji).
Similarly, the truncated traA gene (8124–9836 bp) of pWTY27 was cloned in pET28b to yield pWT371. The 6His-tagged TraA protein was purified by Ni2+ column chromatography and was incubated with the 175-bp (9803–9977) PCR fragment labeled with [γ-32P]ATP at room temperature for 15 min.
The DNase I footprinting assay followed Pan et al. . Primer FTr (5′-TCGAACACGCAACCGAAAGGCCG3′) was end-labeled with [γ-32P]ATP using T4 polynucleotide kinase, and then a 300-bp (321– 620) DNA fragment was PCR-amplified with primers 32PFTr and FTf (5′-CGGCCGCCGTCCGTCTGGTG-3′), followed by purification with the Wizard SV Gel and PCR Clean-Up System (Promega). Ca. 40-ng labeled DNA and different amounts (0.17, 0.43, 0.85 and 2.6 μg) of the purified RepA protein were incubated at room temperature for 10 min in a 56-μl binding buffer (20 mM TrisHCl pH 7.5, 100 mM NaCl, 1 mM ATP-Na, 10% glycerol). 1 Unit DNase I (Promega) was added for 1 min and the reaction was stopped by adding 50 μl stop solution (20 mM EGTA, pH 8.0). DNA was extracted with acid phenol/chloroform solution and precipitated with isopropanol and ethanol. Sequencing ladders were prepared with FTr using the SILVER SEQUENCETM DNA Sequencing Reagents (Promega). The digestion products together with the ladders were analyzed in 6% polyacrylamide (adding 7 M urea) gel. Gels were dried and scanned with the Phosphorimager.
Similarly, to determine the binding sequence of TraA protein and clt sequence, primer Fcltf (5′-CAAGGACTTCATGGACTGGTGCGA-3′,) was end-labeled with [γ-32P]ATP, and then a 406-bp (9671–10077) DNA fragment was PCR-amplified with primers 32PFcltf and Fcltr (5′-CGTGCTCGGCCTGCTCCAGGA-3′). About 40 ng labeled DNA and different amounts (0.6, 1.4, 2.8 and 4.2 μg) of the purified TraA protein were incubated at room temperature for 15min.
Identification of a locus for pWTY27 transfer in Streptomyces lividans
To identify a locus for plasmid conjugal transfer, various pWTY27 fragments around pWTY27.9 were cloned in E. coli plasmids pWT203 which contained the rep/rlrA/rorA genes required for replication and stable inheritance of the non-conjugative Streptomyces plasmid pSLA2 (31) or pWT224 (carrying intact traA). These plasmids were introduced by transformation into S. lividans ZX7 to produce donor strains for conjugation. The recipient strain was S. lividans ZX7 with a chromosomally integrating plasmid pWT181 containing the integrase gene of ΦC31  and selection marker tsr. About equal amount (ca.108) of spores of the donor and recipient strains were mixed and incubated at 30°C for 5 days. Spores were harvested, diluted in water and plated equally on Luria-Bertani (LB) medium (thiostrepton, 50 mg/L), LB (apramycin, 50 mg/L) and LB (thiostrepton + apramycin). The frequency of plasmid transfer = 100 × ratio of colonies on LB (thiostrepton + apramycin) to colonies on LB (apramycin).
Isolation of soil genomic DNA and PCR amplifications of the pWTY27 repA and oriC
Twelve soil samples from 12 cities in nine provinces (Wuhan, Huanggang and Xianning cities of Hubei, Changde and Hengyang of Hunan, Nanjin of Jiangsu, Linyi of Shandong, Anyan of Henan, Xingtai of Hebei, Guiling of Guangxi, Shanghai, and HongKong) in China were collected. Ca. 0.2-g soil sample and 0.5 g glass beads mixed in 1 ml buffer SLX Mlus were vibrated for 5 min and then were lysed in buffer DS at 90°C for 10 min. Crude genomic DNA was isolated by using the E.Z.N.ATM Soil DNA Kit (Omega). To amplify the pWTY27 repA from the soil DNA, nested PCR amplifications were employed . The first round of a PCR reaction was performed using primers (5′-CAGGTCAGGGTGCCCATGCCGTAC-3′, 5′-CGTACCCGCCTTGTACGTCCGCAG-3′) and KOD FX enzyme (Toyoba) under conditions (98°C 10 s, 60°C 30 s, 68°C 40 s for 30 cycles), and then 1 μl PCR product was added for the second round of the PCR reaction with primers (5′-CGGTCGCTCTGCTGCACCCAG-3′, 5′-GCGAGCCCAAGATTCACCGTCTG-3′) under conditions (98°C 10 s, 58°C 30 s, 68°C 30 s for 20 cycles). Similarly, to amplify the Y27 oriC, two primers (5′-ATGCACGCCGACCGCAAGATC-3′, 5′-AYRSGTTGCCGAACAGTGGACA-3′) were used for the first round, and nested primers (5′-CCACGGCCCCGAATCCGCCTC-3′, 5′- GCACAACACCGGCCTGCCTGTG-3′) for the second round of the PCR reactions. To amplify the A3(2) oriC, primers used in the first round reaction were the same as in the Y27 oriC, and new nested primers (5′-GCCTTTCCCATGCCCCT.GGGT-3′, 5′-CCTGCCCTGATGATCCCTCACCAG −3′) for the second round of the PCR reactions.